WO2023042887A1 - 認知機能低下、または過体重もしくは肥満症を処置することに用いるための医薬組成物 - Google Patents
認知機能低下、または過体重もしくは肥満症を処置することに用いるための医薬組成物 Download PDFInfo
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- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
Definitions
- the present invention relates to pharmaceutical compositions containing antagonists of AMPA receptors for use in treating cognitive decline.
- the present invention also relates to pharmaceutical compositions comprising antagonists of AMPA receptors for use in treating diseases or conditions associated with NMDA-refractory NMDN receptors in subjects having NMDA-refractory NMDA receptors.
- the invention further provides pharmaceutical compositions for use in treating overweight and obesity.
- HFD high-fat diet
- AMPA receptors (hereinafter also referred to as "AMPAR") are involved in high-speed excitatory synaptic transmission, and play an important role in learning and memory in cooperation with NMDA receptors.
- the NMDA receptor (hereinafter also referred to as “NMDAR”) is a ligand-gated ion channel belonging to the ionotropic glutamate receptor family.
- NMDA receptors play an important role in excitatory synaptic transmission, learning and memory.
- AMPA receptors are expressed on postsynaptic membranes and induce millisecond-order hyperpolarization in the presence of glutamate (or AMPA), but do not have calcium permeability.
- NMDA receptors are expressed in the post-synaptic membrane and non-synaptic regions, and in the presence of glycine and glutamic acid (or NMDA), are activated with depolarization of the cell membrane to allow cations including calcium ions to flow into the cell.
- NMDA receptors are normally bound and inactivated by magnesium ions. Magnesium blocks of NMDA receptors are released when the AMPA receptors depolarize and raise the membrane potential of the membrane in which the NMDA receptor resides.
- NMDA receptors responsive to glycine and glutamate and lags the depolarization of the AMPA receptors in depolarizing the NMDA receptors and increasing intracellular calcium concentration, thereby generating a signal at the postsynaptic membrane.
- the hippocampus has the lowest firing threshold to stimulation in the brain and plays an important role in acquiring new memories and learning. For example, it is known that Alzheimer's dementia begins with damage to the entorhinal cortex (EC) and is caused by decreased hippocampal function. It can also cause epilepsy due to its low firing threshold. Perampanel is marketed as an AMPA receptor antagonist and is used to treat epilepsy.
- EC entorhinal cortex
- Perampanel is marketed as an AMPA receptor antagonist and is used to treat epilepsy.
- the present invention provides pharmaceutical compositions for use in treating cognitive decline.
- the present inventors discovered a new phenomenon in which the calcium permeability of AMPA receptors is improved in the presence of AMPA, while the activation of NMDA receptors is suppressed in the presence of NMDA.
- the inventors have also found that this new phenomenon occurs in overweight and obese subjects, as well as in subjects with cognitive impairment.
- AMPA receptors are calcium-impermeable, whereas in the examples described later, in the hippocampus of subjects exhibiting overweight and obesity, AMPA in the presence of AMPA It has been demonstrated that the calcium permeability of the receptor is improved and the activation of the NMDA receptor in the presence of NMDA is suppressed, and the control of the AMPA receptor and the NMDA receptor in the hippocampus of the subject is improved. It was shown that something was wrong. Subjects exhibiting overweight and obesity had some degree of cognitive decline.
- AMPA receptor antagonists can be used to inhibit AMPA receptor-mediated calcium influx to induce NMDA receptor release (ie, NMDA receptor activation).
- a pharmaceutical composition for use in treating cognitive impairment in a subject comprising an AMPA receptor antagonist (antagonist).
- the subject has suppressed NMDA receptors in at least one or more regions of the hippocampal body selected from the group consisting of entorhinal cortex (EC), CA1, CA2, CA3, and dentate gyrus (DG);
- EC entorhinal cortex
- CA1, CA2, CA3, and dentate gyrus (DG) entorhinal cortex
- DG dentate gyrus
- the subject has calcium-permeable AMPA receptors in at least one or more regions of the hippocampus, thereby suppressing activation of NMDA receptors in at least one or more regions of the hippocampus;
- [4] The pharmaceutical composition according to any one of [1] to [3] above, wherein the subject has cognitive impairment.
- the cognitive impairment is Alzheimer's dementia.
- [6] The pharmaceutical composition according to any one of [1] to [5] above, wherein the subject has a body mass index (BMI) of 25 or more.
- BMI body mass index
- the pharmaceutical composition according to any one of [1] to [6] above, wherein the subject has a BMI of 30 or more.
- a pharmaceutical composition comprising: [10] The pharmaceutical composition of any one of [1] to [9] above, wherein the AMPA receptor antagonist is perampanel.
- the present invention also provides, for example, the following inventions.
- [1A] A pharmaceutical composition for use in treating overweight or obesity in a subject with overweight or obesity, the pharmaceutical composition comprising an AMPA receptor antagonist.
- [2A] The pharmaceutical composition of [1A] above for use in delaying, suppressing, or halting the development of overweight or obesity in a subject with overweight or obesity.
- [3A] A pharmaceutical composition according to [1A] above for use in reducing body weight in a subject with overweight or obesity.
- [4A] A pharmaceutical composition according to [1A] or [3A] above for use in reducing food intake in a subject with overweight or obesity.
- [5A] The pharmaceutical composition according to any one of [1A] to [4A] above, for use in suppressing the development of diabetes, dyslipidemia, and cardiovascular disease in subjects with overweight or obesity. thing.
- [6A] The pharmaceutical composition of any one of [1A] to [5A] above, wherein the AMPA receptor antagonist is perampanel.
- Figure 1-1 shows that NMDA receptors are suppressed even in the presence of agonists in the hippocampus of model mice with obesity-induced cognitive decline, that AMPA receptors have calcium permeability in the presence of agonists, and We show that the AMPA receptor antagonist perampanel (PER) restores the interaction of AMPAR and NMDAR in the hippocampus of HFD-fed mice.
- Panel A shows AMPA (AMPA receptor agonist) and CTZ (uncoupling inhibitor) in the CD feeding group, HFD feeding group, and PER administration + HFD feeding group (hereinafter sometimes referred to as "HFD group with PER"). Fluo-3 fluorescence images of the hippocampus before and after administration when combined with .
- the right column of panel A is an image showing the difference between the pre-dose image and the post-dose image.
- Mice were 17 weeks old and HFD-fed and HFD-fed with PER groups were HFD-fed or HFD-fed with PER from 8 to 17 weeks of age.
- Panels BD show AMPA-dependent changes in normalized F525 (NF525) in hippocampal CA1, CA2, CA3, DG, and EC regions. F525 signals were measured under the conditions of the CD-fed group (panel B), HFD-fed group (panel C), and PER-administered HFD-fed group (panel D), respectively.
- Panel F shows the Fluo-3 fluorescence images of the hippocampus before and after the application of NMDA and glycine and their difference images in the CD, HFD, and PER-administered HFD-fed groups.
- Panels GI show NMDA-dependent changes of NF525 in hippocampal CA1, CA2, CA3, DG, EC regions. F525 signals were measured under CD (panel G), HFD (panel H), and HFD with PER treatment (panel I) conditions, respectively.
- Figure 1-2 shows that in the hippocampus of Alzheimer's dementia model mice (NL and NLGF), AMPA receptors have calcium permeability in the presence of agonists, and that the AMPA receptor antagonist perampanel (PER) is , shows restoring calcium permeability of AMPA receptors in the hippocampus of HFD-fed mice.
- Figures 1-3 show that NMDA receptors are suppressed even in the presence of agonists in the hippocampus of Alzheimer's disease model mice (NL and NLGF).
- Panel A shows qRT-PCR expression levels of AMPAR subunits, NMDAR subunits, and ADAR2 in hippocampal regions (CA1, CA3, DG, EC) of CD-, HFD-, and HFD-with-PER groups.
- Mice were 17 weeks old and HFD-fed and HFD-fed with PER groups were HFD-fed or HFD-fed with PER from 8 to 17 weeks of age.
- Panel B shows the Ca 2+ permeability index of the hippocampal regions (CA1, CA3, DG, EC) of the CD-fed group, the HFD-fed group, and the HFD-fed group with PER.
- Panel C shows that cell surface (middle column) and intracellular (right column) GluA1 protein expression in the hippocampal CA3 region of the HFD-fed group was increased and decreased in the HFD-fed group with PER, respectively.
- Panel D shows the analysis of Q/R editing in hippocampal regions (CA1, CA2, CA3, DG, EC) in CD- and HFD-fed groups. Mice were 17 weeks old and HFD-fed and HFD-fed with PER groups were HFD-fed or HFD-fed with PER from 6 to 17 weeks of age.
- FIG. 2-2 is a diagram following FIG. 2-1.
- Panels I and J show representative examples of swimming paths to the phantom platform (left of circle) for probe trials (end of trajectory represents starting point) (Panel I) and probe trials with Cue manipulation ( Panel J) shows the results.
- Panel K shows the time to goal of the Morris water maze pattern completion test by test day in the CD-fed group, the HFD-fed group, and the HFD-fed group with PER. Mice were 17 weeks old and HFD-fed and HFD-fed with PER groups were HFD-fed or HFD-fed with PER from 6 to 17 weeks of age.
- FIG. 3 shows morphological changes in the hippocampus in the HFD-fed group and the HFD-fed group with PER.
- Panel A shows brain coronal sections of BL/6 (16 weeks old), HFD-fed and HFD-fed with PER. A coronal section was obtained by MRI. White asterisks and diamonds indicate expansion of the third and bilateral ventricles in the HFD-fed group.
- Right bar graph shows mean volume of total intracranial volume (TIV), total gray matter (GM), and whole brain white matter (WM), and total gray matter (GM), and whole brain white matter (WM) versus TIV. Show the ratio.
- the left Y-axis shows TIV volume (mL) and the right Y-axis shows the volume ratio of GM or WM to TIV, respectively.
- Panel B shows mean volume per TIV of hippocampus CA1, CA3, DG, EC.
- Left and right bar graphs show left and right hippocampal results, respectively.
- the left Y-axis shows the ratio of GM to TIV for CA1, CA3 and DG (GM/TIV), and the right Y-axis shows the ratio of GM to TIV for EC (GM/TIV).
- Panels C and D show 3D images of the hippocampus of Thy-1 mice (17 weeks old).
- the cubic box frame in panel C has a length of 4000 ⁇ m, a width of 3000 ⁇ m and a height of 3000 ⁇ m, and each side of the small cube has a length of 400 ⁇ m.
- Panel D shows the CA1, CA1, The results of observing CA3 and DG are shown.
- Right bar graph shows cell density in Thy-1+-FYP mouse hippocampal CA1, CA3, DG regions.
- Panels EG show Golgi staining of the CA1 and DG regions (panel E, top) and percentage of spines of thin, stubby, mushroom type (panel E, bottom); Immunostaining of MAP2 Ab (antibody), anti-GluA1 Ab, and anti-GluA2 Ab (panel F, top); F, bottom panel); BL/6, HFD-fed group, anti-DCX+ staining in the hippocampal DG area of the group fed with PER (G, left panel), number of DCX+ positive cells/HFP (G, right panel).
- FIG. 4 shows calcium permeable (CP)-AMPA and NMDA receptor interactions, behavioral function, and brain volume in ob/ob mice.
- Panel A shows AMPA and NMDA receptor function (calcium transport capacity) in the EC, CA1, CA2, CA3, DG regions of obese mice (12 weeks old).
- An increase in normalized 525 with AMPA + CTZ administration means that AMPA receptors became calcium permeable, and no change in normalized 525 with NMDA + glycine administration means that NMDA receptors are inhibited.
- Panel B shows the function of AMPA and NMDA receptors in the EC, CA1, CA2, CA3, and DG regions of ob/ob mice (12 weeks old) subjected to PER from 8 to 12 weeks of age.
- No change in normalized 525 with AMPA + CTZ administration means that AMPA receptors are calcium impermeable, and an increase in normalized 525 with NMDA + glycine administration means that inhibition of NMDA receptors is released. do.
- Panel C is a graph of AMPA and NMDA receptor function in the EC, CA1, CA2, CA3, and DG regions of ob/ob and PER-treated ob/ob mice. Each factor in FIG. 4 was used at the following concentrations.
- AMPA 100 ⁇ M
- CTZ 100 ⁇ M
- NMDA 50 ⁇ M
- PER 100 ⁇ M
- glycine 10 ⁇ M
- Panel D Ca permeability index of hippocampal regions (CA1, CA2, CA3, DG, EC) of BL6/J mice, ob/ob mice, ob/ob mice subjected to PER (bottom right column) ).
- Panels EG show the results of behavioral analysis of ob/ob mice and ob/ob mice subjected to PER.
- Panel E shows the results of the running wheel activity test
- panel F shows the results of the 5-trial social memory assay
- panel G shows the results of the pattern-complementary navigation task. Mice were 11 weeks old and PER administration was performed from 6 to 11 weeks. Data are shown as mean ⁇ SE.
- Panel H shows external appearance of BL6 and ob/ob mice (top), gray matter images extracted from T1-weighted images of BL6 and ob/ob mice (middle), and gray matter volumes of BL6 and ob/ob mice (bottom).
- Panel I shows the results of Q/R editing analysis of hippocampal regions (CA1, CA2, CA3, DG, EC) of ob/ob mice (Mouse age: 12 weeks, PER administration: 6 weeks to 12 weeks). indicates Bar graphs show the ratio of molecular numbers of edited and non-edited GluA2 subunits.
- VIC VIC-labeled probe for detecting non-edited sequence
- FAM FAM-labeled probe for detecting edited sequence
- Panel J shows anti-MAP2 Ab (antibody ), showing the immunoreactive areas and GluA2/GluA1 ratios of anti-GluA1 Ab and anti-GluA2 Ab. *: p ⁇ 0.05, ***: p ⁇ 0.001.
- FIG. 5 shows the effect of increased BMI on fluctuations in human memory function and synaptic function.
- Panel A shows an anatomy of the right hippocampal subregion on a T2-weighted image. CA1-CA3, CA4/DG and cerebellum (SUB) are indicated.
- Panels C and D show the correlation between the percentage value of the BOLD signal in the right hippocampus CA3 and the percentage of correct answers in the recognition task.
- Panel E shows the mean percentage of correct answers in the recognition task. The mark is the data of each individual.
- Panels H to J show left and right CA3 activation maps, BOLD signal changes, and hippocampal memory tasks (New, Lure, Same) in the normal weight group (H), overweight group (I), and obese group (J). shows the percentage of correct answers from left to right.
- Color bar T value. *: p ⁇ 0.001.
- FIG. 6 shows the relationship between network connectivity and BMI in humans.
- Panel A shows DMN maps (upper) and anti-correlation maps to DMN (lower; anti.DMN) of the normal-weight group, overweight group, and obese group. Color bars indicate T values.
- Panel B shows functional network connectivity of the normal weight group (left column), overweight group (middle column) and obese group (right column). The diameter of each circle indicates the value of betweenness centrality.
- Panel C shows the mean and standard deviation of betweenness centrality and degree for each node. a. u. : Arbitrary units.
- FIG. 7 shows functional changes in AMPA and NMDA receptors in the HFD-fed group.
- Panel A shows functional changes (calcium transport capacity) of AMPA and NMDA receptors in the EC, CA1, CA2, CA3, and DG regions of HFD-fed mice (7 days from 16 to 17 weeks of age).
- Panel B shows the function of AMPA and NMDA receptors in the EC, CA1, CA2, CA3, DG regions of the HFD-fed group with PER (7 days from week 16 to week 17).
- Panel C is a graph showing the function of AMPA and NMDA receptors in the EC, CA1, CA2, CA3, and DG regions of the CD-fed group, the HFD-fed group, and the HFD-fed group with PER. *: p ⁇ 0.05, t-test.
- Panel D shows the inhibitory effect of 1-naphthylacetylspermine (Naspm) (selective antagonist of AMPA receptors) and GYKI (selective non-competitive AMPA receptor antagonist) in the HFD-fed group (HFD-fed for 7 days).
- Naspm 1-naphthylacetylspermine
- GYKI selective non-competitive AMPA receptor antagonist
- FIG. 8 shows the inhibitory effects of Naspm (a Ca 2+ -permeable AMPA receptor [CP-AMPAR] antagonist) and GYKI (a selective non-competitive AMPAR antagonist) in the hippocampus of ob/ob mice (12 weeks old).
- FIG. 9 shows the results of behavioral analysis of the open field test (panels AC), the elevated plus maze test (panels D, E), and the contextual fear conditioning test (panel F).
- Total travel time panel A
- total distance panel B
- Time panel C
- mean dwell time panel D
- mean number of open and closed arm entries panel E
- mean dwell time (F) are shown.
- FIG. 10-1 shows (A) the effects of AMPA receptor antagonists on CP-AMPA and NMDA receptor functions in the EC, CA1, CA2, CA3, and DG regions of ob/ob mice.
- PER 100 ⁇ M
- Glycine 10 ⁇ M
- APV 50 ⁇ M
- Naspm 10 ⁇ M
- GYKI 100 ⁇ M
- * p ⁇ 0.05, t-test.
- 10-1 shows the effect of leptin on NMDA receptor function in (B) ECs of ob/ob mice with PER, CA1, CA2, CA3, DG regions and (C) ECs of HFD-fed group with PER. , CA1, CA2, CA3, DG effects of leptin on NMDA receptor function.
- PER 100 ⁇ M
- Glycine 10 ⁇ M
- APV 50 ⁇ M
- Naspm 10 ⁇ M
- GYKI 100 ⁇ M
- * p ⁇ 0.05, t-test.
- FIG. 11 shows the results of quantitative analysis of the expression levels of MAP2, GluA1, and GluA2 in the CD-fed group, the HFD-fed group, and the HFD-fed group with PER.
- FIG. 12 shows immunostaining images of anti-MAP2 Ab, anti-GluA1 Ab, and anti-GluA2 Ab in ob/ob mice and 1-week and 12-week PER-administered ob/ob mice. Bar: 100 ⁇ m. Mice were 17 weeks old and PER administration was performed from 6 to 17 weeks.
- FIG. 12 shows immunostaining images of anti-MAP2 Ab, anti-GluA1 Ab, and anti-GluA2 Ab in ob/ob mice and 1-week and 12-week PER-administered ob/ob mice. Bar: 100 ⁇ m. Mice were 17 weeks old and PER administration was performed from 6 to 17 weeks.
- FIG. 13 shows the results of quantitative analysis of MAP2, GluA1, and GluA2 expression in ob/ob mice and 1-week and 12-week PER-treated ob/ob mice. Bar: 100 ⁇ m. Mice were 17 weeks old and PER administration was performed from 6 to 17 weeks.
- FIG. 14 shows the results of anti-DCX+ staining and DCX+ positive cell count/HFP in the hippocampal DG region of ob/ob mice and 1-week and 12-week PER-administered ob/ob mice. Inset shows DCX+ neuronal progenitor cells. Bar: 100 ⁇ m and 20 ⁇ m (inset). Mice were 17 weeks old and PER administration was performed from 6 to 17 weeks.
- FIG. 15 T-value maps of gray matter (GM) volume and voxel intensity in the GM region of the dentate gyrus, hypothalamus, somatosensory cortex, and cerebellum of ob/ob mice (11 weeks old) (panels A and B). , and anatomical ROI maps (MRI images) of the hippocampal dentate gyrus, hypothalamus, somatosensory cortex, and cerebellum of CD mice in MRI images (panel C). Bar: 1mm.
- FIG. 16 shows surface expression levels and intracellular pool levels of hippocampal GluA1 subunits in ob/ob mice.
- Panel A shows GluA1 surface expression (upper row), intracellular pool amount (middle row) and internal control in ob/ob mice (6 or 17 weeks old, or PER administration group (11 weeks to PER administration)). The expression level of a certain actin (lower row) is shown.
- Panel B shows the relative expression levels of GluA1 on the cell surface. For the relative expression level, the expression level of GluA1 was normalized by the expression level of actin.
- Panel C shows the relative expression levels of GluA1 in intracellular pools.
- FIG. 17 shows the relationship between human whole brain GM volume, body weight, BMI, and age. Panels A and B show representative T1-weighted coronal images of normal weight group (panel A) and obese group (panel B).
- FIG. 18 shows the relationship between BMI and percentage of correct answers in new tasks and lure tasks.
- FIG. 18 shows the relationship between BMI and percentage of correct answers in new tasks and lure tasks.
- FIG. 19 shows the relationship between the volume of each region of the human hippocampus by MRI and overweight or obesity.
- Panels A to C show the mean of normal weight group (panel A), overweight group (panel B) and obese (panel C) (*: p ⁇ 0.05). The points in the graph are the data of each individual.
- FIG. 20 shows the volume of each region of the hippocampus of CD-fed and ob/ob mice by volumetric analysis using MRI images.
- Panel A shows whole brain volumes of CD-fed and ob/ob mice (13 weeks old).
- Panel B shows body weights of CD-fed and ob/ob mice (13 weeks old).
- Panel C shows the volume of each region of the left hippocampus of CD-fed and ob/ob mice (13 weeks old).
- Panel D shows the volume of each region of the right hippocampus of CD-fed and ob/ob mice (13 weeks old).
- FIG. 21 shows levels of R/Q editing of GluA2 in CD-fed, HFD-fed, HFD-fed with PER, ob/ob, PER-treated ob/ob. The level of R/Q editing was calculated as a ratio of the total number of reads aligned to the R/Q editing site of GluA2.
- Mice were BL/6J 17 weeks old and fed HFD or HFD with PER from 16 to 17 weeks or from 6 to 17 weeks. PER treatment of ob/ob mice was between 16 and 17 weeks.
- FIG. 21 shows levels of R/Q editing of GluA2 in CD-fed, HFD-fed, HFD-fed with PER, ob/ob, PER-treated ob/ob.
- the level of R/Q editing was calculated as a ratio of the total number of reads aligned to the R/
- Panel 22 shows the quantitative results of Thy1-YFPH+ cells in the hippocampal CA1 region.
- Panel A shows a whole hippocampal image ( ⁇ 10 ⁇ water immersion objective) of the CD-fed group (17 weeks old). Voxel size was X-axis, 4.013 ⁇ m; Y-axis, 4.013 ⁇ m; Z-axis, 5.016 ⁇ m. A portion surrounded by a square is a cut-out region. Bar: 400 ⁇ m.
- Panel B is a panel representation of selected cells.
- Panel C is a 3D reconstruction of the segmented region.
- Panel D is a Bob filter rendered image (average size, 20 ⁇ m, threshold, 5 ⁇ m).
- FIG. 23 shows the effect of perampanel administration on cognitive decline in Alzheimer's dementia model mice.
- FIG. 24 shows photographs of the appearance of wild-type mice, ob/ob mice that are an obesity model, and ob/ob mice administered AMPA receptor antagonists.
- FIG. 25 shows the effect of AMPA receptor antagonist administration on body weight change in HFD-fed groups with equivalent food intake.
- Figure 26 shows the effect of antiepileptic treatment on hippocampus and body weight in a 70 year old female with a BMI value of 38. The woman had a foramen magnum and had quadriplegia and respiratory distress. MRI images of the brain 26 weeks after administration (oral administration) of perampanel (PER) as an antiepileptic treatment after tumor excision are shown.
- PER perampanel
- FIG. 27 shows the changes in treatment, changes in body weight, and changes in lower extremity Fugl-Meyer assessment score (FMA score) for the 70-year-old woman. BMI decreased to 31 after 26 weeks of PER administration.
- FIG. 28 shows that NMDA receptor inactivation in the HFD-fed group is calcium ion dependent.
- FIG. 29 shows the effect of perampanel (PER) administration on the A ⁇ 42/A ⁇ 40 ratio in neurospheres established from Alzheimer's dementia model NL-GF(-/-) mice.
- Figure 30 shows the effect of perampanel (PER) administration on A ⁇ accumulation in neurospheres established from NL-GF(-/-) mice.
- Figure 31 shows the effect of perampanel administration on A ⁇ accumulation in hippocampal dentate gyrus (DG) CA1 and cerebral cortex (Cx) in Alzheimer's dementia model mice (NL-GF(-/-)).
- FIG. 32 shows marked enhancement of epileptiform responses in the CA1 and CA2 regions by biclin (BIC) administration to NL-GF(-/-) mice.
- FIG. 33 shows the effect of administration of perampanel on epileptiform reactions induced by biclin in NL( ⁇ / ⁇ ) mice.
- FIG. 34 shows the effects of memantine administration (mema), exercise load (exercise), and perampanel administration (PER) in a novel object recognition test for NL-GF(-/-) mice.
- FIG. 35 shows the effects of memantine administration (mema), exercise load (exercise), and perampanel administration (PER) in fear conditioning tests on NL-GF(-/-) mice.
- a "subject" is a mammal, such as a primate, such as a human.
- Humans are, for example, 0 to 10 years old, 10 to 20 years old, 20 to 30 years old, 30 to 40 years old, 40 to 50 years old, 50 to 60 years old, 60 to 70 years old, from 70 years old 80 years old, and over 80 years old, and can be any age in the above age ranges. Humans can be male or female.
- cognitive function refers to intellectual functions such as comprehension, judgment, and logic; is understood to include As used herein, a “cognitive dysfunction” is an impairment (or hypofunction, or dysfunction) in higher brain functions, including judgment, calculation, comprehension, learning, orientation, and memory.
- a “cognitive impairment” can be a memory impairment, which in dementia can manifest as memory impairment such as forgetfulness (eg, working memory decline and working memory impairment).
- Cognitive impairment is diagnosed in an environment that is suitable for testing cognitive and memory function, or that allows for concentration or concentration.
- treatment refers to prophylactic treatment and therapeutic treatment.
- therapeutic treatment means treating, curing, preventing or ameliorating the remission of, or reducing the rate of progression of, a disease or disorder.
- prophylactic treatment means reducing the likelihood of developing a disease or condition or delaying the onset of a disease or condition.
- terapéuticaally effective amount means an amount that provides therapeutic or prophylactic treatment.
- NMDA receptor is a kind of ion channel glutamate receptor present in the post-synaptic membrane, and is also called NMDA glutamate receptor. Ionotropic glutamate receptors present in the post-synaptic membrane are broadly classified into AMPA receptors, kainate receptors, and NMDA receptors according to their pharmacological characteristics. NMDA receptors are considered to be involved in memory, learning, cell death after cerebral ischemia, and the like. NMDA receptors, unlike other ionotropic glutamate receptors, are selectively activated by N-methyl-D-aspartate (NMDA; NMDA receptor agonists).
- NMDA N-methyl-D-aspartate
- NMDA receptors are bound to extracellular magnesium ions (Mg 2+ ) at -60 mV to -70 mV, which corresponds to the resting membrane potential, and their channel activity is inhibited.
- Mg 2+ extracellular magnesium ions
- the postsynaptic membrane depolarizes (e.g., the membrane potential is positive or greater than -20 mV, e.g., -20 mV to -10 mV)
- inhibition by magnesium ions is released and NMDA receptors are activated by glutamate in the presence of glycine. and induces the influx of cations from the postsynaptic membrane into the cell, particularly calcium ions (Ca 2+ ), potassium ions (K + ), and sodium ions (Na + ).
- NMDA receptors undergo post-synaptic membrane depolarization (electrical signal; post-synaptic membrane neural activity) and glutamate stimulation (biochemical signal; pre-synaptic terminal neural activity), resulting in channel activity. express.
- AMPA receptor is a type of ionotropic glutamate receptor present in the post-synaptic membrane, and is also called AMPA-type glutamate receptor.
- AMPA receptors are selectively activated by ⁇ -amino-3-hydroxy-5-mesoxazole-4-propionic acid (AMPA; AMPA receptor agonist).
- AMPA receptors are tetramers containing any four of four subunits: GluA1, GluA2, GluA3, and GluA4. Each subunit has a glutamate acceptor site, and channel activity is believed to be induced when glutamate binds to two or more of the four.
- AMPA receptors containing GluA2 are calcium-impermeable, whereas AMPA receptors without GluA2 are calcium-permeable (hereinafter, calcium-permeable AMPA receptors are referred to as "CP-AMPA receptors").
- CP-AMPA receptors calcium-permeable AMPA receptors
- AMPA receptors, including GluA2 are calcium-impermeable because glutamine (Q) in the M2 domain (second hydrophobic domain) of GluA2 is converted to arginine (R).
- Q glutamine
- R arginine
- the editing from Q to R (Q/Reediting) of GluA2 is due to editing at the mRNA level (post-transcriptional regulation).
- the conversion from Q to R is achieved by deaminating the second base (adenine) of the codon specifying glutamine in GluA2 mRNA by adenine deaminase and converting it to inosine; It is generated when the inosine is decoded as guanine by tRNA. Since AMPA receptors containing unedited GluA2 are calcium permeable, the calcium impermeability of AMPA receptors is likely due to the above Q to R conversion of GluA2.
- nucleic acid sequence of human GluA2 is set forth in SEQ ID NO: 1
- an example of the amino acid sequence of unedited human GluA2 is set forth in SEQ ID NO: 2
- an example of the amino acid sequence of edited human GluA2 is set forth in SEQ ID NO: 3.
- the 607th Q in SEQ ID NO: 2 is converted to R by editing. This site is called the Q/R site of GluA2.
- the AMPA receptor component is known to be faster than the NMDA receptor component.
- Example nucleic acid sequences of human GluA1, human GluA3, and human GluA4 are set forth in SEQ ID NOs:4, 6, and 8, and example amino acid sequences are set forth in SEQ ID NOs:5, 7, and 9, respectively.
- the "hippocampus” is the cortex located at the base of the inferior horn of the lateral ventricle in the medial part of the temporal lobe. There is one hippocampus on each side of the brain.
- the hippocampus is part of the limbic system called the hippocampus, which includes the dentate gyrus, hippocampus, subiculum, presubiculum, parasubiculum, and entorhinal cortex. (entorhinal cortex).
- the hippocampus is roughly divided into CA1 to CA3 regions. The hippocampus is believed to be involved in various neuropsychiatric disorders such as epilepsy and Alzheimer's dementia.
- the hippocampus has the lowest seizure threshold in the brain, and in epilepsy animal models much of the electrical activity associated with seizures is recorded from the hippocampus, particularly originating from areas CA2 and CA3.
- An early symptom of Alzheimer's dementia is the inability to acquire new memories.
- the entorhinal cortex is where the pathology of Alzheimer's dementia first appears. In Alzheimer's dementia, it is thought that the information processing ability of the hippocampus is deprived due to damage to the entorhinal cortex. Thus, the hippocampus has a low threshold for firing and has important functions in storing and learning new memories.
- the hippocampus is vulnerable to hypoxia and ischemia, and induces neuronal cell death under hypoxic and ischemic conditions.
- Neuronal cell death is believed to be due to NMDA receptor-mediated excitotoxicity.
- the hippocampus is believed to be essential for the formation of explicit memories such as episodic memories. Areas of the temporal lobe cortex involved in memory formation include the entorhinal cortex, the parasubiculum, the presubiculum, the subiculum, the hippocampus (Ammon's horn), and the dentate gyrus. Inactivation of NMDA receptors in the hippocampus causes cognitive impairment, including memory and learning deficits. Alzheimer's disease includes a condition that eventually progresses to Alzheimer's dementia but has no clinical symptoms (preclinical Alzheimer's disease), mild cognitive impairment (MCI) that progresses to Alzheimer's dementia, and Alzheimer's dementia.
- MCI mild cognitive impairment
- Alzheimer's disease a decrease in A ⁇ 42 levels in the cerebrospinal fluid, an increase in total tau levels, and an increase in phosphorylated tau levels can be seen from the preclinical stage of Alzheimer's disease.
- Decreased levels of A ⁇ 42 in the cerebrospinal fluid are thought to reflect accumulation of A ⁇ 42 in the brain.
- Increased tau accumulation, neurofibrillary tangles, and neuronal loss can lead to mild cognitive impairment.
- Mild cognitive impairment involves lesions in the perihippocampal region. In Alzheimer's dementia, lesions extend to the cerebral neocortex. Dementia of the Alzheimer's type is characterized, for example, by the presence of senile plaques and/or neurofibrillary tangles.
- Dementia of the Alzheimer's type can be diagnosed, for example, based on diagnostic criteria according to the American Psychiatric Association Diagnostic and Statistical Handbook, 5th Edition (DSM-5). That is, dementia of the Alzheimer's type can be diagnosed if: A, meets the criteria of major neurocognitive disorder; B, at least two or more cognitive impairments appear insidiously and progress slowly; C. Evidence from family history or genetic testing for the presence of mutations in the Alzheimer's disease-causing gene OR all of the following: Clear evidence of deterioration in memory/learning and other cognitive functions on detailed medical history and longitudinal neuropsychological testing No other neurodegenerative, cerebrovascular, neurological/psychiatric/systemic disease or condition likely to cause cognitive impairment; D. Clear evidence of deterioration in memory, learning, and other cognitive functions on detailed medical history and neuropsychological testing over time. No other neurodegenerative, cerebrovascular, neurological/psychiatric/systemic disease or condition likely to cause cognitive impairment.
- the term “overweight” refers to humans (especially men and women aged 18 and over) with a Body Mass Index (BMI) of 25 or more and less than 30. BMI is determined by weight (kg)/ ⁇ height (m) ⁇ 2 . The standard BMI value for both adult men and women is about 22.
- “obesity” refers to humans (particularly adult men and women) with a BMI of 30 or higher. Obesity is classified into obesity class I with a BMI of 30 or more and less than 35, obesity class II with a BMI of 35 or more and less than 40, and obesity class with a BMI of 40 or more according to the standards of the World Health Organization (WHO) (WHO standards). It is roughly divided into III.
- WHO World Health Organization
- metabolic syndrome means a BMI of 25 or more, a waist circumference of 85 cm or more for men and 90 cm or more for women, and satisfying two or more of the following (1) to (3).
- Neutral fat is 150 mg/dL or more, or HDL is less than 40 mg/dL.
- Systolic blood pressure is 130 mmHg or higher, or diastolic blood pressure is 85 mmHg or higher.
- Fasting blood sugar is 110 mg/dL or more. Obesity in children can be determined by the degree of obesity.
- obesity in children is defined as a degree of obesity of +20% or more, an increase in body fat percentage of 25% or more for boys, an increase of 30% or more for girls under the age of 11, and an increase of 30% or more for girls aged 11 and over. For females, it means an increase of 35% or more.
- the degree of obesity (%) is obtained by (current body weight ⁇ standard body weight for that age)/standard body weight for that age ⁇ 100.
- Dyslipidemia refers to a condition in which the metabolism of lipids such as triglycerides and cholesterol is abnormal.
- Dyslipidemia is broadly classified into hyper-LDL-cholesterolemia, hypo-HDL-cholesterolemia, and hyper-triglyceridemia.
- Hyper-LDL cholesterolemia can be diagnosed based on whether the blood LDL cholesterol level is 140 mg/dL or higher.
- HypoHDL cholesterolemia can be diagnosed by blood HDL cholesterol levels of less than 40 mg/dL.
- Hypertriglyceridemia can be diagnosed based on whether the blood triglyceride level is 150 mg/dL or more.
- Cardiovascular disease refers to a group of diseases in which arteriosclerosis causes narrowing of the inner walls of blood vessels, resulting in insufficient blood supply to organs.
- Cardiovascular disease includes coronary artery disease, stroke, cerebral infarction, myocardial infarction, and peripheral artery disease. If conditions such as hyperglycemia continue, the inner walls of blood vessels are damaged, and cholesterol accumulates on the walls of the blood vessels. Over time, the buildup increases, constricting the vessel lumen and reducing the blood supply. A decrease in blood flow to the brain can result in a stroke, and a decrease in blood flow to the heart can result in a myocardial infarction. Others can cause coronary artery disease and peripheral artery disease. Dyslipidemia and arteriosclerosis have been cited as risk factors for cardiovascular disease.
- diabetes is a disease in which hyperglycemia continues chronically due to insufficient action of insulin (for example, lack of insulin or lack of responsiveness to insulin). Diabetes is often accompanied by three major complications: retinopathy, nephropathy, and neuropathy. Diabetes is diagnosed by meeting any of (1) to (3) and (4) below: (1) morning fasting blood glucose ⁇ 126 mg/dL, (2) 75 g fluorescent glucose tolerance test. 2-hour value of 200 mg/dL or higher; (3) Blood glucose level measured regardless of time of 200 mg/dL or higher; (4) HbA1c of 6.5% or higher;
- AMPA receptors in the presence of glutamate have enhanced calcium permeability
- NMDA receptors in the presence of glutamate or in the presence of NMDA
- NMDA-refractory a disease or condition associated with NMDA-refractory in a subject having glutamate (or NMDA)-refractory NMDA receptors (hereinafter also simply referred to as "NMDA-refractory”)
- a pharmaceutical composition comprising an AMPA receptor antagonist is provided.
- the hippocampus of NMDA-refractory subjects has NMDA receptors that are suppressed regardless of the membrane potential of postsynaptic membranes in the presence of glycine and NMDA. This results in complete or partial inhibition of NMDA receptors in the hippocampus of NMDA-refractory subjects in the presence of glycine and NMDA, independent of the membrane potential of postsynaptic membranes.
- suppression of NMDA receptors can be suppression of calcium influx into cells (and preferably suppression of elevation of intracellular calcium concentration) via NMDA receptors.
- AMPA receptors have acquired agonist-dependent calcium permeability.
- the disease or condition associated with NMDA refractory can be cognitive impairment, including cognitive decline.
- cognitive impairment can result from overweight or obesity.
- cognitive impairment can result from dementia of the Alzheimer's type.
- BOLD blood oxygen level dependent
- NMDA-refractory subjects may also have a ratio of BOLD responses during rest (no task) to event-related memory tasks that is less than or equal to a second predetermined ratio, as described below.
- Elevated BOLD responses suggest hyperactivity of synaptic function, and depression suggests synaptic dysfunction.
- NMDA unresponsiveness can be assessed by unresponsiveness of brain activity to NMDA administration. In living organisms, when working on a hippocampal function task using functional magnetic resonance imaging (fMRI), the correct answer rate decreases and the BOLD signal becomes persistently low.
- fMRI functional magnetic resonance imaging
- the suppression of NMDA receptors by AMPA receptor antagonists suppresses calcium influx from AMPA receptors (especially hippocampal calcium-permeable AMPA receptors).
- AMPA receptors especially hippocampal calcium-permeable AMPA receptors.
- subjects exhibiting overweight and obesity may have certain cognitive impairments due to the above-described abnormalities in the regulation of AMPA and NMDA receptors in the hippocampus.
- the calcium permeability of AMPA receptors is enhanced in the presence of AMPA, while the activation of NMDA receptors is suppressed in the presence of NMDA.
- a pharmaceutical composition for use in treating cognitive impairment including cognitive decline, hereinafter the same
- the pharmaceutical composition comprising an AMPA receptor antagonist.
- AMPA receptor antagonists include competitive antagonists and non-competitive antagonists (eg, allosteric antagonists).
- a non-competitive antagonist can be preferably used as the AMPA receptor antagonist.
- AMPA receptor antagonists also include AMPA receptor selective antagonists (eg, AMPA receptor selective competitive antagonists and AMPA receptor selective non-competitive antagonists).
- AMPA receptor-selective antagonists have a greater inhibitory effect on AMPA receptors than on other ionotropic glutamate receptors such as NMDA receptors and kainate receptors (e.g., IC 50 for AMPA receptors is higher than other 2-fold or less, 3-fold or less, 5-fold or less, 10-fold or less, 30-fold or less, 50-fold or less, 100-fold or less, 300-fold or less, 500-fold or less, 1000-fold or less of the IC 50 for ionotropic glutamate receptors of , 3000 times or less, 5000 times or less, or 10000 times or less).
- an AMPA receptor selective antagonist can be preferably used.
- AMPA receptor antagonists include, but are not limited to, 2,3-benzodiazepine compounds, 4-(8-chloro-2-methyl-11H-imidazo[1,2-c][2,3]benzodiazepine-6- Benzenamine (GYKI47261), 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI52466), and 1-(4-aminophenyl)-3- methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine (GYKI53655) (Paternain et al., Neuron 1995, 14:185-189), 2, 3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 6 ,
- 1,2-dihydropyridine compounds such as 3-(2-cyanophenyl)-5-(2-pyridyl)-1-phenyl-1,2- Dihydropyridin-2-one (perampanel; E2007) (US 6,949,571B), 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline, [1,2,3,4,- Tetrahydro-7-morpholin-yl-2,3-dioxo-6-(trifluoromethyl)quinoxalin-1-yl]methylphosphonate, 1-(4-aminophenyl)-4-methyl-7,8-methylene-dioxy -5H-2,3-benzodiazepine or (-) 1-(4-aminophenyl)-4-methyl-7,8-methylene-dioxy-4,5-dihydro-3-methylcarbamoyl-2,3-benzodiazepine , [7-(2-aminophenyl)-4-methyl-7,8-methylene
- the pharmaceutical compositions of the present disclosure comprise a therapeutically effective amount of an AMPA receptor antagonist. In some aspects, the pharmaceutical compositions of this disclosure comprise a therapeutically effective amount of an AMPA receptor selective antagonist. In some aspects, the pharmaceutical compositions of this disclosure comprise a therapeutically effective amount of one or more selected from the group consisting of GYKI47261, Naspm, and perampanel.
- the subject is a human.
- the subject is a human (eg, male or female) 18 years of age or older.
- the subject is overweight. In one aspect, the subject is obese. In one aspect, the subject has Class I obesity. In one aspect, the subject is obese class II. In some aspects, the subject has Obesity Class III.
- the subject has cognitive impairment (including cognitive decline). In some embodiments, the subject has dementia and is overweight. In some aspects, the subject has cognitive impairment and is obese selected from the group consisting of obesity classes I-III. In some embodiments, the subject is overweight or obese and has decreased hippocampal memory function of pattern complementation compared to normal subjects. In some embodiments, the subject has cognitive impairment but is not overweight or obese.
- the cognitive impairment is memory impairment (including deterioration of memory function).
- the memory impairment can be social memory impairment.
- the memory impairment can be a reduced ability to complete patterns.
- the cognitive impairment is a learning disability (including a decline in learning function).
- the memory impairment can be a memory impairment in social memory.
- the subject has dementia. In some embodiments, the subject has dementia and is overweight. In some aspects, the subject has dementia and is obese selected from the group consisting of obesity classes I-III. In some embodiments, the subject has dementia but is not overweight or obese.
- the subject has dementia of the Alzheimer's type. In some embodiments, the subject has dementia of the Alzheimer's type and is overweight. In some aspects, the subject has dementia of the Alzheimer's type and is obese selected from the group consisting of obesity classes I-III. In some embodiments, the subject has dementia of the Alzheimer's type but is not overweight or obese. In certain aspects, the patient with dementia of the Alzheimer's type can be a patient with epilepsy. In some embodiments, a patient with dementia of the Alzheimer's type can be a patient without epilepsy. Epilepsy is a disease that causes repeated epileptic seizures, such as sudden loss of consciousness and unresponsiveness.
- a subject with cognitive impairment has a lower gray matter volume than that of a healthy subject.
- central executive network activation is observed in resting brain activity in subjects with cognitive impairment.
- subjects with cognitive impairment exhibit a connection of the central executive network to the default mode network in resting brain activity.
- a subject with cognitive impairment exhibits visual network activation in resting brain activity.
- activation of the central executive network and activation of the visual network is observed in subjects with cognitive impairment.
- a subject with cognitive impairment has a connection between the saliency network and the cerebellar network in resting brain activity.
- a subject with cognitive impairment may particularly be a subject with obesity or overweight.
- the network has been developed as a biomarker of human cognitive function (35). Specifically, the network can be determined by recording a subject's resting brain activity by fMRI and analyzing the connectivity of brain activation (35).
- a subject with cognitive impairment has a change ratio of BOLD responses in the CA3 region of the hippocampus that is greater than or equal to a first predetermined ratio.
- the first predetermined ratio is 1.1 times or more, 1.11 times or more, 1.12 times or more, 1.13 times or more, 1.14 times or more, 1.15 times or more, 1.16 times or more, It can be 1.17 times or more, 1.18 times or more, 1.19 times or more, or 1.2 times or more.
- the upper limit of the first predetermined ratio can be, for example, either 1.3 times or a scaling factor with respect to the first predetermined value indicated above.
- a subject with cognitive impairment has a ratio of change in BOLD responses in the CA3 region of the hippocampus that is less than a second predetermined value.
- the second predetermined ratio is 0.9 times or less, 0.89 times or less, 0.88 times or less, 0.87 times or less, 0.86 times or less, 0.85 times or less, 0.84 times or less, It can be 0.83 times or less, 0.82 times or less, 0.81 times or less, or 0.8 times or less.
- the lower limit of the second predetermined ratio can be, for example, 0.7 times or more, or any of the scaling factors for the second predetermined value indicated above.
- the ratio of change in BOLD responses is determined by measuring BOLD responses by functional MRI (fMRI) in the presence and absence of the behavioral task.
- Behavioral tasks include, for example, event-related memory tasks.
- event-related memory task for example, the same image, a similar image, and a new image can be checked to see if the subject can correctly answer the same image, a similar image, and a new image, respectively.
- the percentage of correct answers can be measured.
- the subject has suppressed NMDA receptors in at least one or more regions of the hippocampal formation selected from the group consisting of entorhinal cortex (EC), CA1, CA2, CA3, and dentate gyrus (DG). wherein the activity of NMDA receptors by NMDA is inhibited in at least one or more regions of said hippocampus, and the subject has cognitive impairment.
- Inhibition of NMDA receptors is inhibition of NMDA receptor activation in the presence of glutamate and glycine (and preferably magnesium ion ions) (ie, under conditions in which NMDA receptors are activated in a physiological environment). Failure to activate NMDA receptors when they should be can result in memory and learning impairments.
- AMPA receptor antagonists are effective in derepressing NMDA receptors. Then, by releasing the suppression of NMDA receptors, the NMDA receptors regain their original functions, and cognitive impairment can be improved.
- pharmaceutical compositions comprising AMPA receptor antagonists of this disclosure may be used in combination with NMDA receptor agonists.
- NMDA receptor agonists include Lapastinel (PCT/US2017/015851).
- hippocampal formation selected from the group consisting of entorhinal cortex (EC), CA1, CA2, CA3, and dentate gyrus (DG). Having suppressed NMDA receptors in the above regions, whereby NMDA-mediated activation of NMDA receptors is suppressed in at least one or more regions of said hippocampus, and the subject may have cognitive impairment.
- the calcium permeability of AMPA receptors can be improved by reducing the expression level of GluA2 and/or by reducing Q/R editing of GluA2.
- GluA2 undergoes post-translational conversion of glutamine at the Q/R site to arginine (subject to Q/R editing).
- Q/R-edited GluA2 renders AMPA receptors calcium-impermeable upon internalization. If the uptake of Q/R-edited GluA2 into AMPA receptors is inhibited, AMPA receptors acquire calcium permeability and allow calcium to flow into cells in response to glutamate.
- the subject's hippocampus has a lower GluA2/AMPA receptor ratio than that in a healthy subject.
- the ratio of unedited form of GluA2 to total GluA2 counts is increased in the subject's hippocampus over that ratio in healthy subjects.
- the ratio of unedited GluA2 to total GluA2 counts in the CA2 region of the hippocampus of the subject is increased by a predetermined ratio or more over the ratio in healthy subjects.
- the predetermined ratio is 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 15 times or more, 20 times or more, 25 times or more. It can be 30 times or more, 35 times or more, 40 times or more, 45 times or more, or 50 times or more.
- the number of Thy-1-positive neurons in any region selected from the group consisting of CA1, CA2, and CA3 of the hippocampus of the subject is higher than the number of cells in a healthy subject. is also decreasing.
- any region (e.g., CA1 region) selected from the group consisting of CA1, CA2, and CA3 of the subject's hippocampus dendritic network tropism (specifically, tropism to CA1) ) is lower than the runnability in healthy subjects.
- the proportion of immature spines in any region of the subject's hippocampal formation eg, DG region
- AMPA receptor antagonists can ameliorate these situations.
- a pharmaceutical composition for use in inhibiting suppression of NMDA receptor activation in a subject having a hippocampus that expresses a calcium-permeable AMPA receptor the pharmaceutical composition comprising an AMPA receptor antagonist goods are provided.
- AMPA receptor antagonists can inhibit glutamate-dependent intracellular calcium transport of AMPA receptors and inhibit suppression of NMDA receptor activation. By inhibiting suppression of NMDA receptor activation, NMDA receptors can induce glutamate and membrane voltage dependent activation.
- the pharmaceutical composition of the present disclosure may contain the AMPA receptor antagonist in the form of a pharmaceutically acceptable salt.
- Pharmaceutically acceptable salts include acid addition salts.
- Acid addition salts include, for example, inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate and phosphate, or citrate, oxalate, phthalate, Fumarate, maleate, succinate, malate, acetate, formate, propionate, benzoate, trifluoroacetate, methanesulfonate, benzenesulfonate, para-toluenesulfonic acid organic acid salts such as salts and camphorsulfonates;
- Pharmaceutical compositions of the present disclosure may include the AMPA receptor antagonist in prodrug form.
- Prodrugs are metabolized in the body and become effective in the body. Prodrugs are often made by esterifying their carboxyl or hydroxy groups. The ester bond is cleaved by esterases present in the body, releasing the active form from the prodrug.
- Pharmaceutical compositions of the present disclosure may further comprise pharmaceutically acceptable excipients in addition to the AMPA receptor antagonist.
- Pharmaceutically acceptable additives include excipients, disintegrants, binders, flow agents, lubricants, coating agents, solubilizers, solubilizers, thickeners, and dispersants, depending on the purpose. , stabilizers, sweeteners, flavors and the like can be used.
- compositions of this disclosure may be formulated for oral or parenteral administration (eg, intravenous administration, etc.).
- the dosage form is not particularly limited, but examples thereof include tablets, capsules, powders, granules, solutions, suspensions, injections, patches, poultices and the like.
- Administration can be, for example, by oral administration or parenteral administration (eg, intravenous administration, intracerebroventricular administration, intracerebrospinal administration, etc.).
- the dose can be, for example, 100-2,000 mg per adult.
- a method of treating a disease or condition associated with NMDA-refractory in an NMDA-refractory subject or a subject with an NMDA-refractory NMDA receptor comprising administering to the subject a therapeutically effective amount of Methods are provided that include administering an AMPA receptor antagonist.
- the subject, the disease or condition associated with NMDA refractory, and the AMPA receptor antagonist can each be as described above.
- An aspect of the present disclosure provides a method of treating cognitive impairment in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an AMPA receptor antagonist.
- the subject, cognitive impairment, and AMPA receptor antagonist can each be as described above.
- a method of inhibiting suppression of NMDA receptor activation in a subject having a hippocampus that expresses a calcium-permeable AMPA receptor comprising administering to the subject an effective amount of an AMPA receptor antagonist.
- Methods are provided that include administering.
- the subject, cognitive impairment, and AMPA receptor antagonist can each be as described above.
- An aspect of the present disclosure provides a method of treating a subject with obesity or overweight comprising administering to the subject an effective amount of an AMPA receptor antagonist.
- An effective amount can be an amount capable of slowing, halting the development of, or ameliorating obesity or overweight.
- the subject may be on a restricted diet.
- the subject can be a non-restricted subject.
- the subject does not or has not been on a diet.
- the subject undergoes or is undergoing dietary therapy.
- cognitive impairment may be ameliorated in the subject.
- obesity or overweight may be ameliorated in the subject.
- Certain aspects of the present disclosure provide methods of treating a subject with dementia of the Alzheimer's type, comprising administering to the subject a therapeutically effective amount of an AMPA receptor antagonist.
- a method of treating dementia of the Alzheimer's type in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an AMPA receptor antagonist.
- the subject can be, for example, a subject with preclinical Alzheimer's disease.
- a subject can be, for example, a subject with an accumulation of A ⁇ in the brain.
- a ⁇ accumulation can be assessed by positron emission tomography (PET).
- PET positron emission tomography
- the subject can be, for example, one with any of decreased A ⁇ 42 levels, increased total tau levels, and increased phosphorylated tau levels in the cerebrospinal fluid.
- the subject can be, for example, a subject with mild cognitive impairment, or moderate cognitive impairment, or severe cognitive impairment.
- the subject can be a subject with neurodegeneration in the hippocampus.
- the subject can be a subject with neurodegeneration in the cerebral neocortex.
- the subject can be, for example, a subject with tau accumulation in the brain. Tau accumulation in the brain can be assessed by PET.
- An aspect of the present disclosure provides an AMPA receptor antagonist for use in the above methods.
- pharmaceutical compositions comprising AMPA receptor antagonists for use in the above methods can be provided.
- Certain aspects of the present disclosure provide for the use of AMPA receptor antagonists in the manufacture of a medicament for use in treating diseases or conditions associated with NMDA-refractory subjects in NMDA-refractory subjects. Also provided in certain aspects of the present disclosure is the use of an AMPA receptor antagonist in the manufacture of a medicament for use in treating cognitive impairment in a subject in need thereof. Certain aspects of the present disclosure provide use of an AMPA receptor antagonist in the manufacture of a medicament for use in inhibiting suppression of NMDA receptor activation in a subject having a hippocampus that expresses a calcium-permeable AMPA receptor. be done.
- the subject, cognitive impairment, and AMPA receptor antagonist can each be as described above.
- An AMPA receptor antagonist can be used in combination with memantine.
- Memantine may be in the form of an addition salt, for example an acid addition salt, preferably a hydrochloride salt.
- memantine can be used in combination, for example, as memantine hydrochloride.
- combination pharmaceutical products are provided that include an AMPA receptor antagonist and memantine.
- a combination pharmaceutical product is provided that includes a pharmaceutical composition comprising an AMPA receptor antagonist and a pharmaceutical composition comprising memantine. Combination pharmaceutical products can be used to treat cognitive impairment.
- mice Male C57BL/6J background mice and ob/ob mice were obtained from CLEA Japan Inc., respectively. and obtained from Japan SLC (3). Female mice were selected because male mice are reported to be more susceptible to HFD-induced body weight gain, metabolic changes, impaired learning and hippocampal synaptic plasticity. The animals were housed 3-4 per cage in a standard 12-hour dark-light cycle room at 25° C. and provided with food and water ad libitum. All animal experiments were conducted in accordance with the guidelines of the University of the Ryukyus Animal Experiment Ethics Committee. Time-series changes in average food consumption and average body weight were monitored in each condition. Human amyloid precursor protein knock-in (APP-KI) mice was provided by Dr.
- APP-KI Human amyloid precursor protein knock-in
- CE-2 diet CLEA Japan, Inc., Tokyo
- the composition of the CE-2 diet was as given in Table 1.
- the CE-2 diet had a total energy of 339.1 kcal/100 g and crude fat of 4.61%.
- F2HFD2 feed Oriental Yeast Co., Ltd., Tokyo, Japan
- F2HFD2 has a total energy of 640 kcal and is composed of 58% lard (wt/wt), 30% fishmeal, 10% skim milk, 2% vitamin-mineral mixture (7.5% carbohydrate, 24.5% protein, corresponding to 60% fat) (7).
- Other components of F2HFD were similar to CE-2.
- Groups of 4-week-old mice were administered HFD (F2HFD2 diet). Control mice were fed a low-fat diet (CE-2 diet).
- HFD mice were orally administered HydroGel (Clear H2O , Portland, ME 04101, USA) at a dose of 5 mg/kg body weight (HFD treatment group with PER).
- CE-2 diet (CLEA Japan, Tokyo) was used for ob/ob mice.
- HFD mice and ob/ob mice were orally administered at a dose of 5 mg/kg body weight to HFD mice and ob/ob mice, one per cage (PER administration group to HFD mice and ob/ob mice), and both groups were administered twice a week.
- body weight and HydroGel intake were monitored and the dose of PER administered was adjusted to 5 mg/kg/body weight.
- a wheeled activity wheel cage (MELQUEST, Japan, Model RWC-15) was used to monitor the activity of individual mice. Wheel rotation was monitored and recorded every 10 min for 14 days as previously reported (45).
- the open field test and elevated plus maze open field test were performed as follows. Mice were allowed to move freely for 5 minutes in a space surrounded by 50 cm square and 40 cm high walls (Muromachi Kikai, Japan), and their trajectories were analyzed using the CompACT VAS/DV video tracking system (Muromachi Kikai, Japan). (46).
- the elevated plus maze test was performed as follows. The space, set at a height of 50 cm from the floor, consisted of two open arms, two closed arms (each 30 cm x 6 cm) and a neutral zone. Mice were placed in the middle of the neutral zone facing the closed arm and allowed to move freely for 3 minutes. The time spent in the open and closed arms and the frequency of visits to different arms were recorded and scored using the CompACT VAS/DV video tracking system (Muromachi Kikai, Japan).
- Novel Object Recognition Test CD, HFD, HFD with PER treatment mice were handled for 5 minutes each day for 5 days before starting the novel object recognition test.
- PER means perampanel, an AMPA receptor antagonist.
- On day 1 they were habituated to a 35 cm square by 25 cm high box for 10 minutes, and on day 2 they were given two identical objects (familiar objects) for 10 minutes.
- On day 3 one of the familiar objects was replaced with a novel object of different shape and color (Fig. 2F).
- the mouse behavior was observed with a video camera for 5 minutes, and the video was digitized and stored in a personal computer.
- the search time for each object was measured by offline analysis (47).
- a contextual-spatial recall Morris water maze task mediated by a pattern-completion test was performed as previously described (49) using CD, HFD, and HFD with PER-treated mice. All experiments were performed at approximately the same time of day. Mice were transported from the colony to the farm and left undisturbed for 30 minutes prior to experimentation. The test was performed in a rectangular dimly lit room with a 120 cm diameter circular pool (Muromachi Kikai, Tokyo, Japan) filled with opaque water with skim milk (Morinaga, Japan) kept at room temperature. Four large objects, illuminated by floor lamps, hung from the black curtains surrounding the pool as extra-maze cues.
- a hidden circular platform 10 cm in diameter was placed 1 cm below the surface of the water and trained to find the platform at intervals of approximately 60 minutes, 4 times a day for 12 days.
- mice were released from four pseudo-randomly assigned starting points (N, S, E, and W) and allowed to swim for 300 seconds. Mice that did not find the platform within 300 seconds were manually guided to the platform and allowed to rest on the platform for 15 seconds.
- a probe test (P1) was performed on day 13 under full-cue conditions. Mice were released in the center of the pool and allowed to swim without a platform for 300 seconds. Following the probe trials, four training trials were performed in the presence of the platform to avoid memory loss that may have occurred during the probe trials.
- the 5-trial social test was performed as previously described (19). In summary, CD, HFD, and HFD with PER treated mice were housed individually starting 7 days prior to testing to establish territory superiority. On the day of testing, female mice were presented to the cage of control male mice (Fig. 2G) and given four consecutive 5-minute trials at 10-minute intervals. On the fifth trial, a new female mouse was presented (Fig. 2G) and the social inquiry time was recorded.
- Contextual Fear Conditioning was applied to published protocols with minor modifications (50). Mice were moved to the animal laboratory and acclimated for at least 30 minutes prior to contextual fear conditioning training. Mice were then placed in a footshock system model MK-450MSQ (Muromachi Kikai Co., Ltd., Japan) and allowed to explore for 2 minutes, followed by 3 electric footshocks (0.8 mA, 2 seconds, 2 minute intervals). gone. After resting for an additional minute, the animal was removed.
- MK-450MSQ Moromachi Kikai Co., Ltd., Japan
- MPRAGE three-dimensional (3D)-prepared rapid gradient-echo sequences were acquired as high-resolution 100 ⁇ m isovoxel images for voxel-based morphometry (matrix size: 280 ⁇ 220 ⁇ 220; field of view: 28 ⁇ 22 ⁇ 22 mm, repetition time: 2000 ms, echo time: 1.78 ms, flip angle: 12 degrees, inversion time: 800 ms, echo train length: 13, averaging times: 2).
- a RARE (rapid acquisition protocol by relaxation enhancement) sequence was used to draw a region of interest on the acquired data image (matrix size: 280 ⁇ 220 ⁇ 220, field of view: 28 ⁇ 22 ⁇ 22 mm; repetition time: 1500 ms; echo time: 25 ms; flip angle: 180°).
- MPRAGE and RARE images were acquired simultaneously in one scan.
- For ex vivo mouse MRI data acquisition four PBS-fixed mouse heads were simultaneously placed in the MRI coil. Four mouse brains were confirmed using MRI raw data.
- GM gray matter
- DPM8 cerebrospinal fluid
- the GM images were segmented using the tissue probability map of the SPMMouse toolbox. These segmented GM images enhanced contrast and normalized image deformation. Finally, these images were smoothed using the 200 ⁇ m isotropic Gaussian kernel method (installed on the SPM8 system). Smoothed images were applied for VBM analysis.
- the volumes of hippocampal subfields (CA1, CA2, CA3, DG and EC) were calculated using ROI files (31,33,51,52).
- the memory task consisted of 108 photographs of 16 lure sets (similar images), 16 repeat sets (same images), and 44 new items (new images). Participants asked whether the photographic stimulus presented on the display was a new item (new), a repeated identical picture (same), or a picture similar but not the same as the previous picture (lure). was instructed to respond with a button. The subject's button operations during the memory task were recorded on a personal computer, and the rate of correct answers for new, same, and lure was calculated. The percentage of correct answers for each task (new task, similar task, same task) was calculated using the following formula.
- Correct answer rate (%) number of correct answers to presented tasks / total number of presented tasks x 100
- the total number of task presentations for the new stimulus was set to 76, and the total number of task presentations for the same stimulus and the lure stimulus was set to 16 for both.
- Anatomical brain images were obtained using a three-dimensional (3D) spoiled gradient recall (SPGR) sequence (slice thickness in sagittal plane: 1 mm, matrix size: 256 ⁇ 256, field of view: 256 ⁇ 256 mm, repetition time: 6.9 ms, echo time: 3 ms, flip angle: 15°).
- SPGR spoiled gradient recall
- a high-resolution T2-weighted fast spin-echo sequence matrix size: 512 ⁇ 512, field of view: 192 ⁇ 192 mm, repeat time: 4300 ms, echo time: 92 ms, in-plane resolution: 0.375 ⁇ 0.375 mm 2 , 23 slices, thickness: 3 mm, space: 0 mm).
- Image acquisition MRI data for functional connectivity analysis were acquired using a GE Medical Discovery MR 750 3T scanner with a 32-channel head coil. Subjects, lying supine on the scanner bed, immobilized the head and neck with foam padding and a Philadelphia neck collar to minimize head movement. Resting fMRI images were acquired using a single-shot EPI sequence covering the whole brain (42 axial slices, 4 mm thickness, no interslice gap, repetition time 2000 ms, echo time 30 ms, flip angle 70°, Matrix size 64 ⁇ 64, field of view 256 ⁇ 256). A total of 150 volumes were shot in one session. Anatomical brain images were acquired using T1-weighted sagittal 3D SPGR sequences.
- Image preprocessing and functional network analysis were performed using SPM12 and CONN toolbox 18. b (www.nitrc.org/projects/conn, RRID: SCR_009550 [Whitfield-Gabrieli and Nieto-Castanon, 2012]). Details are as described in the previous report (32). Images were preprocessed in the order of realignment, slice timing correction, coregistration, normalization, smoothing, and segmentation. Noise in the BOLD signal was removed by linear regression of potential confounding effects in the BOLD signal and temporal bandpass filtering (temporal frequency ⁇ 0.008 Hz or >0.09 Hz).
- the default mode network (DMN) and functional connectivity were analyzed in functional network analysis using the CONN toolbox.
- regions having a positive correlation with BOLD fluctuations in the posterior cingulate cortex (PCC) and precuneus were calculated as DMN maps.
- regions having negative correlation with the DMN map were calculated as anti-correlated DMN maps.
- Mean images of DMN maps and anti-correlated DMN maps for normal-weight, overweight and obese groups were calculated using a one-sample t-test (voxel-level threshold p ⁇ 0.001 (uncorrected), false discovery
- the cluster-level threshold for the rate [FDR] was set at p ⁇ 0.05 (corrected)).
- the functional connectivity analysis we computed the graph theory parameters nodes and edges.
- Correlation coefficients of BOLD signal variation between each ROI were calculated with a seed-based connectivity metric (53) using a 132 ⁇ 132 ROI map (default ROI atlas in CONN toolbox).
- ROI-to-ROI degree the number of edges any node has between each node
- betweenness centrality the number of shortest paths of vertices through any pair of two nodes (j, i) in the graph
- ROI-to-ROI correlation 132 ⁇ 132 matrix established using our 132 ⁇ 132 ROI map.
- Degree is defined at each node as the number of edges from/to each node.
- the degree (di) is defined by the following formula.
- BC i represents a hub connecting other functional modules and nodes (54), and BC i is defined as follows.
- Pj ,k is the number of nodes in the shortest path between each pair of nodes (k,j) (the shortest path when passing through any node defined with i as a variable)
- N is the number of nodes in the ROI map. Total number of nodes in the graph.
- the average value and number of edges for each node were calculated for each of the normal weight group, overweight group, and obese group.
- the anterior cingulate cortex, cerebellar lobule CrusI, and the hippocampus, which is related to hippocampal memory function were set as regions of interest as previously reported (32), and the functional connectivity of the three groups was analyzed.
- a BrainNet viewer https://www.nitrc.org/projects/bnv/) was used to visualize functional connectivity maps for each group (55).
- Hippocampal brain slices were prepared from 17-18 week old male C57BL/6J background mice and mice were divided into CD groups, HFD groups and PER treatment according to previous reports (56, 57). Alternatively, 12-13 week old ob/ob mice were divided into ob/ob and PER-treated ob/ob groups. After cutting slices (thickness, 300-400 ⁇ m) using a linear slicer (PRO 7N, Dosaka EM, Japan), the hippocampal brain slices were treated with a normal Krebs Ringer (125 mM NaCl, 2.5 mM KCl, 10 mM DCl).
- Fluorescence intensity (F525) of Fluo-3 was monitored using a photomultiplier tube under a confocal microscope (excitation wavelength 488 nm; LSM5 PASCAL, Carl Zeiss, Germany).
- Fluo-3 fluorescence images (512 ⁇ 512 pixels) were digitized and stored on a personal computer every 10 seconds for 50 minutes.
- F525 in each region of CA1, CA2, CA3, DG, and EC is the average value of F525 (value 0 to 5 minutes before application of AMPA or NMDA) under offline analysis (Microsoft Excel, Microsoft Corporation, WA) Normalized (NF525).
- brain sections were treated with 125 mM NaCl, 2.5 mM KCl, 10 mM D-glucose, 1.25 mM NaH2PO4 , 26 mM NaHCO3 , 2 mM CaCl2, 1 mM MgCl2 , and 2 mL of extracellular solution containing 1 ⁇ M tetrodotoxin was added in a perfusion chamber (35 mm ⁇ -dish, Ibidi GMBH, Graefelfing, Germany) with continuous bubbling with a gas mixture (95% O 2 ; 5% CO 2 ). /min.
- the concentration of MgCl 2 was 0 mM to prevent Mg 2+ -dependent inhibition of NMDAR (58).
- the maximum value of NF525 was estimated as the maximum value of NF525 within 5 minutes after application of AMPA (Tocris, Bristol, UK) (100 ⁇ M) or NMDA (Tocris, Bristol, UK) (50 ⁇ M).
- Cyclothiazide (CTZ) 100 ⁇ M was used to prevent AMPAR desensitization
- glycine Tocris, Bristol, UK
- 10 ⁇ M was used to activate NMDAR
- perampanel Eisai Co, Ltd., Tokyo
- GYKI GYKI47261
- AAV (2R)-amino-5-phosphonovaleric acid
- NASPM 1-Naphthylacetylspermine
- CA1, CA2, CA3, DG, and EC regions were treated with ice-cold artificial cerebrospinal fluid (ARTCEREB; Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 2 mM BS3 (Thermo Fisher Scientific, Wilmington, DE, USA). It was added to a filled Eppendorf tube (Eppendorf, Hamburg, Germany). Incubation was performed on ice for 30 minutes. Crosslinking was terminated by quenching the reaction with 100 mM glycine (10 min at 4°C).
- ARTCEREB Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan
- 2 mM BS3 Thermo Fisher Scientific, Wilmington, DE, USA
- Hippocampal subregions were lysed in ice-cold lysis buffer containing protease and phosphatase inhibitors (25 mM HEPES, pH 7.4, 500 mM NaCl, 2 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF). , 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 1 ⁇ protease inhibitor mixture [Sigma-Aldrich, St Louis, Mo.], and 0.1% Nonidet P-40 [v/v]]). Cloudy and rapidly homogenized by sonication for 5 seconds. Total protein concentration of lysates was determined using the Lowry method (61).
- Membranes were then incubated overnight at 4° C. with anti-GluA1 (1:1000, Merck Millipore, Burlington, Mass.) and actin (1:5000, Protein Tech, Chicago, IL, USA). Membranes were incubated with HRP-labeled anti-rabbit IgG (1:3,000, Cell Signaling Technology, Danvers, Mass., USA) for 30 minutes and washed extensively again with TBS-T. After immersing the membrane in the chemiluminescent detection substrate Chemi-Lumi One (Nacalai Tesque, Tokyo, Japan) for 1 minute, luminescence was detected using a LuminoGraph1 imaging system (Atto, Tokyo, Japan). The surface and intracellular bands of each lane were analyzed using CS Analyzer (Atto, Tokyo, Japan).
- Mouse CA1, CA2, CA3, DG, EC regions were isolated and homogenized in disposable BioMasher2TM tubes (Nippi, Nippi, USA) using 0.5 mL of TRIzol RNA isolation reagent (Thermo Fisher Scientific, Wilmington, DE). Tokyo, Japan). Total RNA was extracted from each region separately according to the rest of the TRIzol RNA isolation reagent protocol provided by the manufacturer (Thermo Fisher Scientific, Wilmington, Del.). 1 ⁇ g of total RNA was reverse transcribed using PrimeScript RT Reagent Kit (Takara, Shiga, Japan).
- Relative expression levels of target genes in each sample were determined using a standard curve and normalized to the expression level of ⁇ -actin in the same samples.
- the sequences of primers and probes for these genes are shown in Supplementary Table 2.
- the ratio of mRNA amount of GluA2/(mRNA amount of GluA1+GluA3+GluA4)" was used as a Ca 2+ permeability index.
- the template for Sanger sequencing of the Sanger sequencing GluA2 editing site is cDNA was amplified and prepared using a forward primer (GAGGAATTTGAAGATGGAAGAGA: SEQ ID NO: 44) and a reverse primer (AGGAGGAGATGATGATGAGAGGGT: SEQ ID NO: 45).
- PCR products (10%) were confirmed by agarose gel electrophoresis.
- the PCR products were purified using the innuPREP PCRpure Lite Kit (Analytik Jena, Jena, Germany) according to the manufacturer's instructions prior to direct sequencing.
- Cycle sequencing was performed using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA) and each primer described above. Sanger sequence analysis was performed using ABI3500 (Applied Biosystems, CA, USA) and sequence viewer software 4Peaks.
- the Q/R editing status of GluA2 was analyzed using a QuantStudio TM 3D digital PCR system equipped with a TaqManTM custom SNP genotyping assay (Thermo Fisher Scientific, Wilmington, Del.). TaqMan probes were synthesized by Thermo Fisher Scientific, and VIC-labeled probes were used to detect unedited (Q) sequences, and FAM-labeled probes were used to detect edited (R) sequences (5′ CATCCTTGCTGCATAAA 3′: SEQ ID NO: 46, 5′ ATCCTTGCCGCATAAA 3′: SEQ ID NO: 47).
- Mouse forward primer 5' TTGGGATTTTTAATAGTCTCTGGTTTTCCTT 3' (SEQ ID NO: 48) and mouse reverse primer: 5' GACCAACCTTGGCGAAATATCG 3' (SEQ ID NO: 49).
- 5-50 ng of cDNA was mixed with a digital PCR master mix (Thermo Fisher Scientific, Wilmington, Del.) and the mixed TaqMan probes were loaded onto a QuantStudio TM 3D digital PCR 20K chip (Thermo Fisher Scientific, Wilmington, Del.) using an automated chip loader according to the manufacturer's instructions. , Wilmington, DE, USA).
- the loaded chip was amplified using the Gene Amp 9700 PCR system (Thermo Fisher Scientific, Wilmington, DE, USA) under the following conditions. 39 cycles of 10 min at 96°C, 2 min at 56°C, 30 sec at 98°C were followed by a final extension step of 2 min at 60°C. After amplification, the chips were imaged using the QuantStudio 3D Instrument (Thermo Fisher Scientific, Wilmington, DE, USA). For chip data analysis, QuantStudio 3D Analysis Suite Cloud Software (Thermo Fisher Scientific, Wilmington, Del.) was used for relative and quantitative data analysis.
- Golgi staining and image acquisition of the whole hippocampus Four coronal sections (5 mm wide, including the whole hippocampus from dorsal to ventral and medial to lateral) of the mouse brain from Bregma ⁇ 0.94 mm to Bregma ⁇ 4.04 mm were cut. % paraformaldehyde for 2 days and then stained with the Golgi Cox staining system (FD Rapid GolgiStain TM Kit, MD 21041, USA).
- the impregnated tissue was cut into 100 ⁇ m sections, counterstained with crystal violet, and the total number of 15 ⁇ m spines in the apical dendrites of CA1 and DG and the percentage of morphological spines (thin, stubby, mushroom) were evaluated using Axio. Observer Z1 (Carl Zeiss, Germany) was used.
- Another set of paraformaldehyde-fixed, 5-mm-wide coronal sections of the brain, including the entire hippocampus, were processed using the passive CLARITY technique (PACT) (62) and analyzed using a 10x clear objective as described in Lightsheet Z. et al.
- Thy1-YFPH transgenic wild-type mice (Carl Zeiss, Germany) and Thy1-YFPH transgenic wild-type mice (The Jackson Laboratory, stock number: 003709, strain name: B6.Cg-Tg.(Thy1-YFP)16Jrs/J) (63, 64). ), HFD-fed mice, and 3D images of the whole hippocampus of HFD-fed mice with PER treatment were acquired, respectively.
- the Z-stack function allows you to acquire a series of XY images at different focus positions and create a Z-stack.
- the specimens yielded dorsal-to-lateral and medial-to-lateral 400 ⁇ m ⁇ 400 ⁇ m 3D datasets of CA1, CA3 and DG, respectively.
- the gallery view displays images from the Z-stack in chronological order.
- Mouse brains were perfusion-fixed with 4% paraformaldehyde, embedded in paraffin, cut into 4-micrometer-thick sections, and treated with a monoclonal antibody against DCX (E-6 monoclonal, 1:50, Santa Cruz Biotechnology). Inc, Dallas, USA), MAP2ab (AP-20 monoclonal, 1:100, SIGMA-ALDRICH, St. Louis, USA), GluA1 (AB1540 polyclonal, 1:100, EMD Millipore Corp., Burlington, USA), GluA2 ( AB1768-1 polyclonal, 1:5, EMD Millipore Corp., Burlington, USA) was used for immunohistochemical analysis.
- DCX E-6 monoclonal, 1:50, Santa Cruz Biotechnology. Inc, Dallas, USA
- MAP2ab AP-20 monoclonal, 1:100, SIGMA-ALDRICH, St. Louis, USA
- GluA1 AB1540 polyclonal, 1:100, EMD Millipore Corp., Bur
- Quantitative Immunostained Areas with ZEISS ZEN Inicis Software Acquisition of immunostained areas in the expression areas of MAP2, GluA1 and GluA2 was performed using a ZVI format AxioVision (Carl Zeiss, Germany) microscope (20X objective). Generation of data image processing with image segmentation was performed using machine learning with ZEN Inicis software. Over 1000 cells were examined in each group. A one-way analysis of variance was used to test the variance among the three groups, excluding data with 3 SD or more. Then, the following sample numbers were used.
- the Bonferroni method was used for post hoc testing to analyze significant differences between groups.
- RNA-seq analysis Sequenced raw RNA-seq fastq reads were analyzed using HISAT2 (v.2.2.0) for mouse GRCm38 (ensemble release 104, http://ftp.ensemble.org/pub/release -104/fasta/mus_musculus/dna/) (65). The average mapping rate for all samples was 94.21% (range 88.70-96.30%). Aligned reads were quantified using Salmon (v.0.14.2) (66) and TPM values were calculated using StringTie (v.2.1.2) (67). Variant calling was performed using GATK package (v.3.8) (70) according to GATK Best Practices for RNAseq short variant discovery (68, 69).
- RNA-editing levels were calculated as the ratio of the total number of reads aligned to the R/Q editing site of GluA2 (chr3: 80706912) and the number of reads TC converted at this site.
- HFD stimulates AMPAR-mediated calcium influx in the mouse hippocampus.
- Direct measurements of intracellular calcium concentration [Ca 2+ ]i) were taken to track hippocampal calcium signaling (Fig. 1A, Supplementary Data Movie 1) in acute brain slices of the hippocampus with HFD.
- 100 ⁇ M AMPAR with 100 ⁇ M CTZ cyclothiazide; channel desensitizer
- EC entorhinal cortex
- CA1 cornu ammonis 1
- CA2, CA3, and DG dentate gyrus
- HFD alters the expression of glutamate receptor subunits.
- AMPAR-mediated [Ca 2+ ]i changes were observed in mice fed HFD for at least 7 days (Fig. 7A, B, C).
- these mice maintained NMDAR-mediated activation in EC, DG, CA3, CA2, CA1 even when bathing with 50 ⁇ M NMDA plus 10 ⁇ M glycine.
- these pathological AMPAR-mediated increases in [Ca 2+ ]i were completely abolished by the addition of 100 ⁇ M PER, similarly 100 ⁇ M GYKI47261 (Tocris, UK, Bristol) and Ca 2+ permeabilization.
- CP-AMPARs Ca 2+ -permeable AMPARs
- ALS amyotrophic lateral sclerosis
- 13 neuropathic pain
- AMPAR- and NMDAR-mediated alteration of calcium dynamics in [Ca 2+ ]i may play an important role in obesity with cognitive decline.
- AMPAR mediates the fastest rate of excitatory neurotransmission.
- AMPAR is composed of four subunits GluA1-4 that determine the functional properties of AMPAR channels, and AMPAR diversity is due to the abundance of these subunits.
- the GluA2 subunit is essentially Ca 2+ impermeable, whereas the other subunits of GluA1, GluA3, and GluA4 are Ca 2+ permeable.
- the Ca 2+ permeability of AMPARs is regulated by the relative amount of GluA2 expression. That is, an increase in the relative amount of the GluA2 subunit would decrease the Ca 2+ permeability of AMPAR, and a decrease in the relative amount would increase the Ca 2+ permeability of AMPAR.
- GluA2 is edited at the Q/R site within the reentrant M2 membrane loop region by adenosine deaminase (ADAR2) acting on RNA type 2, involved in the editing of double-stranded RNA from adenosine (A) to inosine (I).
- ADAR2 adenosine deaminase
- RNA type 2 involved in the editing of double-stranded RNA from adenosine (A) to inosine (I).
- qRT-PCR Real-time quantitative reverse transcription-polymerase chain reaction
- RNA in hippocampal subregions (CA1, CA3, DG, EC) was isolated and quantitative analysis of mRNA for GluA1, GluA2, GluA3, GluA4 for AMPAR, GluN1, GluN2A, GluN2B, GluN2C for NMDAR was performed qRT- Performed by PCR.
- the results showed significantly elevated expression of GluA1, GluN1, and GluN2B in the CA3 region associated with HFD-fed mice, and elevated expression of GluN2B in the DG (p ⁇ 0.05 each), suggesting HFD with PER.
- the ratio of GluA2 to other GluA subunits was determined. In addition, the ratio was taken as the Ca 2+ permeability index (Ca 2+ -permeability index).
- NMDA receptors Activation of NMDA receptors is not required for pattern completion during association in memory retrieval (22), and our data suggest that a change in AMPA receptors to a calcium-permeable form is responsible for the ability of HFD mice to complete pattern completion. It was suggested to play an important role in disability.
- Golgi staining showed that PER restored the complete runnability loss of the dendritic network in CA1 of HFD mice (Fig. 3E, upper left panel).
- staining with an anti-MAP2 antibody showed that dendrite motility was lost in the CA1 region, and PER completely restored dendrite motility (Fig. 3E, upper left panel).
- the CA1 GluA1 immunostaining reaction was significantly enhanced in HFD mice, but was reduced by PER treatment and the GluA2/GluA1 ratio was increased.
- AD model mice NL-GF mice begin to accumulate A ⁇ in the brain from 8 weeks of age (Nat. Neurosci. 2014 17, 661-4 ).
- the results of the novel object recognition test of mice at 13 weeks of age were as shown in FIG. As shown in FIG.
- AD model mice cannot distinguish between mice in the same room and mice in the first meeting, whereas in the perampanel (PER) administration group, mice in the same room and mice in the first meeting have a significant difference. made a distinction between This result indicates that CA induced dysfunction in NL-GF mice, whereas AMPA receptor antagonists suppressed the development of this dysfunction.
- AMPA receptor antagonists suppressed the development of this dysfunction.
- Leptin 25,26
- NMDAR-mediated synaptic transmission plays a role (27).
- HFD-fed mice generally initially show elevated leptin, one of the hallmarks of obesity, and over time, chronic obesity develops leptin resistance, resulting in an inability to suppress appetite and promote energy expenditure.
- All of Gyki, Naspm, and PER showed significant inhibitory effects on CP-AMPAR in ob/ob mice in all regions of EC, CA1, CA2, CA3, and DG (Fig. 10-1A ).
- AMPA was used at 100 ⁇ M
- CTZ was used at 100 ⁇ M
- NMDA was used at 50 ⁇ M
- PER was used at 100 ⁇ M
- glycine was used at 10 ⁇ M
- APV was used at 50 ⁇ M
- Naspm was used at 10 ⁇ M
- Gyki was used at 100 ⁇ M
- leptin was used at 100 nM. * indicates p ⁇ 0.05 in the t-test.
- Ob/ob mice were analyzed as a leptin-deficient obese model and a leptin-resistant obese model. Specifically, we analyzed these leptin-deficient/resistant models on high-resolution MRI, tissue morphology in staining for MAP2, GluA1, GluA2, qRT-PCR, and behavioral analysis. This revealed whether the leptin-deficient/resistant state affected hippocampal morphology (Fig. 4). We demonstrated CP-AMPAR-mediated [Ca 2+ ]i elevations in HFD-fed mice as well (Fig. 1A–E) and in all hippocampal slices (EC, CA1, CA2, CA3, and DG) of ob/ob mice.
- Ob/ob mice and their littermates with wild-type traits were obtained as offspring of ob+/- mice. These mice were fed ad libitum and some of the ob/ob mice were fed a diet containing 5 mg/kg body weight perampanel from 8 to 12 weeks of age. Appearance and body weight of these mice were compared at 12 weeks of age. The results were as shown in FIG. As shown in FIG. 24, compared to ob/ob mice, perampanel-treated mice were prevented from overweight and exhibited a leaner appearance closer to wild type. This result suggests that administration of perampanel is effective in treating obesity and overweight. In addition, it was suggested that cognitive impairment may lead to an increase in food intake, and that the amount of contact may be suppressed by reducing cognitive impairment by administering an AMPA receptor antagonist.
- the amount of food fed was uniform (see lower panel of Figure 25)
- the body weights were monitored. bottom.
- significant weight loss was observed in the PER-containing HFD-fed group than in the HFD-fed group. This suggested that in overweight or obese subjects, AMPA receptor antagonists produced weight loss effects even without reducing food intake.
- fMRI behavioral tasks 35, 36 analyze variations in blood oxygenation level dependent (BOLD) response, hippocampal memory function of pattern complementation, and pattern separation ability (formation of distinct representations of similar inputs).
- BOLD blood oxygenation level dependent
- hippocampal memory function of pattern complementation hippocampal memory function of pattern complementation
- pattern separation ability formation of distinct representations of similar inputs.
- BMI blood oxygenation level dependent
- the structural network that supports pattern completion is based on CA3 and CA1.
- a central role for this region in ensemble dynamics (dynamic synchronization) was observed in healthy volunteers (37) (Fig. 5H).
- the overweight group did not reveal significant cognitive decline compared to the control group (Fig. 5I).
- a functional connectivity map for each group was generated by seed-based analysis, with the posterior cingulate cortex and precuneus set as default mode networks, and the anterior cingulate cortex and hippocampal memory cerebellar lobule CrusI functioned as regions of interest (35). ).
- the different network connections among subjects in each classified group are shown in FIG. 6B. Specific alternations between larger networks and centrality mapping associated with disruption of modular autonomy are evident in the overweight and obese groups compared to the normal BMI group, leading to failure of correct memory retrieval. rice field. Independent network formation of the default mode network (DMN), saliency network (SN), affective network (EN), and cerebellar network (CBN) was observed in the normal weight group.
- DN default mode network
- SN saliency network
- EN affective network
- CBN cerebellar network
- a Central Enforcement Network (CEN) was added to that of the normal weight group, demonstrating new connectivity between the DMN and CEN. Endogenous connections within ENs and CBNs also increased. Intrinsic connectivity of the CEN and visual network (VN) was enhanced in the obese group, with bilateral insular cortices emerging in the SN. Centrality between the anterior cingulate cortex and cingulate gyrus was enhanced over the other two groups. Connectivity between different functional networks (DMN, SN, CEN, CBN and VN) was enhanced in the obese group. The total number of nodes in the normal-weight, overweight, and obese groups was estimated to be 17, 21, and 23, respectively (Fig. 6B,C).
- AMPA Receptor Antagonists to Severely Obese Women
- body weight of a 70-year-old woman with a BMI of 38 were investigated.
- Antiepileptic therapy was administered after tumor resection.
- 2 mg/day (later changed to 4 mg/day) of perampanel was orally administered. Changes in body weight and lower extremity Fugl-meyer assessment score of the woman were observed.
- the size of the hippocampus was also assessed from MRI of the woman's brain 26 weeks after surgery. The results were as shown in FIG. As shown in FIG. 26, administration of PER clearly increased the size of the hippocampus (see the frame in the upper photograph of the figure). The numerical values were as shown in Table 6 below.
- AMPA receptor antagonists increased hippocampal volume in women. Furthermore, as shown in Figure 27, the women's body weight decreased steadily after administration of the AMPA receptor antagonist. The results are similar to those shown in Figures 24 and 25.
- NMDA receptors were inactivated.
- Expression of calcium ion-dependent NMDA receptors and reactivity to NMDA and glycine were confirmed in mice (2w HFD) fed with HFD for 2 weeks. The results were as shown in panel A of FIG.
- Brain slices of the 2w HFD group were treated in the presence of 3 ⁇ M BAPTA-AM or under calcium ion-free conditions for 60 minutes, after which the treated brain slices were subjected to calcium ion imaging.
- addition of NMDA and glycine did not activate NMDA receptors in any brain region, and NMDA receptors were inactivated.
- D-( ⁇ )-2-amino-5-phosphonovaleric acid (APV) 50 ⁇ M
- Panel B of FIG. 28 shows a comparison of maximum normalized fluorescence intensity (calcium ion concentration).
- HFD central role of CP-AMPAR in obesity-related cognitive decline
- HFD stimulates hippocampal calcium dynamics via AMPAR, subunit structure rearranges from non-CP-AMPAR to CP-AMPAR, and NMDAR calcium signaling was shown to downregulate Ca 2+ influx by altering .
- Inactivation of transmission by NMDAR function is thought to correspond to cognitive decline.
- CP-AMPAR caused a decrease in hippocampal size and cell number (particularly CA3), decreased dendritic integrity of CA1, and spine maturation in the DG region. It was found that ingestion of HFD for 7 days or longer affected hippocampal memory circuits via calcium signals. This short-term intake preserved NMDAR-mediated calcium dynamics, but revealed AMPAR-mediated calcium entry in the hippocampus.
- NMDAR-mediated calcium entry Long-term intake over 8 weeks after weaning completely reduced NMDAR-mediated calcium entry, accompanied by changes in AMPAR-mediated calcium dynamics.
- AMPAR and NMDAR are involved in high-speed transmission of glutamate-based synapses, are localized in postsynaptic membranes, and play important roles in human cognition such as learning and memory.
- Inhibition of calcium influx into synapses by NMDARs is regulated by intracellular calcium concentrations regardless of the calcium source, including AMPARs, voltage-gated calcium channels, internal stores, and NMDARs (9).
- AMPARs co-localize with NMDARs and mediate excitatory synaptic responses on the tenth millisecond timescale, whereas HFDs increase calcium entry of these receptors and ultimately NMDARs. decreased the magnitude of calcium currents through In addition to inducing synaptic plasticity (LTP/LTD) (36), NMDARs have been reported to play an important role in regulating appetite and food preferences, which can lead to rebound from overeating, overeating, failure of dietary restriction, It is thought to trigger a vicious cycle of increased obesity (37), ultimately further accelerating cognitive decline. Attempts to restore neuronal function by activating NMDARs through AMPAR-mediated hyperstimulation have also been shown to promote neuronal injury (72).
- Altered glutamatergic synaptic transmission affects functional network connectivity in resting fMRI data.
- High BMI is associated with cognitive impairment in the young (38), and overweight and obesity in the middle-aged. May increase future risk of cognitive decline in old age.
- HFD induces obesity and causes hypertension, type 2 diabetes, cardiovascular events, and patients with these diseases are at increased risk of cognitive decline (40, 41).
- Effective treatment and disease control are presumably hampered by the inability to provide adequate lifestyle management, including dietary self-regulation. In medicine, developing effective interventions for cognitive decline associated with obesity is key to better disease management.
- AMPAR is an Attractive Target for Cognitive Decline
- a high frequency of occurrence of hydrocephalus was seen among early-onset obese mouse groups.
- obesity begins in early childhood, from 2 to 6 years old (46), and most children who are obese at that age were also obese in adolescence.
- Appropriate and effective interventions are urgent challenges for human health and disease.
- application of HFD with PER treatment restored dysregulated calcium signaling through both AMPARs and NMDARs, and at the same time induced restoration of brain size and behavioral memory capacity. Preventing weight gain due to obesity has health benefits.
- an AMPA receptor antagonist suppresses the deposition of ⁇ -amyloid.
- perampanel was used as an AMPA receptor antagonist. It was found to significantly increase the A ⁇ 40 ratio, reduce A ⁇ deposition, and suppress A ⁇ accumulation in the hippocampus and cerebrum of model mice.
- Biomarkers of Alzheimer's disease include CSF (cerebrospinal fluid) and plasma (plasma) levels of A ⁇ 42/A ⁇ 40 ratio, phosphorylated tau (P-tau) at threonine 181 and 217, neurofilament light (NfL) (Curr Opin Neurol 2021 1 266-274). It has been suggested that the A ⁇ 42/A ⁇ 40 ratio can predict A ⁇ accumulation in PET (Doecke JD et al., Neurology 2020). NL-GF (-/-) mouse hippocampus on embryonic day 17-19 was removed, cultured by the Neurosphere method of Brewer GJ et al. The serum was measured by the EIA method.
- mice treated with perampanel showed decreased A ⁇ accumulation.
- AD Alzheimer's disease
- Epilepsy occurs more frequently in AD patients than in control populations (Pascal E 2012), with an incidence of epilepsy of 7-21% in patients with sporadic AD (Amatniek et al., 2012). 2006; Hauser et al., 1986; Mendez and Lim, 2003), increasing to 30% in early-onset familial AD (FAD) (Palop and Mucke, 2009; Larner and Doran, 2006).
- FAD early-onset familial AD
- Early insidious hippocampal hyperexcitability was detected in AD patients (Lam AD et al., Nat Med 2017). As shown in FIG.
- Perampanel inhibits glutamate-induced activation of AMPA receptors on the postsynaptic membrane and is a non-competitive AMPA receptor antagonist for epileptic patients aged 12 years and older with partial-onset seizures (including secondary generalized seizures) during tonic seizures. It is indicated for primary seizures and partial seizures (including secondary generalized seizures) in epileptic patients aged 4 and over. seems to be suitable.
- AMPA receptor antagonists work with memantine to improve cognitive function of dementia subjects. Memantine and exercise load, memantine and perampanel, and perampanel alone (5 mg/kg or 10 mg/kg) were administered and subjected to behavioral experiments. As behavioral tests, a novel object recognition test and a hippocampal function-dependent fear conditioning test were performed. Memantine is an antagonist of the NMDA receptor, but it has different properties than other NMDA receptor antagonists (see Hokama Y., et al., Neuro Oncology, 2022, noac162). Thus, memantine is a drug that only works when there is an excess of glutamate in the brain. Memantine also acts more favorably on non-synaptic NMDA receptors (extrasynaptic NMDARs) than on synaptic site NMDA receptors (synaptic NMDARs).
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Abstract
Description
[1]対象において、認知機能障害を処置することに用いるための医薬組成物であって、AMPA受容体アンタゴニスト(拮抗剤)を含む、医薬組成物。
[2]対象が、嗅内皮質(EC)、CA1、CA2、CA3、および歯状回(DG)からなる群から選択される海馬体の少なくとも1以上の領域において抑制されたNMDA受容体を有し、これにより、前記海馬の少なくとも1以上の領域においてNMDAによるNMDA受容体の活性が抑制され、かつ、対象が認知機能障害を有する、上記[1]に記載の医薬組成物。
[3]対象が、前記海馬体の少なくとも1以上の領域においてカルシウム透過型AMPA受容体を有し、これにより、前記海馬の少なくとも1以上の領域においてNMDA受容体の活性化が抑制されている、上記[2]に記載の医薬組成物。
[4]対象が、認知機能障害を有する対象である、上記[1]~[3]のいずれかに記載の医薬組成物。
[5]認知機能障害が、アルツハイマー型認知症である、上記[4]に記載の医薬組成物。
[6]対象が、25以上の体格指数(BMI)を有する、上記[1]~[5]のいずれかに記載の医薬組成物。
[7]対象が、30以上のBMIを有する、上記[1]~[6]のいずれかに記載の医薬組成物。
[8]対象が、事象関連記憶課題により血液中酸素化レベル依存(BOLD)応答が安静時に対して1.1倍以上の変動を示す、上記[1]~[7]のいずれかに記載の医薬組成物。
[9]NMDA不応性の対象またはNMDA不応答性のNMDA受容体を有する対象においてNMDA不応性に関係する疾患または状態を処置することに用いるための医薬組成物であって、AMPA受容体アンタゴニストを含む医薬組成物。
[10]AMPA受容体アンタゴニストが、ペランパネルである、上記[1]~[9]のいずれかに記載の医薬組成物。
[1A]過体重または肥満症を有する対象において過体重または肥満症を処置することに用いるための医薬組成物であって、AMPA受容体アンタゴニストを含む医薬組成物。
[2A]過体重または肥満症を有する対象において過体重または肥満症の進展を遅延させる、抑制する、または停止させることに用いるための、上記[1A]に記載の医薬組成物。
[3A]過体重または肥満症を有する対象において体重を減少させることに用いるための
医薬組成物であって、上記[1A]に記載の医薬組成物。
[4A]過体重または肥満症を有する対象において摂食量を減少させることに用いるため
の医薬組成物であって、上記[1A]または[3A]に記載の医薬組成物。
[5A]過体重または肥満症を有する対象において、糖尿病、脂質異常症、および心血管疾患の発症を抑制することに用いるための、上記[1A]~[4A]のいずれかに記載の医薬組成物。
[6A]AMPA受容体アンタゴニストが、ペランパネルである、上記[1A]~[5A]のいずれかに記載の医薬組成物。
A、major neurocognitive disorderの基準を満たし;
B、少なくとも2つ以上の認知機能障害が潜行性に出現し、徐々に進行し;
C、家族歴か遺伝子検査からアルツハイマー型認知症の原因遺伝子の変異が存在する証拠がある、または、以下のすべてを満たす:
・詳細な病歴や経時的な神経心理検査で、記憶・学習と他の認知機能の低下の明確な証拠がある
・認知機能が長く安定していることがなく、確実に、徐々に悪化している
・認知機能障害の原因となりそうな他の神経変性疾患、脳血管疾患、神経/精神/全身疾患や状態がない;
D、詳細な病歴や経時的な神経心理検査で、記憶・学習と他の認知機能の低下の明確な証拠がある
認知機能が長く安定していることがなく、確実に、徐々に悪化している
認知機能障害の原因となりそうな他の神経変性疾患、脳血管疾患、神経/精神/全身疾患や状態がない。
(1)中性脂肪が150mg/dL以上、または、HDLが40mg/dL未満である。(2)収縮期血圧が130mmHg以上、または拡張期血圧が85mmHg以上である。(3)空腹時血糖が110mg/dL以上である。
子どもの肥満は、肥満度により判定され得る。具体的には、子どもの肥満は、肥満度が+20%以上であり、体脂肪率が、男子では25%以上増加した状態、11歳未満の女子では30%以上増加した状態、11歳以上の女子では35%以上増加した状態である場合をいう。肥満度(%)は、(現在の体重-その年齢の標準体重)/その年齢の標準体重×100で求められる。
雄のC57BL/6Jバックグラウンドのマウスとob/obマウスは、それぞれCLEA Japan Inc.と日本SLCから入手した(3)。雌マウスを選択した理由は、雄マウスは、HFDによる体重増加、代謝変化、学習や海馬のシナプス可塑性の障害などの影響を受けやすいことが報告されているためである。これらの動物は、25℃の標準的な12時間の暗-明サイクルの部屋で、自由に利用できる食物と水が提供される環境下において、1ケージあたり3~4匹で飼育した。すべての動物実験は,琉球大学の動物実験倫理委員会のガイドラインに従って行われた。各条件において平均摂餌量と平均体重の時系列変化をモニターした。
ヒトamyloid precursor protein knock-in (APP-KI) mice は理化学研究所の西道隆臣博士のご供与による。このマウスは、ヒトAPP遺伝子にSwedish KM670/671NL mutation(NL), the Artic E693G mutation (G) and the Iberian I716F (F) mutation (F)の変異を入れたものであり、実験には変異一つのNLマウスと変異すべてを有するNL-G-Fマウスを用いた。これらマウスはいずれも、human amyloid beta (Aβ)40とAβ42 oligomersを産生する(Saito et al., Single App knock-in mouse models of Alzheimer’s disease. Nat. Neurosci. 2014 17,661-4)。上記ノックインマウスは2ヶ月(8週)を過ぎてから脳内でのAβの蓄積が認められる。
マウスの対照食(CD)としては、CE-2飼料(日本クレア株式会社、東京都)を使用した。CE-2飼料の組成表は表1にされる通りであった。CE-2飼料の総エネルギーは339.1 kcal/100gで、粗脂肪は4.61%であった。一方、高脂肪食(HFD)としてはF2HFD2飼料(オリエンタル酵母株式会社、東京、日本)を使用した。F2HFD2の総エネルギーは640 kcalで、ラード58%(wt/wt)、魚粉30%、スキムミルク10%、ビタミン・ミネラル混合物2%で構成されている(炭水化物7.5%、タンパク質24.5%、脂肪60%に相当)(7)。F2HFDの他の成分はCE-2と同様であった。4週齢のマウス群にHFDを投与した(F2HFD2飼料)。対照マウスには低脂肪食を与えた(CE-2飼料)。
HFDマウスにはHydroGel(Clear H2O, Portland, ME 04101, USA)を5mg/kg体重の用量で経口投与した(PERを伴うHFD処置群)。ob/obマウスには、CE-2飼料(日本クレア、東京)を使用した。HFDマウス、およびob/obマウスを1ケージに1匹ずつ、PERを5mg/kg体重の用量で経口投与し(HFDマウス、およびob/obマウスにPER投与群)、両群とも週に2回、体重およびHydroGelの摂取量をモニターして、PERの投与用量を5mg/kg/体重に調整した。
ホイールケージ(MELQUEST, Japan, Model RWC-15)を用いて、個々のマウスの活動をモニターした。車輪の回転は、以前に報告したように、14日間にわたって10分ごとにモニターし、記録した(45).
オープンフィールドテストは以下の通りに行った。50cm四方、高さ40cmの壁(室町機械、日本)で囲まれた空間で、マウスを5分間自由に行動させ、その軌跡をCompACT VAS/DVビデオトラッキングシステム(室町機械、日本)を用いて解析した(46)。
高架式プラス迷路試験は、以下の通りに行った。床から50 cmの高さに設置された空間は、2本のオープンアーム、2本のクローズドアーム(各30 cm×6 cm)、ニュートラルゾーンで構成された。マウスは、クローズドアームに向けて、ニュートラルゾーンの中央に配置され、3分間自由に行動させた。オープンアームとクローズドアームに滞在した時間、および異なるアームへの訪問頻度を記録し、CompACT VAS/DVビデオトラッキングシステム(日本、室町機械)を用いてスコア化した。
CD、HFD、PER処理を伴うHFD(PERを伴うHFD)のマウスは、新規物体認識試験開始前の5日間、毎日5分間ハンドリングした。PERは、AMPA受容体アンタゴニストであるペランパネルを意味する。1日目に35 cm四方、高さ25 cmの箱に10分間慣れさせ、2日目に2つの同じ物体(馴染みの物体)を10分間与えた。3日目には,慣れ親しんだ物体の1つを、形と色の異なる新規の物体と交換した(図2F)。マウスの行動をビデオカメラで5分間観察し,その映像をデジタル化してパソコンに保存した。各物体の探索時間は、オフライン解析により測定した(47)。
記憶障害は、以前に記述したように、Morris水迷路試験を用いて評価した(48)。要約すると、直径120 cmの水迷路プール(室町機械、日本、東京)には、不透明な水(室温)が入っており、水面下2 cmにプラットフォーム(直径10 cm)が沈んでいた。隠れプラットフォーム課題は、4~7日間(1日2回、3時間間隔)で行い、その間、1日2回(15分間隔)の試行を行った。プラットフォームの位置は一定に保ち、エントリーポイントは試行の間に半ランダムに変更した。隠れプラットフォーム課題の最終日から24時間後に、プラットフォームを使わずに1分間のプローブ試行を行った。プローブ試行のエントリーポイントは、ターゲット象限とは反対側の象限に設定した。記憶保持は、隠されたプラットフォーム課題において、脱出用プラットフォームが置かれた正しい象限にいた時間で評価した。パフォーマンスは、CompACT VAS/DVビデオトラッキングシステムを用いてモニターした。
モリス水迷路課題は、CD、HFD、およびPER処理を伴うHFDのマウスを用いて、前述のように実施した(49)。すべての実験は、一日のうちのほぼ同じ時間帯に行った。マウスはコロニーから飼育場に運ばれ、実験の前の30分間は邪魔されないようにした。試験は、室温に保たれたスキムミルク(森永、日本)を用いた不透明な水で満たされた直径120cmの円形プール(室町機械,東京,日本)を備えた長方形の薄暗い部屋で行った。フロアランプで照らされた4つの大きな物体が、迷路外の手がかりとしてプールを囲む黒いカーテンに吊るされていた。水面下1cmのところに直径10cmの隠れた円形のプラットフォームを置き、12日間、1日4回、約60分の間隔でプラットフォームを見つけるように訓練した。訓練中、マウスは擬似ランダムに割り当てられた4つのスタート地点(N、S、E、およびW)から放たれ、300秒間泳がされた。300秒以内にプラットフォームを見つけられなかったマウスは、手動でプラットフォームまで誘導され、15秒間プラットフォーム上で休息させられた。
13日目に手がかりが揃っている(full-cue)条件でプローブ試験(P1)を行った。マウスをプールの中央に放し、プラットフォームのない状態で300秒間泳がせた。プローブトライアルに続いて、プラットフォームの存在下で4回のトレーニングトライアルを行い、プローブトライアル中に生じた可能性のある記憶の消滅を回避した。その後、1日に1回、迷路の手がかりを追加したプローブ試行を4回行い、プローブ試行の間は再訓練を行わなかった。1手がかりのプローブ試行(P2)では、プラットフォームからより遠くにある1つの手がかりを残し、他の3つの手がかりを周囲のカーテンから外した。2手がかりのプローブ試行(P3)では、プラットフォームに近い位置にある1つの手がかりと1手がかりのプローブ試行で使用した手がかりを残し、他の2つの手がかりを周囲のカーテンから外しました。手がかりなしのプローブ試行(P5)では、4つの余分な迷路手がかりをすべて取り除いた。トレーニングおよびプローブトライアルのデータは,CompACT VAS/DVビデオトラッキングシステムソフトウェアを用いて収集し、分析した。隠れたプラットフォームへの逃避時間(ゴール到達時間)を測定した。
5試行の社会性テストは、以前に記載したように行った(19)。要約すると、CD、HFD、およびPER処理を伴うHFDの対象マウスは、テリトリーの優位性を確立するために、試験の7日前から個別に収容した。試験当日、雌マウスを対象となる雄マウスのケージ(図2G)に提示し、5分間の試行を10分間の間隔で4回連続して行った。5回目の試行では、新規の雌マウスを提示し(図2G)、社会的調査の時間を記録した。
文脈的恐怖条件付けは、公表されているプロトコルに若干の修正を加えて適用した(50)。マウスを動物実験室に移動させ、文脈的恐怖条件付けトレーニングの前に少なくとも30分間順応させた。次に、マウスをフットショックシステムモデルMK-450MSQ(室町機械株式会社,日本)に入れ、2分間探索させた後、3回の電気フットショック(0.8 mA,2秒,2分間隔)を行った。さらに1分間放置した後、動物を取り出した。
Bruker BioSpec 117/11 11.75 Tesla MRI scanner (Bruker BioSpin GmbH, Ettlingen, Germany)を用いて、CD、HFD、PER処理を伴う8匹のex vivoマウスの解剖学的脳画像を取得した。MPRAGE(three-dimensional (3D)-prepared rapid gradient-echo)シーケンスは、ボクセルベースモルフォメトリーの高解像度100μmアイソボクセル画像として取得された(マトリックスサイズ:280×220×220;視野:28 × 22 × 22 mm、繰り返し時間:2000 ms、エコー時間:1.78 ms、フリップアングル:12 度、インバージョン時間:800 ms、エコートレイン長:13、平均回数:2)。
RARE(rapid acquisition protocol by relaxation enhancement)シーケンスを用いて,取得したデータ画像に関心領域を描画した(マトリクスサイズ:280 × 220 × 220,視野:28 × 22 × 22 mm;繰り返し時間:1500 ms;エコー時間:25 ms;フリップアングル:180°)。1回のスキャンでMPRAGE画像とRARE画像を同時に取得した。
ex vivoマウスのMRIデータ取得は、PBSで固定した4匹のマウスの頭部を同時にMRIコイルに入れて取得した。4匹のマウスの脳は、MRIの生データを用いて確認した。
SPM8解析ツール(Wellcome Department of Clinical Neurology, London; http://www.fil.ion.ucl.ac.uk)およびSPMMouseツールボックス(http://www.spmmouse.org/)を用いて、MPRAGEおよびRARE画像のボクセルベースモルフォメトリー(VBM)解析および前処理を行った。
生データは4枚のマウス頭部MRI画像に分割して別々に保存した。分割した画像の3D(x-y-z-)座標をSPM標準座標系に変換し、3D座標の原点をブレグマ点とした。次に、セグメンテーションツール(SPM8システムにインストールされている)を用いて、脳画像を灰白質(GM)、白質、脳脊髄液にセグメンテーションした。SPMMouseツールボックスの組織確率マップを用いて、GM画像に分割しました。これらの分割されたGM画像は、コントラストを向上させ、画像の変形を正規化した。最後に、これらの画像を200μmの等方性ガウスカーネル法(SPM8システムにインストールされている)を用いて平滑化した。平滑化した画像をVBM解析に適用した。
海馬のサブフィールド(CA1、CA2、CA3、DG、およびEC)の体積は、ROIファイルを用いて算出した(31、33、51、52)。CD、HFD、およびPER処置を伴うHFDの全脳体積の平均値間の差は、一元配置分散分析(ANOVA)によって検定した。ANOVAで3群間に有意差があった場合(p<0.05)、CD群とHFD群;CD群とPER処理を伴うHFD群;HFD群とPER処理を伴うHFD群の間でScheffe post hoc analysis(Scheffe)を行った。
本研究の参加者は,117名の健康なボランティア(平均年齢37.8 ± 19.6歳,女性65名,男性52名)と5名の良性腫瘍患者(平均年齢55.5 ± 9.6歳,女性2名,男性3名)で、参加者全員が本研究に参加することに書面で同意し、事象関連記憶課題と3T MRIによるT1強調画像の撮影を行った。参加者は、WHO基準の体格指数(BMI)に基づいて、普通体重(BMI<25)、過体重(BMI≧25かつ<30)、肥満(BMI≧30)の3群に分けた。正常体重群は84名(平均BMI 20 ± 1.8)、過体重群は27名(平均BMI 26 ± 1.2)、肥満群は11名(平均BMI 32±2.3)であった。すべての実験は、琉球大学の人を対象とした医学・健康研究に関する倫理委員会の承認を得て行われた。
本研究で用いた事象関連記憶課題のfMRI実験の詳細は,既報(32)に記載されている。記憶課題は、ルアーセット16個(類似画像)、リピートセット16個(同一画像)、新規アイテム44個(新規画像)の108枚の写真で構成されていた。参加者は、ディスプレイに表示された写真刺激が、新規アイテム(new)、繰り返しの同一写真(same)、あるいは前回の写真と似ているが同じではない写真(類似;lure)のいずれであるかをボタンで応答するよう指示された。記憶課題中の被験者のボタン操作をパーソナルコンピュータに記録し、new,same,lureの正答率を算出した。各課題(新規課題、類似課題、同一課題)の正答率は、以下の式で算出した。
正答率(%)=提示された課題の正答数/提示された課題の総数×100
今回の実験では、新しい刺激に対するタスクの提示総数を76個、同じ刺激とルアー刺激に対するタスクの提示総数をともに16個に設定した。
3T-MRI(Discovery MR750; General Electric, Milwaukee, WI)を用いて、脳の機能画像および構造画像を取得した。BOLDコントラストを測定するための機能画像には、エコー・プラナー・イメージング(EPI,繰り返し時間:1500 ms,エコー時間:25 ms,フリップアングル:70°,マトリクスサイズ:128 × 128,視野:192 × 192,面内分解能:1.5 × 1.5 mm2,23スライス,3 mm厚,0 mmスペース)を用いた。解剖学的脳画像は、3次元(3D)スポイルド・グラディエント・リコール・エコー(SPGR)シーケンス(矢状断面におけるスライス厚:1 mm,マトリックスサイズ:256×256、視野:256×256mm、繰り返し時間:6.9ms、エコー時間:3ms、フリップアングル:15°)を用いて取得した。海馬の構造を可視化するために、および3D SPGR画像とEPI機能画像のコレジストレーションを行うために、高分解能T2強調高速スピンエコーシーケンス(マトリクスサイズ:512×512,視野:192×192 mm,繰り返し時間:4300 ms,エコー時間:92 ms、面内分解能:0.375×0.375 mm2、23スライス、厚さ:3 mm、スペース:0 mm)を取得した。
SPM12を用いて、機能画像の再配置、時間的相関、空間的正規化、空間的平滑化の前処理を行い、解析した。海馬アトラス(Duvernoy社)に基づき、高解像度の冠状T2強調画像上に、海馬の下位領域(CA3、CA1、DG)、海馬周辺領域(傍海馬回、周囲皮質、嗅内皮質)をペンタブレットを用いて手書きで描画した。各被験者の海馬領域におけるBOLD応答の信号変化の割合をMarsBarツールボックスを用いて抽出した。3D-SPGR画像をボクセルベースモルフォメトリーの解析に使用した。T1強調画像は,SPM 12セグメンテーションツールを用いて、GM、白質、脳脊髄液の各画像にセグメンテーションした。脳全体のGMの体積は、GM画像の空間的な正規化と調節の後に計算された。
MRIデータは、GE Medical Discovery MR 750 3Tスキャナー(32チャンネルヘッドコイル付)を用いて取得した。仰臥位でスキャナのベッドに横たわった被験者は、フォームパッドとフィラデルフィア製ネックカラーで頭と首を固定し、頭の動きを最小限に抑えた。安静時fMRI画像は、全脳をカバーするシングルショットEPIシーケンスを用いて取得した(42軸スライス,厚さ4 mm,スライス間ギャップなし,繰り返し時間2000 ms,エコー時間30 ms,フリップアングル70°,マトリクスサイズ64 × 64,視野256 × 256)。1回のセッションで,合計150個のボリュームを撮影しました。解剖学的脳画像は、T1強調矢状3D SPGRシーケンスを用いて取得した。
画像の前処理および機能ネットワーク解析は、SPM12およびCONN toolbox 18.b(www.nitrc.org/projects/conn、RRID: SCR_009550 [Whitfield-Gabrieli and Nieto-Castanon, 2012])を用いて行った。詳細は既報(32)のとおりである。画像は、リアライメント、スライスタイミング補正、コレジストレーション、ノーマライゼーション、スムージング、セグメンテーションの順に前処理を行った。BOLD信号のノイズは、BOLD信号に含まれる潜在的な交絡効果の線形回帰と,時間的なバンドパスフィルタリング(時間周波数が0.008Hz以下または0.09Hz以上)によって除去した。
CONNツールボックスを用いた機能的ネットワーク解析において、デフォルトモードネットワーク(DMN)と機能的結合性を解析した。まず、個別解析として、後部帯状皮質(PCC)と楔前部のBOLD変動と正の相関を持つ領域をDMNマップとして算出した。次に、DMNマップと負の相関を持つ領域を反相関DMNマップとして算出した。正常体重群、過体重群、肥満群のDMNマップと反相関DMNマップの平均像を、1サンプルt検定を用いて算出した(ボクセルレベルの閾値はp<0.001(補正なし)、偽発見率[FDR]のクラスターレベルの閾値はp<0.05(補正あり)とした)。
機能的結合解析では、グラフ理論パラメータのノードとエッジを計算した。各ROI間のBOLD信号の変動の相関係数は、132 × 132 ROIマップ(CONN toolboxのデフォルトROIアトラス)を用いたシードベースの接続性測定法(53)で計算した。ROI-to-ROI degree (任意のノードが各ノード間に持つエッジの数)とbetweenness centrality (グラフ内の任意の2つのノードのペア( j, i )に通る頂点の最短パスの数)は,我々の132 × 132 ROIマップを用いて確立したROI-to-ROI correlation 132 × 132 matrixによって計算した。
度合い(Degree)は、各ノードにおいて、各ノードから/へのエッジの数として定義されます。
度合い(di)は以下の式で定義されます。
i = 1,2,3.....,132; j = 1,2,3....,132
Betweenness centrality(BCi)は、他の機能モジュールやノードをつなぐハブを表しており(54)、BCiは以下のように定義される。
17-18週齢の雄のC57BL/6Jバックグラウンドマウスから海馬脳スライスを調製し、これまでの報告(56, 57)に従って、マウスをCD群、HFD群、PER処理を施したHFD(PERを伴うHFD)群に分け、あるいは12-13週齢のob/obマウスをob/ob群、PER処理を施したob/ob群に分けた。リニアスライサー(PRO 7N, Dosaka EM, Japan)を用いてスライス(厚さ, 300-400μm)を切り出した後、海馬脳スライスを通常のKrebs Ringer(125 mM NaCl, 2.5 mM KCl, 10 mM D-glucose, 1.25 mM NaH2PO4, 26 mM NaHCO3, 2 mM CaCl2, 1 mM MgCl2, 混合ガス[95% O2; 5% CO2]で連続的にバブリング)中で、室温で60分間インキュベートし、切断ダメージを回復させた。Ca2+インジケーターであるFluo-3 AM(励起波長:508 nm,発光波長:525 nm,Kd,0.4 μmol/L)(5 μM)(同仁堂,熊本,日本)を脳スライス標本に90分間ロードした。[Ca2+]iをモニターするために、共焦点顕微鏡下で光電子増倍管を用いてFluo-3の蛍光強度(F525)をモニターした(励起波長488 nm;LSM5 PASCAL, Carl Zeiss, Germany)。今回の実験では、低倍率(2.5倍)の対物レンズ(FLUAR 2.5×,NA=0.12,Carl Zeiss, Germany)を用いて、海馬スライス全体のCaイメージングを行った(図1)。Fluo-3蛍光画像(512×512ピクセル)をデジタル化し、10秒ごとに50分間、パーソナルコンピュータに保存した。CA1、CA2、CA3、DG、ECの各領域のF525は、オフライン解析(Microsoft Excel, Microsoft Corporation, WA)のもと、F525の平均値(AMPAまたはNMDA印加の0~5分前の値)で正規化(NF525)した。Caイメージングの間、脳切片は、125 mM NaCl,2.5 mM KCl,10 mM D-glucose,1.25 mM NaH2PO4,26 mM NaHCO3,2 mM CaCl2,1 mM MgCl2,および1 μM テトロドトキシンを含む細胞外溶液を、灌流チャンバー(35 mm μ-dish, Ibidi GMBH, Graefelfing, Germany)内で混合ガス(95% O2; 5% CO2)により連続的にバブリングしながら2 mL/minで灌流した。なお、NMDAを適用するケースでは、MgCl2の濃度は、NMDARのMg2+依存性阻害を防ぐために0 mMとした(58)。NF525の最大値は、AMPA(Tocris, Bristol, UK)(100 μM)またはNMDA(Tocris, Bristol, UK)(50μM)印加後5分以内のNF525の最大値として推定した。AMPARの脱感作を防ぐためにシクロチアジド(CTZ)(100μM)、NMDARを活性化するためにグリシン(Tocris, Bristol, UK)(10μM)をそれぞれ使用し、ペランパネル(Eisai Co,Ltd., Tokyo, Japan)(100μM),GYKI47261(Tocris, Bristol, UK;以下単に「GYKI」ともいう)(100μM),または(2R)-アミノ-5-ホスホノ吉草酸(APV)(Tocris, Bristol, UK)(50μM)をそれぞれAMPARまたはNMDARのアンタゴニストとして使用した。また、Ca2+透過性のAMPARのアンタゴニストとして、1-ナフチルアセチルスペルミン(NASPM)(Tocris, Bristol, UK)(20μM)を用いた(59)。
ここで行われたBS3手順を用いた架橋は、以前に記載された方法に基づいて行われた(60)。17~18週齢の雄のC57BL/6Jバックグラウンドマウス(CD、HFD、およびPER処理を伴うHFD)、または12~13週齢のob/obマウス(ob/ob、およびPER処理を伴うob/ob)の海馬スライスを、以前に報告されたように調製した(56、57)。海馬スライスを準備して回収した後、解剖用ナイフを用いて海馬スライスからCA1、CA2、CA3、DG、EC領域を分離した。CA1、CA2、CA3、DG、EC領域を、2 mM BS3(Thermo Fisher Scientific, Wilmington, DE, USA)を添加した氷冷した人工脳脊髄液(ARTCEREB;大塚製薬株式会社、東京、日本)を入れたエッペンドルフチューブ(Eppendorf, Hamburg, Germany)に加えた。インキュベーションは氷上で30分間行った。架橋は100mMグリシンで反応をクエンチすることで終了させた(4℃で10分)。海馬のサブ領域を、プロテアーゼおよびホスファターゼ阻害剤を含む氷冷した溶解バッファー(25 mM HEPES, pH7.4, 500 mM NaCl, 2 mM EDTA, 1 mM DTT, 1 mM フェニルメチルスルホニルフルオリド, 20 mM NaF, 1 mM オルトバナジン酸ナトリウム, 10 mM ピロリン酸ナトリウム, 1×プロテアーゼ阻害剤混合物[Sigma-Aldrich, St Louis, MO]、および0.1% Nonidet P-40 [v/v]])に再懸濁し、5秒間の超音波処理で急速にホモジナイズした。ライセートの総タンパク質濃度は、Lowry法(61)を用いて測定した。サンプルを分注し(マウス1匹につき約15に分割)、後の分析のために-80℃で保存した。BS3サンプルは、精製せずに直接SDS-PAGEで分析し、表面バンドと細胞内バンドを同じレーンで測定したため、正規化の必要がなく、サンプルのスループットが向上した。全タンパク質ライセート(40μg)をロードし、還元条件下で5%-10% Tris-HClゲル(Thermo Fisher Scientific, Wilmington, DE)上で電気泳動し、タンパク質をニトロセルロース膜(Thermo Fisher Scientific, Wilmington, DE)に移してイムノブロッティングを行った。メンブレンはブロッキング試薬Blocking One(Nacalai Tesque, Tokyo, Japan)を用いて室温で30分間ブロッキングした。その後、メンブレンを抗GluA1(1:1000、Merck Millipore, Burlington, MA)およびアクチン(1:5000、Protein Teck, Chicago, IL, USA)とともに4℃で一晩インキュベートした。膜をHRP標識抗ウサギIgG(1:3,000、Cell Signaling Technology, Danvers, MA, USA)と30分間インキュベートし、TBS-Tで再度広範囲に洗浄した。膜を化学発光検出基質Chemi-Lumi One(Nacalai Tesque, Tokyo, Japan)に1分間浸した後、LuminoGraph1イメージングシステム(Atto, Tokyo, Japan)を用いて発光を検出した。各レーンの表面および細胞内のバンドをCS Analyzer(Atto, Tokyo, Japan)を用いて解析した。
17-18週齢の雄のC57BL/6Jバックグラウンドマウス(CD、HFD、およびPER処理を伴うHFD)または12-13週齢のob/obマウス(ob/obおよびPER処理を伴うob/ob)マウスの海馬スライスを、以前に報告されたように準備した(56, 57)。海馬スライスを準備して回収した後、57BL/6Jバックグラウンドマウスとob/obマウスからそれぞれ解剖用ナイフを用いて海馬スライスからCA1、CA2、CA3、DG、EC領域を分離した。マウスのCA1,CA2,CA3,DG,EC領域を分離し、0.5 mLのTRIzol RNA単離試薬(Thermo Fisher Scientific, Wilmington, DE)を用いて、使い捨てのホモジナイズチューブBioMasher2(商標)(Nippi, Tokyo, Japan)で直ちに溶解させた。トータルRNAは、メーカー(Thermo Fisher Scientific, Wilmington, DE)が提供するTRIzol RNA isolation reagentの残りのプロトコールに従って、各領域から個別に抽出した。PrimeScript RT Reagent Kit(Takara, Shiga, Japan)を用いて、1μgのtotal RNAを逆転写した。得られたcDNAのアリコートを1:10に希釈し、TB Green Premix ExTaq(Takara, Shiga, Japan)のマスターミックスに加え、7500 Fast Real-Time PCRシステム(Applied Biosystems, Foster City, CA, USA)を用いて、製造者の指示に従ってリアルタイムPCRを行った。リアルタイムPCRの条件は以下の通りであった。95℃で30秒、次いで95℃で5秒と60℃で34秒とを40サイクル。アニーリングステップごとに蛍光強度を測定し、7500ソフトウェアを用いて閾値サイクルタイムを決定した。さらに、相対的なβ-アクチンの発現レベルをそれぞれの対照(ビヒクル群)に対して正規化した。各サンプルにおける標的遺伝子の相対的な発現レベルは、標準曲線を用いて決定し、さらに同じサンプルのβ-アクチンの発現レベルに対して正規化した。これらの遺伝子のプライマーおよびプローブの配列を補足表2に示す。GluA2のmRNA量/(GluA1+GluA3+GluA4のmRNA量)の比率」をCa2+透過性指標とした。
GluA2編集部位のサンガーシークエンス用テンプレートは、
フォワードプライマー(GAGGAATTTGAAGATGGAAGAGA:配列番号44)とリバースプライマー(AGGAGGAGATGATGATGAGGGT:配列番号45)を用いてcDNAを増幅して調製した。PCR産物(10%)をアガロースゲル電気泳動で確認した。適切な予想サイズを確認した後、PCR産物を直接シークエンスの前にinnuPREP PCRpure Lite Kit(Analytik Jena, Jena, Germany)を用いてメーカーの指示に従って精製した。サイクルシーケンスは、BigDye Terminator v3.1 Cycle Sequencing Kit(Applied Biosystems, CA, USA)と上述の各プライマーを用いて行った。サンガーシークエンス解析は、ABI3500(Applied Biosystems, CA, USA)とシーケンスビューアソフトウェア4Peaksを用いて行った。
GluA2のQ/R編集状態は、TaqMan(商標)カスタムSNPジェノタイピングアッセイ(Thermo Fisher Scientific, Wilmington, DE)を搭載したQuantStudioTM 3DデジタルPCRシステムを用いて解析した。TaqManプローブはThermo Fisher Scientific社が合成したもので,非編集(Q)配列の検出にはVIC標識のプローブを、編集(R)配列の検出にはFAM標識のプローブを用いた(それぞれ5’ CATCCTTGCTGCATAAA 3’:配列番号46,5’ ATCCTTGCCGCATAAA 3’:配列番号47)。PCR反応には以下の増幅用プライマーペアを使用した:
マウスフォワードプライマー:5’ TTGGGATTTTTAATAGTCTCTGGTTTTCCTT 3’(配列番号48)および
マウスリバースプライマー:5’ GACCAACCTTGGCGAAATATCG 3’(配列番号49)。5-50 ngのcDNAをデジタルPCRマスターミックス(Thermo Fisher Scientific, Wilmington, DE)と混合し、混合したTaqManプローブをメーカーの指示に従って自動チップローダを用いてQuantStudioTM 3DデジタルPCR 20Kチップ(Thermo Fisher Scientific, Wilmington, DE, USA)にロードした。ロードしたチップは、Gene Amp 9700 PCRシステム(Thermo Fisher Scientific, Wilmington, DE, USA)を用いて、以下の条件で増幅した。96℃で10分、56℃で2分、98℃で30秒のサイクルを39回繰り返した後、60℃で2分間の最終伸長ステップを行った。増幅後、QuantStudio 3D Instrument(Thermo Fisher Scientific, Wilmington, DE, USA)を用いてチップを画像化しました。チップデータの解析には、QuantStudio 3D Analysis Suite Cloud Software (Thermo Fisher Scientific, Wilmington, DE) を用いて、相対的および定量的なデータ解析を行った。
Bregma -0.94 mmからBregma -4.04 mmまでのマウス脳の冠状切片(背側から腹側、内側から外側の全海馬を含む、幅5 mm)を4%パラホルムアルデヒドで2日間固定した後、Golgi Cox染色システム(FD Rapid GolgiStainTM Kit, MD 21041, USA)で染色した。含浸させた組織を100μmの切片に切り、クリスタルバイオレットでカウンターステインし、CA1とDGの先端樹状突起にある15μmのスパインの総数と、形態的なスパイン(thin, stubby, mushroom)の割合をAxio Observer Z1(Carl Zeiss, Germany)を用いて調べた。海馬全体を含む脳のパラホルムアルデヒド固定された5mm幅の冠状切片の別のセットを、受動的CLARITY技術法(PACT)(62)を用いて処理し、10倍クリアー対物レンズを用いてLightsheet Z.1(Carl Zeiss, Germany)を通して調べ、Thy1-YFPHトランスジェニック野生型マウス(The Jackson Laboratory, stock number: 003709, strain name: B6.Cg-Tg.(Thy1-YFP)16Jrs/J)(63, 64)、HFD給餌マウス、PER処理を伴うHFD給餌マウスの海馬全体の3D画像をそれぞれ取得した。Z次元、時間次元、チャンネル次元に沿って最大強度の投影画像を取得し、特定の画素位置でスタック内の全画像に対する最大値を含む画素を持つ出力画像を作成した。Z-stack機能では、異なるフォーカス位置にある一連のXY画像を取得し、Z-stackを作成することができます。このようにして、標本から、CA1、CA3、DGのそれぞれの背側から外側、および内側から外側の400μm×400μmの3Dデータセットが得られた。ギャラリービューでは、Z-スタックからの画像を時系列で表示している。
3D再構成は、Arivis Vision 4次元(4D)ソフトウェアを用いて行った。Blob finder(フィルター)を使用して、ノイズの多い画像の中から、球状の形状に近い、丸めた2Dおよび3Dセグメントに用いられた。ガウス尺度を用いて、物体のシードを見つけ、分水嶺アルゴリズムで物体の境界を特定した。関心構造の平均サイズを20μm、閾値を5に設定した。 高解像度レンダリングは、現在の視界をより高い画像やデータの解像度で視覚化するアプローチである。
マウスの脳を4%パラホルムアルデヒドで灌流固定し、パラフィンを埋め込み、4マイクロメートル厚の切片に切断して、DCXに対するモノクローナル抗体(E-6モノクローナル、1:50、Santa Cruz Biotechnology Inc,Dallas, USA), MAP2ab (AP-20 monoclonal, 1:100, SIGMA-ALDRICH, St. Louis, USA), GluA1 (AB1540 polyclonal, 1:100, EMD Millipore Corp., Burlington, USA), GluA2 (AB1768-1 polyclonal, 1:5, EMD Millipore Corp., Burlington, USA)の免疫組織化学分析に用いた。
MAP2、GluA1、GluA2の各発現領域における免疫染色領域の取得は、ZVIフォーマットのAxioVision(Carl Zeiss, Germany)顕微鏡(対物レンズ20X)を用いて行った。画像分割を伴うデータ画像処理の生成は、ZEN Intellesisソフトウェアを用いた機械学習を用いて行った。各グループで1000個以上の細胞を調べた。3SD以上のデータを除いた3群間の分散の検定には、一元配置分散分析を用いた。そして、以下のサンプル数を用いた。
HFDシリーズ
MAP2 CD n=731、HFDs n=288、HFD+PER n=225;GluA1 CD n=220, HFD n=156, HFD+PER n=94;GluA2 CD n=78, HFD n=114, HFD+PER n=182;GluA2/GluA1 CD n=78, HFD n=94, HFD+PER n=94
ob/obシリーズ
MAP2 ob/ob対照 n=849, ob/ob PER 1w n=967, ob/ob+PER 12 w n=511; GluA1 ob/ob対照 n=163, ob/ob PER 1w n=124, ob/ob+PER 12 w n=242。GluA2 ob/ob対照 n=39, ob/ob PER 1w n=44, ob/ob+PER 12 w n=369, GluA2/GluA1 ob/ob対照 n=54, ob/ob PER 1w n=41, ob/ob+PER 12 w n=234.
グループ間の有意差を分析するための事後検定にはBonferroni法を用いた。
シークエンスされた生のRNA-seq fastqリードは,HISAT2(v.2.2.0)を用いて,マウスGRCm38(アンサンブルリリース104,http://ftp.ensembl.org/pub/release-104/fasta/mus_musculus/dna/)のゲノムにアラインメントした(65)。全サンプルの平均マッピング率は94.21%(範囲88.70-96.30%)であった。整列したリードはSalmon (v.0.14.2) (66)を用いて定量化し、TPM値はStringTie (v.2.1.2) を用いて算出した(67)。バリアントコールは、GATK パッケージ(v.3.8)(70)を用いて,GATK Best Practices for RNAseq short variant discovery(68,69)に従って行った。リードグループ情報の追加、ソート、重複のマーク、インデックスの作成は、Picard社のツールを用いて行った。GATKツールのSplit N Cigar Readsは、リードをエクソンセグメントに分割し、イントロン領域にはみ出した配列をハードクリッピングするために使用した。バリアントコーリングとフィルトレーションは、それぞれGATK HaplotypeCallerとVariantFiltrationを用いて行った。出力されたバリアントの機能アノテーションは、SnpEff (v.4.3) (71)を用いて行った。RNA-editingレベルは、GluA2のR/Q編集部位(chr3: 80706912)にアライメントされたリードの総数と、この部位でT-C変換されたリードの数との比率として算出した。
動物モデル実験では、一元配置のANOVAとBonferroniの多重比較検定および/または両側t検定を用いて統計解析を行った。統計的有意性はp < 0.05とした。ヒトの実験では、BMI、全脳のGM容積、体重と、課題条件(新規、類似、同一)に対する正答率との関係を偏相関分析で解析した。また、3つの課題条件(新規、類似、同一)における平均脳活動と正答率の差を一元配置分散分析で解析した。
HFDはマウス海馬のAMPARを介したカルシウム流入を刺激する
最初に、離乳後12週間のHFD飼育に伴うマウス肥満モデル(表1に示されている食餌の組成(7)を用いた海馬周辺領域を有する海馬の急性脳スライスにおいて細胞内カルシウム濃度([Ca2+]i)の直接測定を行い海馬のカルシウムシグナル伝達(図1A、補足データ動画 1)を追跡した。HFDを与えられたマウスは、EC(嗅内皮質)、CA1(cornu ammonis 1)、CA2、CA3、およびDG(歯状回)に100μM CTZ(サイクロサイアザイド;チャネル脱感作剤)を含む100μM AMPARを適用した。適用前後で正規化されたF525を比較した。正規化されたF525(NF525)の最大値を示標として評価すると、HFD群では、AMPA型受容体を介したカルシウムシグナル伝達の顕著な活性化を認めた(図1B、C、D、E、および表3)。
対照的に、10μM グリシン(チャネルコアクチベーター)を含む50μM NMDA(N-メチル-D-アスパラギン酸)を介したカルシウムシグナル伝達は、HFD飼育マウスに由来する海馬では不活性化されたが、CD(control diet: 高脂肪飼料ではない通常の飼料)を与えられたマウスは海馬を介した活性化を示した(図1F、G、H、IおよびJ、表3)。
HFDとペランパネル(PER)(Fycompa(商標)、エーザイ、日本)(5mg/kg/日、ハイドロゲルにより経口投与)を与えられたマウス由来の海馬スライスで、新規の非競合的AMPAR拮抗薬(8)で処置したマウスは、AMPARを介した伝達は正常化されたることを同定した(図1D、表3)。またNMDARを介したシグナル伝達が同時に回復(図1I、表3、および補足ムービー3)した。HFDを与えられたマウスに由来する急性スライスにおけるAMPARを介した[Ca2+]iの上昇の誘導は、100μM PERによって完全に消失した(図1B-D)。これらの事実は、AMPARを介したカルシウム流入がNMDA受容体(NMDAR)の不活性化を誘導したことを示唆する。その結果、HFD摂取マウスでは、CD摂取マウスと比較して、EC、DG、CA3、CA2、CA1において、100μMのAMPARと100μMのCTZの印加による[Ca2+]iの有意な増加が観察され、同時に50μMのNMDAと10μMのグリシンによる有意な不活性化も観察された(図1E、J)。これらの結果は、HFDがグルタミン酸受容体サブユニットの発現を変化させることを強く示唆するものであった。AMPARを介した[Ca2+]iの変化は、少なくとも7日間、HFDを与えたマウスで観察された(図7A, B, C)。興味深いことに、これらのマウスは、EC、DG、CA3、CA2、CA1において、10μMのグリシンを加えた50μMのNMDAを浴中に適用しても、NMDARを介した活性化が維持された。この場合も、これらの病的なAMPARを介した[Ca2+]iの増加は、100μMのPERの添加によって完全に消失し、同様に、100μMのGYKI47261(Tocris, UK, Bristol)やCa2+透過性AMPAR(CP-AMPAR)の選択的アンタゴニストである20μMのNASPM(Tocris, UK, Bristol)などの他のAMPARアンタゴニストについても同様に病的なAMPARを介した[Ca2+]iの増加は消失した(図7A-D)。インビトロの人工的な系においては、カルシウム透過型AMPARを介したCa2+の侵入が[Ca2+]iによるNMDARの阻害をきたすことが提案されていたが(9)、その生理的意義や疾患との関連については不明であった。上記結果は、過体重または肥満症の対象において、カルシウム透過型AMPA受容体によるNMDARの抑制を見出し、これをAMPA受容体アンタゴニストにより解消し得ることを示す。
Ca2+透過性AMPAR(CP-AMPAR)は脳腫瘍(10)、筋萎縮性側索硬化症(ALS)(11)、コカイン中毒(12)、神経因性疼痛(13)、てんかん(14)などさまざまな神経疾患の背後にある病態生理に関連している。病態生理学の背後にある分子の多様性は、各疾患で異なり十分解明されていない。しかし、上記結果に基づけば、AMPARおよびNMDARを介した[Ca2+]iにおけるカルシウム動態の変容が認知機能低下を伴う肥満に重要な役割を果たす可能性がある。
AMPARは、最も早い速度の興奮性神経伝達を仲介する。AMPARは、AMPARチャネルの機能特性を決定する4つのサブユニットGluA1-4で構成され、AMPARの多様性はこれらのサブユニットの豊富度による。具体的には、GluA1、GluA3、およびGluA4の他のサブユニットがCa2+透過性であるのに対して、GluA2サブユニットは本質的にCa2+不透過性である。そして、AMPARのCa2+透過性は、GluA2発現の相対量によって調節される。すなわち、GluA2サブユニットの相対量が増加すれば、AMPARのCa2+透過性は低下し、相対量が減少すれば、AMPARのCa2+透過性は増加すると考えられる。
GluA2は、二本鎖RNAのアデノシン(A)からイノシン(I)への編集に関与するRNAタイプ2に作用するアデノシンデアミナーゼ(ADAR2)によって、リエントリーM2膜ループ領域内のQ/R部位において編集される(レビュー、15、16および17参照)。リアルタイム定量的逆転写ポリメラーゼ連鎖反応(qRT-PCR)、AMPARとNMDARのサブユニット組成を明らかにすることを試みた。海馬のサブ領域(CA1、CA3、DG、EC)でのトータルRNAを分離し、AMPARに関してはGluA1、GluA2、GluA3、GluA4、NMDARに関してはGluN1、GluN2A、GluN2B、GluN2CのmRNAの定量分析をqRT-PCRにより行った。その結果、HFDを与えたマウスに関連するCA3領域でのGluA1、GluN1、およびGluN2Bの発現の有意な上昇、およびDGでのGluN2Bの発現上昇(それぞれp<0.05),PERを伴うHFDを与えたマウスにおけるCA1でのGluA2の有意な上昇を見出した(p<0.05)(図2A)。各サブユニットのmRNA量に基づいて、他のGluAサブユニットに対するGluA2の比率(図2B)を求めた。また、当該比をCa2+透過性指標(Ca2+-permeability index)とした。
対照食(CD)、高脂肪食(HFD)、またはPER(Fycompa(商標)、エーザイ、日本)(5mg/kg/日)を含むHFDで維持された離乳後(6週間)の食餌摂取量とマウスの体重を18wまで監視した(図2、EおよびF)。食事のカロリー量は3つのグループ間で有意差は認められなかったが、PER投与グループによるHFDの体重の有意な抑制は、HFDと比較して16wから始まり、HFDの有意な体重増加はCDと比較して8wから始まった。認知行動に対するHFDの効果を明らかにするために、オープンフィールド試験(図9、AC)高架式十字迷路試験(図9、DE)、文脈的恐怖条件付け試験(図9、F)、新規物体認識テスト(図2G)と5回試行のダイレクトインターラクションテスト(図2H)を実施した。これらのテストの中で、後者の2つのテストでは、CD群とHFD群の間にそれぞれ有意差があることがわかった(p<0.005)。海馬NMDARは自発的物体認識記憶の検索に重要な役割を果たすと報告されているため(18、19、20)、行動分析を実施した。その結果、HFD群では、物体認識記憶の検索能が低下した(図2G)。この結果はHFDマウスの海馬NMDA受容体機能の変化とよく一致していた。また、HFDマウスのCA1およびCA3でNR2B RNAレベルの上昇が見られた(図2A)。これは、AMPARによるNMDARの阻害によるリバウンド現象の可能性がある。海馬CA2領域は社会性記憶に不可欠な役割を果すと報告されている(21)。以上の通り、高脂肪食によりカルシウム不透過型AMPARから透過型-AMPARに誘発されることで社会性記憶に異常をきたすこと(図2H)や、および海馬CA2領域のNMDA受容体機能が阻害されることを見いだした。(図1)。確かに、PER治療は、CA2の[Ca2+]i信号の正常化に伴って、HFDを与えられたマウスの社会性記憶障害を回復させた。HFD飼育マウスで認められたパターン補完能力(部分的な手がかりから保存された記憶全体を復元する能力)の低下がPER投与にて回復していることが判明した(図2I、J、K)。NMDA受容体の活性化は、記憶想起における連想中のパターン補完には必要なく(22)、私たちのデータからは、AMPA受容体がカルシウム透過型に変化することがHFDマウスのパターン補完能力の障害に重要な役割を果たすことが示唆された。
NL-GFマウスは、8週齢からAβの脳内に蓄積し始める(Nat. Neurosci. 2014 17,661-4)。本実施例では、上記ADモデルマウス(n=5)に対して8週齢から13週齢まで5mg/kg体重/日の用量のペランパネルを含むCD食を摂取させ、マウスを新規物体認識テストに供した。陰性対照(n=5)では、ペランパネルを含まない通常のCD食を摂取させた。13週齢におけるマウスの新規物体認識テストの結果は図23に示される通りであった。図23に示されるように、ADモデルマウスは、同室のマウスと初対面のマウスとを区別できないのに対して、ペランパネル(PER)投与群では、有意差を持って同室のマウスと初対面のマウスとを区別した。この結果は、NL-GFマウスではCAが機能障害を誘発するのに対して、AMPA受容体アンタゴニストはこの機能障害の発症を抑制したことを示す。このように、アルツバイマー型認知症におけるAMPA受容体のカルシウム透過性の向上とこれによるNMDA受容体の抑制は、AMPA受容体アンタゴニストにより処置でき、かつ、当該処置はアルツハイマー型認知症における脳機能障害を回復させた。
食欲不振の摂食行動によって刺激されるエネルギー消費を調節するホルモンであるレプチン(25、26)も、NMDARを介したシナプス伝達に重要な役割を果たす(27)。HFDを与えられたマウスは一般に、最初に肥満の特徴の1つであるレプチンの上昇を示し、徐々に慢性肥満はレプチン耐性を形成し、食欲を抑制しエネルギー消費を促進することが出来なくなる(28)。ob/obマウスにおけるCP-AMPARに対して、Gyki、Naspm、及びPERのいずれも、EC、CA1、CA2、CA3、及びDGのいずれの領域においても有意な抑制効果を示した(図10-1A)。PERを経口投与したob/obマウスにおいて、leptin(レプチン)は、EC、CA1、CA2、CA3、及びDGのいずれの領域においても有意にNMDARの機能を増強させた(図10-2パネルB)。PERを経口投与したHFDマウスにおいても同様に、leptin(レプチン)は、EC、CA1、CA2、CA3、及びDGのいずれの領域においても有意にNMDARの機能を増強させた(図10-2パネルC)。これらの結果は、海馬グルタミン酸作動性シナプス伝達におけるレプチンの重要性を示している。なお、これらの実験では、AMPAは100μMで用いられ、CTZは100μMで用いられ、NMDAは50μMで用いられ、PERは100μMで用いられ、グリシンは10μMで用いられ、APVは50μMで用いられ、Naspmは10μMで用いられ、Gykiは100μMで用いられ、レプチンは100nMで用いられた。*は、t検定においてp<0.05であることを示す。
体格指数(BMI)と脳灰白質量の相関関係を調査するために、122人の日本人被験者(84人(BMI:25未満)、27人(BMI:25以上30未満)、11人(BMI:30以上)を含む)(表5)の脳のT2磁気共鳴(MR)イメージングによって得られたデータから容量分析を適用することによって解析した。
特に海馬の記憶機能におけるヒト認知のバイオマーカーに対する安静時fMRIの重要性が高まっている(35)。安静時脳活動を計測したfMRIデータでは組織化された大規模ネットワークの中で、デフォルトモードネットワークが健康な個人の顕著性と中央執行ネットワークを動的に制御することを示唆している。肥満の個人ではこのネットワークが乱れて認知機能の低下をもたらした(図6A)。最後に、正常、過体重、および肥満の検査官のグラフ理論に基づく人間のネットワークの定量分析を適用した。シードベースの分析によって各グループの機能的接続マップを作成し、後帯状皮質と楔前部をデフォルトモードネットワークに設定し、前帯状皮質と海馬記憶の小脳小葉CrusIを関心領域として機能させた(35)。分類された各グループの被験者間で異なるネットワーク接続を図6Bに示した。モジュールの自律性の崩壊に関連するより大きなネットワークと中心性マッピングの間の特定の交代は、通常のBMIグループと比較して過体重と肥満のグループで明らかになり、正しい記憶想起の失敗をもたらした。デフォルトモードネットワーク(DMN)、顕著性ネットワーク(SN)、感情的ネットワーク(EN)、および小脳ネットワーク(CBN)の独立したネットワーク形成が正常体重群で観察されました。過体重のグループでは、中央執行ネットワーク(CEN)が通常の体重群のネットワークに追加され、DMNとCENの間の新たな接続性が実証された。ENとCBN内の内因的な接続も増加した。肥満グループではCENおよび視覚ネットワーク(VN)の固有の接続性が強化され、両側の島皮質がSNに出現した。前帯状皮質と帯状回の間の中心性は、他の2つのグループよりも増強された。異なる機能ネットワーク(DMN、SN、CEN、CBN、およびVN)間の接続は、肥満グループで拡張された。正常体重、過体重、および肥満のグループのノードの総数は、それぞれ17、21、および23と推定された(図6B、C)。
38のBMI値を有する70歳女性の海馬および体重に対するAMPA受容体アンタゴニストの効果を検討した。70歳女性は、大孔腫瘍により四肢麻痺および呼吸障害を有した。腫瘍摘出後、抗てんかん治療を施した。抗てんかん治療としては、2mg/日(その後、4mg/日に変更)のペランパネルを経口投与した。前記女性の体重および下肢のFugl-meyer assessmentスコアの推移を観察した。また、術後26週目の前記女性の脳のMRIから海馬の大きさを評価した。結果は、図26に示される通りであった。図26に示されるように、PER投与により明らかに海馬(図の上部写真の枠内参照)の大きさが増大した。数値化すると以下表6の通りであった。
HFDがAMPARを介して海馬のカルシウム動態を刺激し、サブユニット構造が非CP-AMPARからCP-AMPARに再配列し、NMDARカルシウムシグナル伝達を変化させてCa2+流入をダウンレギュレートすることを示した。NMDARの機能による伝達の不活性化は、認知機能の低下に対応すると考えられる。CP-AMPARは、海馬のサイズと細胞数(特にCA3)の減少、CA1の樹状突起の完全性の低下、DG領域のスパインの成熟を引き起こした。HFDを7日以上摂取すると、カルシウムシグナルを介して海馬の記憶回路に影響を与えることがわかった。この短期間の摂取では、NMDARを介したカルシウムの動態は維持されていたが、AMPARを介したカルシウムの侵入が海馬で明らかになった。離乳後8週間以上の長期摂取では、AMPARを介したカルシウム動態の変化に伴い、NMDARを介したカルシウムの侵入は完全に減少した。AMPARとNMDARは、グルタミン酸系シナプスの高速伝達に関与し、シナプス後膜に局在して、学習や記憶などのヒトの認知に重要な役割を果たしている。NMDARによるシナプスへのカルシウム流入の抑制は、AMPAR、電位依存性カルシウムチャネル、内部貯蔵庫、NMDARなど、カルシウムの供給源にかかわらず、細胞内のカルシウム濃度によって調節される(9)。とりわけ、AMPARはNMDARと共局しており、10分の1ミリ秒の時間スケールで興奮性シナプス反応を司っているが、HFDはこれらの受容体のカルシウム進入を増加させ、最終的にNMDARを介したカルシウム電流の大きさを減少させた。NMDARは、シナプス可塑性の誘導(LTP/LTD)(36)に加えて、食欲や食嗜好の制御に重要な役割を果たしていることが報告されており、これが暴飲暴食、食事制限の失敗による反動、肥満の増加(37)という悪循環を引き起こし、最終的には認知機能の低下をさらに加速させると考えられる。AMPAR介在性亢奮によりNMDARを活性化して神経機能を回復させる試みは、却って神経傷害を促進することも示されている(72)。
BMIが高いと、若年層では認知障害と関連し(38)、中年層では、過体重と肥満が、老年期の認知機能低下の将来的なリスクを高め得る。高齢者では、BMIが高いほど認知機能が向上し、死亡率が低下するという逆説的な現象が認められる(39)。HFDは肥満を誘発し、高血圧、2型糖尿病、心血管イベントを引き起こし、これらの疾患を持つ患者は、認知機能低下のリスクが高まりる(40, 41)。おそらく、食行動の自己規制を含む適切なライフスタイル管理ができないために、効果的な治療や疾患のコントロールが妨げられている。医療においては、肥満に伴う認知機能の低下に対する効果的な介入方法を開発することが、より良い疾病管理の鍵となる。体重過多の人と肥満の人の脳では、楔前部と後部帯状皮質の安静時活動の低下と、背外側前頭前野と島皮質の活動低下が同時に起こるという相関関係が崩れていた(図6)。同様の乱れは、メタボリックシンドロームの被験者でも観察された(42)。さらに、グラフ理論を応用した安静時fMRIデータの結合性解析では、過体重の脳から肥満の脳にかけて、構造的・機能的な結合性が乱れており、ノードやエッジを伴うネットワークの数が増え、それらが一体となって右下前頭葉が位置する階層的なネットワークを形成しているのに対し、正常体重群では中心性のない単純なモジュール形成が見られた。トップダウンの干渉を受けずに自律的に働く少数の分散した並列のモジュールネットワークが高効率につながる。
注目すべきことに、高頻度の水頭症の出現は、早期発症の肥満マウスグループの間に見られた。ヒトでは、肥満は2歳から6歳までの幼児期に始まり(46)、さらに、その年齢で肥満であるほとんどの子供は青年期に肥満であった。適切で効果的な介入は、人間の健康と病気にとって喫緊の課題である。重要なことに、PER治療を伴うHFDの適用は、AMPARとNMDARの両方を介した調節不全のカルシウムシグナル伝達を回復し、同時に、脳のサイズと行動記憶能力の回復を誘発した。肥満による体重増加を防ぐことは健康上の利益をもたらす。側坐核でCP-AMPARを誘発すると報告されている食物制限(47)は、通常、食物制限はリバウンドし、体重増加と過食に終わる。これらを総合すると、AMPARはヒトの肥満に関連する認知機能低下の魅力的な治療標的であることが示唆される。
本実施例では、AMPA受容体アンタゴニストとしてペランパネルを用いて、ペランパネルはアルツハイマー型モデルマウス由来神経幹細胞培養系において培養上清のAβ42/Aβ40比を有意に上げ、Aβの沈着を低下させること、およびモデルマウスの海馬と大脳のAβの蓄積を抑制することを明らかにした。
てんかんは対照集団よりもAD患者で頻繁に発生し(Pascal E 2012)、てんかんの発生率は散発性AD患者で7~21%(Amatniekら、2006;Hauserら、1986;Mendez and Lim、2003)、早期発症家族性AD(FAD)では30%まで増加する(Palop and Mucke、2009;Larner and Doran、2006)。AD患者では、早期の潜行性海馬の過興奮性が検出された(Lam AD et al., Nat Med 2017)。図32に示すように、Aβ凝集を促進するトランスジェニック動物では、1つの変異を有するNL(-/-)マウスでも3つの変異を有するNL-G-F(-/-)マウスでも、野生型(WT)マウスに比べ、てんかん様反応の増強が確認された。特に、NL-G-F(-/-)マウス(15-17週齢)ではBicculine(ビクリン;BIC)によるCA1およびCA2領域のてんかん様反応の著しい増強が確認された(図32参照)。これらの結果は、海馬の過活動が背景にある臨床的背景と一致している。アミロイドβが蓄積したADの動物モデルでは、てんかん様反応が亢進することが報告されている(Busche, 2008; 2012)。本実験でも、NL(-/-)マウス、NL-G-F(-/-)(15-17週齢(W))ともにビクリンによるてんかん誘発が対照マウスと比較して増強していた。また、17W NL(-/-)マウスの脳ではアミロイドβの蓄積を検出できなかったことから、アミロイドβの蓄積はてんかんの興奮性そのものとは直接関係ないことが示唆された。
認知症モデルであるNL-G-F(-/-)マウスに対して、メマンチン(mema)、運動負荷(exsercise)、メマンチンと運動負荷、メマンチンとペランパネル、ペランパネル単独(5mg/kgまたは10mg/kg)を投与し、行動実験に供した。行動試験としては、新規物体認識テストと海馬機能依存の恐怖条件付け試験を実施した。メマンチンは、NMDA受容体のアンタゴニストであるが、他のNMDA受容体アンタゴニストとは異なる性質を有する(Hokama Y., et al., Neuro Oncology, 2022, noac162参照)。すなわち、メマンチンは、脳内でグルタミン酸が過剰に存在する場合にのみ作動する薬剤である。メマンチンはまた、シナプスを形成していないNMDA受容体(extrasynaptic NMDAR)に対してシナプス形成している部位のNMDA受容体(synaptic NMDAR)に対するよりも優位に作用する。
Claims (6)
- 過体重または肥満症を有する対象を処置することに用いるための、AMPA受容体アンタゴニストを含む医薬組成物。
- 過体重または肥満症を有する対象において過体重または肥満症の進展を遅延させる、抑制する、または停止させることに用いるための、請求項1に記載の医薬組成物。
- 過体重または肥満症を有する対象において体重を減少させることに用いるための、請求項1に記載の医薬組成物。
- 過体重または肥満症を有する対象において摂食量を減少させることに用いるための、請求項1または3に記載の医薬組成物。
- 過体重または肥満症を有する対象において、糖尿病、脂質異常症、動脈硬化、および心血管疾患の発症を抑制することに用いるための、請求項1~4のいずれか一項に記載の医薬組成物。
- AMPA受容体アンタゴニストが、ペランパネルである、請求項1~4のいずれか一項に記載の医薬組成物。
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