WO2011005978A2 - Procédés de manipulation de la signalisation cellulaire - Google Patents

Procédés de manipulation de la signalisation cellulaire Download PDF

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WO2011005978A2
WO2011005978A2 PCT/US2010/041392 US2010041392W WO2011005978A2 WO 2011005978 A2 WO2011005978 A2 WO 2011005978A2 US 2010041392 W US2010041392 W US 2010041392W WO 2011005978 A2 WO2011005978 A2 WO 2011005978A2
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light
cell
protein
transducing
generating
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WO2011005978A3 (fr
WO2011005978A9 (fr
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Ute Hochgeschwender
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Duke University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • the disclosure generally relates to methods of monitoring and/or modulating cell signaling. More particularly, the disclosure relates to polynucleotides, polypeptides, cells, assays, and methods that incorporate a light-generating protein and a light-transducing protein that can be used to manipulate, monitor, and study cell signaling in various cell types.
  • the genetic targeting of light-activated proteins is only one component of optogenetic methods.
  • the other component includes the hardware, such as integrated fiberoptic and solid-state optical tools, to allow for control in specific cell types, even at locations deep within the brain of a living subject (e.g., mammal) [Aravanis AM, et al., "An optical neural interface: in vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology,” J Neural Eng 4: S143-56, 2007].
  • Optical fibers can deliver light deep into the brain region of interest, and for superficial brain areas such as the cerebral cortex, either optical fibers or LEDs can be directly mounted to the surface of the animal's brain.
  • the disclosure provides an isolated polynucleotide comprising a sequence that encodes a light-generating protein, a sequence that encodes a light-transducing protein, and a promoter sequence operably connected to the sequences that encode the light- transducing protein and the light-generating protein.
  • the disclosure provides a vector comprising an isolated polynucleotide comprising a sequence that encodes a light-generating protein, a sequence that encodes a light-transducing protein, and a promoter sequence operably connected to the sequences that encode the light-transducing protein and the light-generating protein.
  • the disclosure provides a recombinant cell comprising a polynucleotide comprising a sequence that encodes a light-generating protein, a sequence that encodes a light- transducing protein, and a promoter sequence operably connected to the sequences that encode the light-transducing protein and the light-generating protein.
  • the disclosure provides a method of modulating cell signaling comprising, providing a cell comprising a polynucleotide that comprises a sequence encoding a light-generating protein, a sequence encoding a light-transducing protein, and a promoter sequence operably connected to the sequences that encode the light-transducing protein and the light-generating protein; and contacting the cell with a substrate capable of inducing emission of a photon by the light generating protein; wherein the emission of the photon by the light generating protein is capable of inducing a response in the light-transducing protein that is effective to modulate cell signaling.
  • the disclosure provides a method of monitoring cell signaling comprising, providing a cell comprising a polynucleotide having a sequence that encodes a light-generating protein, a sequence that encodes a light-transducing protein, and a promoter sequence operably connected to the sequences that encode the light-transducing protein and the light-generating protein; contacting the cell with a substrate capable of inducing emission of a photon by the light generating protein, wherein the emission of the photon by the light generating protein is capable of inducing a response in the light-transducing protein that is effective to induce or inhibit cell signaling; and wherein the emission of the photon by the light-generating protein provides a detectable signal for monitoring spatial light emission in a cell, a cell population, an organ, or an animal.
  • the disclosure provides a genetically modified non-human organism comprising a polynucleotide having a sequence that encodes a light-generating protein, a sequence that encodes a light-transducing protein, and a promoter sequence operably connected to the sequences that encode the light-transducing protein and the light-generating protein.
  • the disclosure provides a genetically modified non-human transgenic animal comprising a polynucleotide encoding a light transducing protein operably connected to a first promoter; and a polynucleotide encoding a substrate-inducible light- generating protein operably connected to a second promoter.
  • the disclosure provides a method of modulating cell signaling comprising, providing a cell comprising a first polynucleotide encoding a light-transducing protein operably connected to a first promoter; and a second polynucleotide encoding a light- generating protein operably connected to a second promoter; and contacting the cell with a substrate capable of inducing emission of a photon by the light generating protein, wherein the emission of the photon by the light generating protein is capable of inducing a response in the light-transducing protein that is effective to modulate cell signaling.
  • the disclosure provides a method of modulating cell signaling comprising, providing a first cell comprising a first polynucleotide encoding a light- transducing protein operably connected to a first promoter; providing a second cell comprising a second polynucleotide encoding a light-generating protein operably connected to a second promoter; and contacting the second cell with a substrate capable of inducing emission of a photon by the light-generating protein, wherein the emission of the photon by the light generating protein is capable of inducing a response in the light-transducing protein that is effective to modulate cell signaling.
  • the disclosure provides a recombinant cell comprising a polynucleotide encoding a non-native light-transducing protein operably connected to a first promoter; and a polynucleotide encoding a non-native substrate-inducible light-generating protein operably connected to a second promoter.
  • the disclosure provides a population of cells comprising a polynucleotide encoding a non-native light transducing protein operably connected to a first promoter; and a polynucleotide encoding a non-native substrate-inducible light-generating protein operably connected to a second promoter.
  • the disclosure provides a population of recombinant cells, wherein the population comprises two sub-populations of recombinant cells, the first sub-population comprising a first polynucleotide encoding a non-native light-transducing protein operably connected to a first promoter, and the second sub-population comprising a second polynucleotide encoding a non-native substrate-inducible light-generating protein operably connected to a second promoter.
  • the disclosure provides an isolated fusion protein comprising a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence comprises a functional light-generating protein and the second amino acid sequence comprises a functional light-transducing protein, .
  • the disclosure provides an isolated fusion protein comprising a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence is selected from a functional light-generating protein and a functional light-transducing protein, and the second amino acid sequence comprises a dimerizing domain.
  • the disclosure provides a method for identifying a neural pathway involved in a disease state comprising providing a control cell comprising a neuron; providing a test cell comprising a recombinant cell as described herein; providing a type of stimulus to the control cell; providing at type of stimulus to the test cell, wherein the stimulus is the same as provided to the control cell; and measuring a response from both the control cell and the test cell, wherein a difference in the measured responses between the control cell and the test cell indicate that the neural pathway is involved in a disease state.
  • the disclosure provides a method for identifying a neural pathway involved in a disease state comprising providing a non-human transgenic test animal according to the description herein; providing a non-human control animal; providing a type of stimulus to the control animal; providing at type of stimulus to the test animal, wherein the stimulus is the same as provided to the control animal; and measuring a response from both the control animal and the test animal; wherein a difference in the measured responses between the control animal and the test animal indicates that the neural pathway is involved in a disease state.
  • the disclosure provides a method of potentiating insulin release in a pancreatic beta cell comprising introducing to the pancreatic beta cell a polynucleotide comprising a first sequence that encodes a light-transducing protein, a second sequence that encodes a light-generating protein, and a promoter sequence operably connected to the first and second sequences; exposing the cell to conditions that allow for the expression of the polynucleotide; and contacting the cell with an amount of substrate capable of inducing emission of a photon by the light-generating protein, wherein the amount of substrate induces the emission of photons in an amount effective to potentiate insulin release.
  • the disclosure provides an assay for identifying an agent capable of modulating cell signaling activity comprising providing a cell according to the description herein; contacting the cell with an amount of a substrate that is effective to induce emission of a photon by the light-generating protein; measuring the cell signaling activity in response to the substrate; contacting the cell with an amount of a candidate agent; and measuring the cell signaling activity in response to the candidate agent, wherein a measured change in the cell signaling activity in response to the contacting with the candidate agent relative to the contacting with the substrate indicates that the candidate agent is capable of modulating cell signaling activity.
  • the disclosure provides assays that include the polynucleotide, protein, cells, and/or methods of the above-described aspects.
  • Figure IA is a schematic of a mouse brain highlighting different nuclei and pathways.
  • PFC prefrontal cortex
  • NAc nucleus accumbens
  • AMG amygdala
  • VTA ventral tegmental area.
  • Stippled lines indicate neuronal pathways; various shades colors indicate expression of particular genes (black, red, green, red + green).
  • Figure IB is a schematic of a transgenic mouse line expressing ChR2 from the "green” (NAc) promoter and luciferase (GLuc) from the
  • Figure 2 is a schematic of the pcDNA3.1/hChR2(H134R)-EYFP construct (SEQ ID NO: 1
  • FIG 3A is a schematic of the individual components in their natural state, specifically native Channelrhodopsin (ChR2) is a transmembrane protein with its N terminus
  • native fluorescence protein (here the modification YFP) is expressed in granules; native Gaussia
  • Luciferase is a secreted protein.
  • Figure 3B is a schematic of the engineered fusion protein with GLuc connected to the N-terminal end of ChR2, and YFP fused to the C-terminal end of ChR2, resulting in GLuc-ChR2-YFP.
  • FIG. 4A is a schematic of native ChR2 transmembrane protein with its N terminus
  • FIG. 4B is a schematic of modified versions of CHR2 and GLuc.
  • GLuc is anchored in the membrane by adding a transmembrane region (TM).
  • TM transmembrane region
  • CH2-CH3- ⁇ l hinge region of the IgG heavy chain is added to co-localize both proteins through dimerization (CH2-CH3 hinge).
  • both proteins are fused to a fluorescent reporter (Venus, Cherry) with wavelenths which do not interfere with the wavelength emitted by GLuc and responded to by ChR2.
  • Figure 5 is a schematic of the pcDNA3.1/GLuc-hChR2(H134R)-EYFP fusion construct (SEQ ID NO: 1).
  • Figure 6 are images of cells showing similar transfection efficiencies of PC 12 cells with ChR2 and GLuc-ChR2.
  • Figure 6A is a transmittant image of cells transfected with 0.2 ⁇ g of pcDNA3.1/hChR2(Hl 34R)-EYFP (SEQ ID NO: 2).
  • Figure 6B is a YFP fluorescence image of cells transfected with 0.2 ⁇ g of pcDNA3.1/hChR2(H134R)-EYFP.
  • Figure 6C is a transmittant image of cells transfected with 0.2 ⁇ g of pcDNA3.1/GLuc-hChR2(H134R)-EYFP
  • Figure 6D is a YFP fluorescence image of cells transfected with 0.2 ⁇ g of pcDNA3. l/GLuc-hChR2(H134R)-EYFP.
  • Figure 7A is wide-field fluorescent image showing YFP-tagged ChR2 expression in a transfected PC 12 cell.
  • Figure 7B is a transmittant light image of the same field as in Figure
  • Figure 7A wherein the cell was whole-cell voltage-clamped by a glass pipette (left).
  • Figure 7C is a schematic of a family of photocurrents activated by various intensity of light in a PC 12 cell expressing ChR2.
  • Figure 7D is a graph of the relationship between photocurrent amplitude and light intensity obtained from the same cell depicted in Figures 7A-C.
  • Figure 7E is wide- field fluorescent image showing GLuc-ChR2 expression in a transfected PC 12 cell.
  • Figure 7F is a transmittant light image of the same field as in Figure 7E, wherein the cell was whole-cell voltage-clamped by a glass pipette (left).
  • Figure 7G is a schematic of a family of photocurrents activated by various intensity of light in a PC 12 cell expressing GLuc-ChR2.
  • Figure 7H is a graph of the relationship between photocurrent amplitude and light intensity obtained from the same cell depicted in Figures 7E-G.
  • Figure 8 are graphs of relative luminescence for PC 12 cells transfected with the fusion construct (GLuc-ChR2), a 1 :1 mixture of native secreted GLuc and ChR2-YFP (GLuc + ChR2), or with ChR2-YFP alone (ChR2), as indicated next to the graphs.
  • Figure 9A is a transmittant light image of a PC 12 cell transfected with GLuc.
  • Figure 9B is a wide-field fluorescent image of the same field showing YFP-tagged ChR2 expression, wherein cell was whole-cell voltage-clamped by a glass pipette (right).
  • Figure 9C is a wide- field fluorescent image of the same field showing YFP-tagged ChR2 expression after administration of the substrate (CTZ).
  • Figure 9D is a schematic of GLuc-generated blue light (upper, blue trace) evoking a strong inward current (lower trace).
  • Figure 10 shows light induced current in PC 12 cells transfected with ChR2 alone (no GLuc); and (B) control demonstrating lack of current in the same cells with application of CTZ substrate only, indicating that the currents seen in GLuc-ChR2 transfected cells upon substrate application are specific to the bio luminescence elicited by CTZ and not to CTZ alone.
  • Figure 1 IA is a graph of the decay of CTZ-induced current (red trace) compared to arc-lamp-induced (green trace) current.
  • Figure HB is a graph of the rise of CTZ-induced current (red trace) compared to arc-lamp-induced (green trace) current.
  • Figure 12A is a transmitted light image of transfected HEK cells.
  • Figure 12B is a wide-field fluorescent image of the same field showing YFP-tagged GLuc-ChR2 expression.
  • Figure 12C is a luminescence image after application of substrate CTZ.
  • Figure 12D is a schematic showing administration of the substrate (CTZ; arrow) leading to a sharp increase in luminescence (upper, blue trace) and GLuc-generated blue light evoking a strong inward current (lower trace).
  • Figure 13 are graphs of luminescence (blue/light gray trace) versus current (red/dark gray trace) in HEK cells transfected with the GLuc-ChR2 fusion construct.
  • Figure 14 is a graph of substrate concentration versus luminescence for HEK cells.
  • Figure 15A is an ICR mouse implanted with HEK cells transiently transfected with the GLuc-ChR2 fusion construct.
  • Figure 15B is an ICR mouse implanted with HEK cells alone.
  • Figure 15C is a graph of the total photon count per second calculated for a constant number of HEK cells exposed to varying concentrations of CTZ.
  • Figure 15D is a graph of photon count per second calculated for a constant number of HEK cells exposed to 50 and 5 micrograms of coelenterazine over time.
  • Figure 16 is a schematic of the pLenti-Synapsin-Gluc-hChR2(Hl 34R)-EYFP-WPRE construct (SEQ ID NO: 4).
  • Figure 17 are graphs of the Gaussia Luciferase emission spectrum and the channelrhodopsin activation spectra.
  • Figure 18 is a schematic of the pcDNA3.1/GLuc-VChRl-EYFP construct (SEQ ID NO: 6).
  • Figure 19A is a transmittant light image of a VChRl -transfected HEK cell.
  • Figure 19B is a wide-field fluorescent image of the same field showing YFP-tagged VChRl expression,w herein the cell was whole-cell voltage-clamped by a glass pipette (visible right in A).
  • Figure 19C is a wide-field fluorescent image of the same field showing YFP-tagged VChRl expression and a sharp increase in luminescence after administration of the substrate (CTZ).
  • Figure 19D is a graph of current and luminescence versus time, showing GLuc- generated blue light (blue trace) evoking a strong inward current (black trace).
  • Figure 2OA is a wide-field fluorescent image of a VChRl -transfected primary neuron showing YFP-tagged VChRl expression.
  • Figure 2OB is a VChRl -transfected primary neuron after administration of the substrate (CTZ).
  • Figure 2OC is a graph of current versus time, showing GLuc-generated blue light (blue trace) evoking a strong inward current when voltage- clamped (black trace).
  • Figure 2OD is a graph of current versus time, showing a difference in electrical potential when current-clamped.
  • Figure 21 A is a graph of luminescence versus time for VChRl -transfected primary neurons after administration of the substrate (CTZ).
  • Figure 2 IB is a graph of the input-output (I-O) curves before and after CTZ application.
  • the disclosure relates to polynucleotides and methods for manipulating and/or monitoring cell activity wherein the polynucleotides and methods include a light-generating protein and a light-transducing protein.
  • the polynucleotides can be incorporated into excitable cells and used to manipulate or monitor cell signaling activity in response to addition of a substrate molecule that is capable of inducing emission of light from the light-generating protein.
  • the polynucleotides encode for fusion proteins comprising a light-generating protein and a light-transducing protein.
  • the method includes identifying active agents that can act as inducers or inhibitors of cell signaling activity.
  • the cells comprising the polynucleotides can be used in various assays to study cell signaling (e.g., in response to contacting with an agent), or the cells can be within a genetically modified non-human organism.
  • compositions and methods for manipulating cell signaling particularly of excitable cells.
  • nucleic acid molecules including fusion genes that encode fusion proteins comprising light-generating proteins and light-transducing proteins can be genetically targeted to certain cell populations.
  • the experiments described herein demonstrate that a light signal from the light-generating protein can be captured by the light-transducing protein, which is used to activate or silencing neuronal cell signaling.
  • the methods may be used with a wide variety of excitable cells, including but not limited to, neurons, cardiomyocytes, muscle fibers, pancreatic beta cells, bone cells, zygotes, and other cell types that respond to ion flux (e.g., sodium, calcium, or chloride flux).
  • ion flux e.g., sodium, calcium, or chloride flux.
  • Calcium signaling is important for a variety of cellular processes, including but not limited to, neuronal signaling, immune system cellular activation, egg activation after fertilization in mammals, control of cell survival, regulation of proliferation, inducing cell death (e.g., through induction of apoptosis) and cytoskeletal organization.
  • Light-emitting protein as used herein relates to a functional luciferase.
  • the luciferase can be derived from any source, such as those from Gaussia, Renilla reniformis, and firefly, and include derivatives that are able to emit a photon of light in response to contact with a substrate molecule.
  • Substrates include, but are not limited to, luciferin, coelenterazine and other modified substrates.
  • the substrate is matched with the light generating protein.
  • firefly luciferase uses luciferin as a substrate while Renilla luciferase uses coelenterazine as a substrate.
  • substrates may be chosen to affect the kinetics, membrane permeability, turn over or signal strength of the emission.
  • Luciferases oxidize luciferin to produce oxyluciferin and light energy. The chemical reaction can occur intracellularly, extracellualrly, or be membrane anchored.
  • Light- generating proteins can also be expressed as part of a fusion protein. Those skilled in the art will appreciate that the light-generating protein may be expressed as a fusion protein with the light transducing protein. The proteins may be expressed and methods may be used in vitro, ex vivo or in vivo.
  • Gaussia Luciferase (GLuc) emits blue light with a wavelength at 477nm.
  • Other known luciferases emit green, red, infrared, or yellow light after contact with the appropriate substrate.
  • a light-generating protein can be matched with the light-transducing protein to result in maximal efficiency of signal generation after addition of the substrate.
  • the kinetics of activation and decay of the light- generating protein must be considered.
  • Some light-generating proteins have flash kinetics which include rapid decay, while others with longer decay times may be advantageous when studying longer term kinetics such as calcium flux in cells.
  • the luciferase is Gaussia luciferase as described by SEQ ID NO: 9, and encoded by SEQ ID NO: 8 or SEQ ID NO: 16.
  • Light-transducing protein as used herein include any protein that can covert light energy (i.e., photons) into an effector function in a single component system (e.g., opsins from microbes) or in a more complex multi component signaling cascade, (e.g., G protein-coupled receptors) and signaling pathways (e.g. human rhodopsin).
  • opsins from microbes
  • signaling pathways e.g. human rhodopsin
  • a non-limiting example of a class of light-transducing protein include the microbial opsins, for example Chlamydomonas channelrhodopsin-2 (ChR2), or Volvox channelrhodopsin-1 (VChRl).
  • the light- transducing protein comprises Chlamydomonas channelrhodopsin-2 (ChR2), or Volvox channelrhodopsin-1 (VChRl).
  • Channelrhodopsins relate to the subfamily of opsin proteins that function as light- gated ion channels. They can function as sensory photoreceptors to controlling phototaxis, intracellular acidity, calcium influx, electrical excitability, and other cellular processes.
  • Channelrhodopsins that are characterized and known include Channelrhodopsin-1 (ChRl), Channelrhodopsin-2 (ChR2), and Volvox Channelrhodopsin (VChRl).
  • the channelrhodopsins are nonspecific cation channels, conducting H + , Na + , K + , and Ca 2+ ions.
  • Channelrhodopsin-2 (ChR2) of the green alga Chlamydomonas is a light-gated ion channel, enabling the use of light to control intracellular acidity, calcium influx, and electrical excitability. It is a seven-transmembrane protein like rhodopsin, and contains the light- isomerizable vitamin A derivative all-trans -retinal. Most vertebrate opsins are G-protein coupled receptors that open other ion channels indirectly via messengers, but channelrhodopsins form the channel pore. This makes cellular depolarization extremely fast, robust, and useful for bioengineering and neuroscience applications, including photostimulation. Peak absorbance of the Channelrhodopsin-2 retinal complex is about 470 nm (blue light).
  • halorhodopsin may be used to inhibit neural activity rather than stimulate the activity.
  • the light-transducing protein may also be chosen based on the kinetics of the protein. Light- transducing proteins that are channels or pumps are likely to have faster kinetics than those that are capable of activating other channels or pumps indirectly. Combinations of various pairs of light-generating and light-transducing proteins may allow precise multi-color activation and silencing of cellular signals, such as neural activity. Light-transducing proteins may be expressed at the cell surface (e.g.
  • the light- transducing proteins may be made as fusion proteins or may dimerize with other proteins such as the light-generating proteins.
  • the polynucleotides disclosed herein can encode light-generating and light- transducing proteins that comprise a naturally-occurring amino acid sequence, as well as a modified amino acid sequence that can alter, for example, the light emission wavelength or the light absorbance wavelength. Further, the polynucleotides can comprise a sequence that is codon-optimized for expression in a particular organism or cell type, while retaining the naturally-occurring sequence, or the modified amino acid sequence. Codon usage and optimization is known in the art.
  • the light-generating and the light-transducing proteins may be expressed in cells or as transgenes in transgenic organisms, such as transgenic mice.
  • the light-generating and light- transducing proteins may be expressed on the same cell or on different cells in close proximity to each other.
  • Those of skill in the art will appreciated that a variety of methods may be utilized to allow expression of the proteins.
  • polynucleotides encoding the proteins may be operably connected to promoters which allow expression of the protein and cells transformed or transfected with the resultant construct.
  • the promoters can be any promoter sequence known in the art and selected to allow for expression on a particular subset of cells, or in a particular organism.
  • the light-transducing and light-generating protein may be expressed from polynucleotides operably connected to the same promoter or distinct promoters. Use of distinct promoters may help define a limited subset of cells or a limited signaling pathway for study. Alternatively, the light-transducing and light-generating protein may be expressed as a fusion protein. An additional level of regulation of expression may be added by encoding a STOP cassette flanked by loxP or fit sites between the promoters and the polynucleotides encoding the light-generating and/or light-transducing proteins. In this system, gene expression would be further limited to cells expressing Cre or FIp recombinase.
  • the cell types capable of expressing the light-generating and light-transducing protein can be very limited or more general.
  • protein expression may be limited by physical location.
  • the light- transducing or light-generating proteins or polynucleotides encoding the proteins may be stereotactically injected into a physical location in an animal.
  • the methods described for localization of the proteins may be used in any combination or in combination with other methods available to those skilled in the art.
  • the light-generating protein is constitutively "off” but is switched “on” in the presence of substrate.
  • photon emissions are stimulation dependent, i.e., they only occur when substrate is given.
  • these methods use proteins for both - light signal production as well as light signal transduction - and proteins can be genetically targeted to specific cellular (e.g., neuronal) populations, the interrogation of the circuits can be targeted to the intersection of different cellular subsets defined by the intersection of the expression of two different genes, thus making it highly cell-type specific.
  • the methods allow cell-type-specific and temporally precise control of cellular function within intact cells in living animals.
  • the method allows particular neuronal signal pathways to be isolated and studied in live animals.
  • cells expressing the light-generating proteins are then contacted with the substrate of the light-generating protein to induce production of light and subsequent activation of the light-transducing protein.
  • the substrate may be added by any means, including any means available to those skilled in the art.
  • the substrate may be added to cell culture or may be administered to a mouse or other transgenic animal expressing the light-generating protein.
  • the substrate may be administered acutely, continuously, intermittently or repeatedly.
  • the substrate may be administered by any means available, including but not limited to, intraperitoneal, intramuscular, intravenous, subcutaneous, or intracerebral.
  • the dose of the substrate and length of administration may also be altered to provide information regarding sensitivity of the signaling pathway, single action potential assays or excitotoxic insult and possible degeneration.
  • the signal generated by administration of the substrate to the cells or animals may be monitored in a variety of ways. Signaling may be measured directly or indirectly. For example, cell signaling in response to addition of the substrate and generation of light may be measured through production of an action potential in the cells expressing the light transducing protein, i.e. monitoring may be electrophysiological. Alternatively, the change in signaling may be monitored indirectly by assessing specific behaviors of the animal induced by signaling of the light-transducing protein.
  • the methods described herein may be used to study normal animals.
  • the methods may be used to help break down and understand complex neural circuits in the brain such as those regulating behavior.
  • the methods may also be used to study or obtain a better understanding of disease states, including but not limited to, depression, schizophrenia, stroke, manic-depressive disorder, addiction, and Parkinson's disease.
  • FIG. IA distinct neural pathways connect cells in disparate areas of the mouse brain and these cells express distinct genes.
  • the methods described herein, and illustrated in Figure IB allows for a specific pathway to be activated by generating a transgenic animal in which expression of a light-transducing protein is controlled by one promoter and expression of a light-generating protein is controlled by a second promoter such that when substrate is provided only the pathway in which both the light-generating and light- transducing proteins are expressed will be activated.
  • non-human transgenic animals and cells including a first promoter operably connected to a polynucleotide encoding a non-native light transducing protein and a second promoter operably connected to a polynucleotide encoding a non-native substrate-inducible light-generating protein are provided.
  • the animals and cells described herein are useful in the methods as described above.
  • the disclosure provides an isolated polynucleotide comprising a sequence that encodes a light-generating protein, a sequence that encodes a light-transducing protein, and a promoter sequence operably connected to the sequences that encode the light- transducing protein and the light-generating protein.
  • the light-transducing protein comprises an opsin.
  • the opsin comprises channelrhodopsin-2, channelrhodopsin-1, Volvox channelrhodopsin-1, or halorhodopsin.
  • the light-generating protein comprises a luciferase.
  • the lucif erase comprises Gaussia luciferase.
  • the isolated polynucleotide is a fusion gene and encoding a fusion protein.
  • the fusion gene encodes a fusion protein that comprises a light-generating protein attached to the N-terminus of the light-transducing protein.
  • the polynucleotide encodes for a light-generating protein having an emission wavelength and a light-transducing protein having an absorbance wavelength, wherein at least a portion of the emission and absorbance wavelengths overlap. In further embodiments, the peak emission wavelength and the peak absorbance wavelength overlap.
  • the polynucleotide can include additional sequences such as promoters, enhancers, or regions that encode for amino acid sequences including dimerization domains, transmembrane regions, fluorescent proteins, and the like.
  • the isolated polynucleotide comprises SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 17, or SEQ ID NO: 19.
  • the disclosure provides an isolated polypeptide or fusion protein comprising a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence comprises a functional light-generating protein, and the second amino acid sequence comprises a functional light-transducing protein.
  • the fusion protein is encoded by a polynucleotide as described herein.
  • light- transducing protein comprises an opsin, or a functional fragment thereof.
  • the opsin comprises channelrhodopsin-2, channelrhodopsin-1, Volvox channelrhodopsin-1, or halorhodopsin, or a functional fragment thereof.
  • the light-generating protein comprises a luciferase.
  • the luciferase comprises Gaussia luciferase.
  • the fusion protein comprises a functional light-transducing protein selected from channelrhodopsin-2, channelrhodopsin-1, Volvox channelrhodopsin-1, and halorhodopsin, or a functional fragment thereof, and the functional light-generating protein comprises Gaussia luciferase, or a functional fragment thereof.
  • the fusion protein comprises a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence comprises a functional light-transducing protein, and the second amino acid sequence comprises a dimerizing domain.
  • the first amino acid sequence comprises a functional light-generating protein
  • the second amino acid sequence comprises a dimerizing domain.
  • the fusion protein can further include a third amino acid sequence that comprises a transmembrane domain.
  • the functional light-transducing protein can comprise an opsin selected from channelrhodopsin-2, channelrhodopsin-1, Volvox channelrhodopsin-1, and halorhodopsin, or a functional fragment thereof.
  • any known type of domain that can dimerize can be used.
  • the dimerizing domain comprises the hinge- CH2-CH3 ( ⁇ l) heavy chain region of human IgGl.
  • the dimerizing domain can include specific binding pair, such as biotin-streptavidin, with one member of the binding pair being associated with the light-transducing protein and the other being associate with the light- generating protein. Typically the association will be in the form of fusions or post- translational modification.
  • the fusion protein can further comprise a transmembrane domain.
  • transmembrane domain e.g., amyloid precursor protein, B lymphocyte activation antigen B7, and the like
  • dimerizing e.g., hinge-C2-C3 of IgGl, specific binding pairs, and the like domains are well known in the art and can be used to target the fusion protein to the cell membrane and help to associate the light-generating and light-transducing proteins so that transfer of light energy can occur.
  • the polypeptide or fusion protein can include an amino acid linker sequence.
  • the linker sequence can be of various lengths (e.g., between 1 and about 50 amino acids in length) and can be located between the various distinct regions of the polypeptide of fusion protein, such as between the light-generating and light-transducing proteins, or other regions (e.g., transmembrane domain, dimerizing domain, fluorescent protein or other amino acid tags).
  • polypeptide comprises SEQ ID NO: 18, or SEQ ID NO:
  • polypeptide is encoded by a polynucleotide sequence comprising SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 17, or SEQ ID NO:
  • Some embodiments relate to methods, polynucleotides, polypeptides, cells, and assays that include functionally-active fragments of the light-generating and/or the light- transducing protein. These embodiments provide an amino acid sequence that comprises less than the full length amino acid sequence of either the light-generating or the light-transducing protein, or both, as described herein. Such a fragment can result from a truncation at the amino terminus, a truncation at the carboxy terminus, and/or an internal deletion of one or more amino acid residues from the amino acid sequence(s).
  • Naturally occurring fragments may result from alternative RNA splicing, from in vivo processing such as removal of the leader peptide and propeptide, and/or from protease activity.
  • the fragments can be tested for activity by identifying function (e.g., light emission, signaling activity, or both).
  • Embodiments provide an amino acid sequence comprising a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar to the amino acid sequence of the fusion protein, or the individual light-generating and light-transducing proteins, and which retains functional activity.
  • embodiments provide an amino acid sequence comprising a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the various fusion proteins described herein, and which retains functional activity for cell signaling.
  • the polynucleotide comprises a sequence that is at least 70 percent identical to the nucleotide sequence encoding the native light-generating protein or native light-transducing protein, or both, or comprises a nucleotide sequence encoding polypeptides that are at least 70 percent identical to the native light-generating protein or native light- transducing protein, or both.
  • the nucleotide sequences can be at least 75 percent, or about 80 percent, or about 85 percent, or about 90 percent, or about 95 percent identical to any of the nucleotide sequence encoding the native light-generating protein or native light-transducing protein, or both, or the nucleotide sequences that encode polypeptides that are about 75 percent, or about 80 percent, or about 85 percent, or about 90 percent, or about 95 percent identical to the native light-generating protein or native light-transducing protein, or both.
  • Nucleic acid molecules also include fragments of the above nucleic acid molecules which are at least about 10 contiguous nucleotides, or about 15, or about 20, or about 25, or about 50, or about 75, or about 100, or greater than about 100 contiguous nucleotides.
  • Related nucleic acid molecules also include fragments of the above native light-generating and/or native light- transducing polynucleotide molecules which encode an amino acid sequence of a light- generating protein and/or a light-transducing protein of at least about 25 amino acid residues, or about 50, or about 75, or about 100, or greater than about 100 amino acid residues of bioremediase protein.
  • the isolated nucleic acid molecules include those molecules which comprise nucleotide sequences which hybridize under moderate or highly stringent conditions as defined below with any of the above nucleic acid molecules.
  • the nucleic acid molecules comprise sequences which hybridize under moderate or highly stringent conditions with a nucleic acid molecule encoding a polypeptide, which polypeptide comprises a sequence as shown in any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 19, or with a nucleic acid fragment as defined above, or with a nucleic acid fragment encoding a polypeptide as defined above.
  • related nucleic acid molecules include sequences which are complementary to any of the above nucleotide sequences.
  • high stringency conditions refers to those conditions that (1) employ low ionic strength reagents and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% NaDodSO4 (SDS) at 5O 0 C, or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1%.
  • a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1%.
  • Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 may be used with 750 mm NaCl, 75 mm sodium citrate at 42 0 C.
  • Another example is the use of 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sulfate at 42 0 C, with washes at 42 0 C in 0.2x SSC and 0.1% SDS.
  • moderate stringency conditions refers to conditions which generally include the use of a washing solution and hybridization conditions (e.g., temperature, ionic strength, and percent SDS) less stringent than described above.
  • a non-limiting example of moderately stringent conditions includes overnight incubation at 37 0 C in a solution comprising: 20% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ l/ml denatured sheared salmon sperm DNA, followed by washing the filters in Ix SSC at about 37-5O 0 C.
  • Those skilled in the art will recognize how to adjust the temperature, ionic strength and other parameters as necessary in order to accommodate factors such as nucleic acid length and the like.
  • Embodiments provide nucleic acid constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide encoding a light-transducing and/or a light-generating protein or functional fragments thereof, and a suitable promoter region.
  • Suitable vectors can be chosen or constructed, which contain appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as desired.
  • Vectors can be plasmids, phage (e.g. phage, or phagemid) or viral (e.g. lentivirus, adenovirus, AAV) or any other appropriate vector.
  • phage e.g. phage, or phagemid
  • viral e.g. lentivirus, adenovirus, AAV
  • identity refers to a relationship between the sequences of two or more amino acid sequences or two or more nucleic acid molecules, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between amino acid or nucleic acid molecule sequences, as the case may be, as determined by the match between strings of nucleotide or amino acid sequences.
  • Identity measures the percent of identical matches between two or more sequences with gap alignments addressed by a particular mathematical model or computer programs (i.e., "algorithms").
  • similarity is a related concept, but in contrast to "identity”, refers to a measure of similarity which includes both identical matches and conservative substitution matches. Since conservative substitutions apply to polypeptides and not nucleic acid molecules, similarity only deals with polypeptide sequence comparisons. If two polypeptide sequences have, for example, 10/20 identical amino acids, and the remainder are all non- conservative substitutions, then the percent identity and similarity would both be 50%. If in the same example, there are 5 more positions where there are conservative substitutions, then the percent identity remains 50%, but the percent similarity would be 75% (15/20). Therefore, in cases where there are conservative substitutions, the degree of similarity between two polypeptide sequences will be higher than the percent identity between those two sequences.
  • Non-limiting methods for determining identity and/or similarity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux, et al, Nucleic Acids Research 12:387 [1984]; Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215:403-410 [1990]).
  • the BLAST X program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul] et al., NCB NLM NIH Bethesda, Md. 20894; Altschul et al., J. MoI. Biol. 215:403-410 [1990]).
  • NCBI National Center for Biotechnology Information
  • the well known Smith Waterman algorithm may also be used to determine identity.
  • GAP Genetics Computer Group, University of Wisconsin, Madison, Wis.
  • two polypeptides for which the percent sequence identity is to be determined are aligned for optimal matching of their respective amino acids (the "matched span", as determined by the algorithm).
  • a gap opening penalty (which is calculated as 3. times, the average diagonal; the "average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal " is the score or number assigned to each perfect amino acid match by the particular comparison matrix)
  • a gap extension penalty which is usually 1/10 times the gap opening penalty
  • a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm.
  • a standard comparison matrix (see Dayhoff et al., in: Atlas of Protein Sequence and Structure, vol. 5, supp. 3 [1978] for the PAM250 comparison matrix; see Henikoff et al., Proc. Natl. Acad. Sci USA, 89:10915-10919 [1992] for the BLOSUM 62 comparison matrix) is also used by the algorithm.
  • the GAP program is useful with the above parameters.
  • the aforementioned parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps) using the GAP algorithm.
  • Preferred parameters for nucleic acid molecule sequence comparison include the following:
  • the GAP program is also useful with the above parameters.
  • the aforementioned parameters are the default parameters for nucleic acid molecule comparisons.
  • the disclosure provides a vector comprising the isolated polynucleotide as described herein.
  • the vector can be any type of vector that finds use as a vehicle to transfer foreign genetic material into a cell.
  • Non-limiting examples of vectors include plasmids, viral vectors (e.g., derived from lentivirus, adenovirus, adeno-associated virus (AAV), retrovirus, etc.), bacteriophage, cosmids, and artificial chromosomes.
  • the vector can be an expression (or expression constructs) for driving expression of the polynucleotide in a target cell. Vectors and methods for inserting them into a target cell are known in the art [See, e.g., Sambrook et al., 1989].
  • the disclosure provides recombinant cells that comprise the polynucleotides described herein.
  • the cells include any variety of excitable cells, including but not limited to, neurons, cardiomyocytes, muscle fibers, pancreatic beta cells, bone cells, zygotes, and other cell types that respond to ion flux (e.g., sodium or calcium flux).
  • the cell comprises polynucleotide comprising a sequence that encodes a light- generating protein, a sequence that encodes a light-transducing protein, and a promoter sequence operably connected to the sequences that encode the light-transducing protein and the light-generating protein.
  • the cell comprises a polynucleotide encoding a non-native light-transducing protein operably connected to a first promoter; and a polynucleotide encoding a non-native substrate-inducible light-generating protein operably connected to a second promoter.
  • the cell includes a population of cells comprising a polynucleotide encoding a non-native light transducing protein operably connected to a first promoter; and a polynucleotide encoding a non-native substrate-inducible light-generating protein operably connected to a second promoter.
  • the disclosure provides a genetically engineered non-human organism comprising the isolated polynucleotides as described herein.
  • Genetically engineered non-human organisms can be generated by any technique known in the art, such as by electroporation of naked DNA into the animal, infection and incorporation of DNA using any viral vector (e.g., lentivirus, retrovirus, adenovirus), DNA introduced into embryonic stem cells which are then transplanted into tissues of the animal, and germline introduction of DNA by microinjection into zygotes or by microinjection of embryonic stem cells into embryos.
  • the genetically engineered non-human organism is used in the various methods described herein.
  • the disclosure provides a method of modulating cell signaling comprising, providing a cell comprising a polynucleotide as described herein; and contacting the cell with a substrate capable of inducing emission of a photon by the light generating protein; wherein the emission of the photon by the light generating protein is capable of inducing a response in the light-transducing protein that is effective to modulate cell signaling.
  • the disclosure provides a method of monitoring cell signaling comprising, providing a cell comprising a polynucleotide as described herein; contacting the cell with a substrate capable of inducing emission of a photon by the light generating protein, wherein the emission of the photon by the light generating protein is capable of inducing a response in the light-transducing protein that is effective to induce or inhibit cell signaling; and wherein the emission of the photon by the light-generating protein provides a detectable signal for monitoring spatial light emission in a cell, a cell population, an organ, or an animal.
  • the emission of a photon of light by the light-generating protein can be detected using any of the methods described or specifically exemplified herein, or that are otherwise known in the art.
  • the disclosure provides a method for identifying a neural pathway involved in a disease state comprising: providing a control cell comprising a neuron; providing a test cell comprising a recombinant cell as described herein; providing a type of stimulus to the control cell; providing at type of stimulus to the test cell, wherein the stimulus is the same as provided to the control cell; and measuring a response from both the control cell and the test cell; wherein a difference in the measured responses between the control cell and the test cell indicate that the neural pathway is involved in a disease state.
  • the disease state is a disorder of the brain that does not manifest itself by a specific phenotype or molecular signature.
  • the disease state is a psychiatric disorder or an addiction disorder, such the as non-limiting examples of schizophrenia, autism, depression, relapse behavior, and anxiety-related disorders.
  • observation of cell signaling can be performed by any method or using any apparatus known in the art.
  • whole animal imaging in vivo imaging
  • a detectable light-generating protein such as luciferase
  • non-invasive visualization of the signal can be made in the live animal using, for example, a sensitive CCD camera.
  • This technique has been employed in animal models for applications in tumorigenesis, stem cell migration, and gene expression.
  • the disclosure provides a method of potentiating insulin release in a pancreatic beta cell comprising introducing to the pancreatic beta cell a polynucleotide comprising a first sequence that encodes a light-transducing protein, a second sequence that encodes a light-generating protein, and an optional a promoter sequence operably connected to the first and second sequences; exposing the cell to conditions that allow for the expression of the polynucleotide; and contacting the cell with an amount of substrate capable of inducing emission of a photon by the light-generating protein; wherein the amount of substrate induces the emission of photons in an amount effective to activate the light-transducing protein and thereby potentiate insulin release.
  • the method is used to treat dysfunction of the pancreatic beta cells.
  • the dysfunction is related to improper insulin production and/or release in association with diabetes mellitus type 1.
  • the disclosure provides an assay for identifying an agent capable of modulating cell signaling activity comprising: providing a recombinant cell as described herein; contacting the cell with an amount of a substrate that is effective to induce emission of a photon by the light-generating protein; measuring the cell signaling activity in response to the substrate; contacting the cell with an amount of a candidate agent; and measuring the cell signaling activity in response to the candidate agent; wherein a measured change in the cell signaling activity in response to the contacting with the candidate agent relative to the contacting with the substrate indicates that the candidate agent has is capable of modulating cell signaling activity.
  • the measured chance in the cell signaling activity is from at least about 10% to 100% or more.
  • aspects that identify an agent capable of modulating cell signaling activity include embodiments relating to pharmaceutical compositions including the agents identified by the assays and methods disclosed herein, in combination with a pharmaceutically acceptable formulation agent.
  • the compounds can be provided as pharmaceutically acceptable salts such as, for example, basic or acidic addition salts. See, e.g., Remington: The Science and Practice of Pharmacy, 21 st ed., Lippincott Williams & Wilkins, A Wolters Kluwer Company, Philadelphia, Pa (2005).
  • aspects provide methods for modulating cell signaling in a subject that include administering to a subject in need of treatment at least one compound identified by the methods described herein.
  • the method treats or prevents a disease state associated with improper pancreatic function, such as diabetes.
  • the method treats or prevents a disease state associated with a neuropsychiatric disorder, such as schizophrenia, autism, depression, anxiety-related disorders, addiction, and relapse behavior.
  • aspects provide medicaments that include a compound identified by the methods described herein, for use in the treatment of a condition associated with improper cell signaling activity.
  • the condition includes improper pancreatic function, such as diabetes.
  • the condition is a disease state associated with a neuropsychiatric disorder, such as schizophrenia, autism, depression, anxiety-related disorders, addition, and relapse behavior.
  • the plasmid pcDNA3.1/hChR2(H134R)-EYFP (SEQ ID NO: 2) was kindly provided by Dr. Karl Deisseroth (Stanford University, Stanford, CA).
  • the CMV promoter drove a mammalian codon-optimized channelrhodopsin 2 gene with the histidine-134 to arginine mutation, which increased peak and steady state photo currents relative to wild-type ChR2.
  • Yellow fluorescent protein was fused to the carboxy terminus of ChR2 (see Figure 2).
  • ChR2 is a directly light-switched cation-selective ion channel. Per photocycle the channel opened rapidly after absorption of one photon to generate a large permeability for monovalent and divalent cations. Ch R2 desensitized in continuous light; peak current restored after desensitization in a process with a time constant of around 7 sees. Calculations for a single channel current for one ChR2 molecule ran at 5 fA for a single-channel event.
  • ChR2 channels have to open in a given cell. With physical light sources, this was mainly achieved by adjusting the light intensity. For wide-field illumination (as demonstrated in Figure 7), the peak amplitude of photocurrents increased as a function of light intensity. As shown in Figure 7 illumination evoked photocurrents in a ChR2 -positive PC 12 cell. A family of photocurrents activated by various intensity of light as shown in Figure 7C, wherein the peak amplitude of photocurrents (lower trace) increased as a function of light intensity (upper trace). An incident light from a mercury lamp was attenuated by ND filters and then filtered by 480/30 nm band-pass filter. The cell was held at -60 mV.
  • Shown in Figure 7D is the relationship between photocurrent amplitude and light intensity obtained from the same cell. Fitting of the Hill equation yielded the maximum current of -180 pA, the Hill coefficient of 0.97, and the half-maximum light intensity of 230 uW/mm 2 .
  • Focused laser illumination as used in fiberoptic stimulation of ChR2 in the brain of moving animals, evoked ChR2 current which increased sublinearly with increasing laser intensity: due to the Gaussian intensity profile of a focused laser beam and scattering of excitation light, out-of-focus membrane became increasingly illuminated as the focal intensity was increased, increasing the total photocurrent. However, this effect was counteracted by saturation of the ChR2 photocycle in the center of the beam. Moreover, the area of a cell "hit" by the laser light represented only a small fraction of the total membrane of the cell, since most surface area was contained in the extensive dendritic and axonal arborization.
  • GauSiSia Luciferase (Gl uc) is a photon-generating enzyme. The specific activity of this luciferase in the presence of 10 ⁇ M concentration of its substrate coelenterazine was extremely high: 1.24 x 10 16 Qps/mg (Quanta per second per milligram). This translated into one photon produced every 2 seconds by each luciferase.
  • the luciferase will be designed to be physically near the rhodopsin channel on the cell surface.
  • High levels of GLuc can be targeted to the mammalian cell surface by proper selection of a transmembrane (TM) domain.
  • TM transmembrane
  • proteins can be expressed on the cell surface as dimers. This can be accomplished by inserting dimerization domains between the respective proteins and their transmembrane regions. Again, different domains can be selected.
  • IgGl heavy chain [Venus]
  • FIG. 4 Native and engineered Channelrhodopsin (ChR2) and Gaussia Luciferase (GLuc) are shown in Figure 4.
  • native ChR2 is a transmembrane protein with its N terminus (N) at the cell surface (out), and its C terminus (C) towards the inside of the cell (in).
  • Native GLuc is a secreted protein.
  • Figure 4B shows modified versions of both molecules. GLuc is anchored in the membrane by adding a transmembrane region (TM). To both GLuc and ChR2 the CH2-CH3- ⁇ l hinge region of the IgG heavy chain is added to co-localize both proteins through dimerization (CH2-CH3 hinge). Finally, both proteins are fused to a fluorescent reporter (Venus, Cherry) with wavelengths which do not interfere with the wavelength emitted by GLuc and responded to by ChR2.
  • TM transmembrane region
  • the codon-optimized GLuc (pCMV-GLuc) was purchased from Nanolight, Inc.
  • the codon-optimized CHR2 (see above) was generously provided by Dr. Karl Deisseroth.
  • the TM of B7 and the hinge-CH2-CH3 ( ⁇ l) region were generated as PCR fragments.
  • the fluorescent proteins were originally provided by Dr. Roger Tsien (mCherry) and by the NCRR Yeast Resource Center, University of Washington (Venus).
  • PC 12 cells will be stably infected with ChR2 first; cells expressing ChR2-Venus at high levels will be identified by fluorescence-activated cell sorting (FACS). Localization of ChR2 in the cell membrane will be confirmed by fluorescence and confocal microscopy. These PC12-ChR2 cells will then be stably trans fected with GLuc-mCherry. Again, clones expressing high levels of GLuc-mCherry will be isolated by FACS, and cell surface expression will be verified by microscopy.
  • FACS fluorescence-activated cell sorting
  • PC12-ChR2-GLuc cells (and wild-type PC 12 cells, as well as PC12-ChR2 cells and PC12-GLuc cells as controls) will be analyzed extensively electrophysio logically to determine basic and kinetic properties of luciferase-induced current responses. Specifically, the amplitude of currents elicited, latencies of currents, correlation with substrate concentration, as well as light-evoked postsynaptic responses will be assessed. These experiments will determine if the above design of the constructs positions ChR2 and GLuc in the cell membrane to permit efficient photon activation of ChR2 by GLuc resulting in depolarization.
  • Example 3 Determination of the range of protein expression levels compatible with efficient current generation.
  • PC 12 cells will be stably transfected with CMV-ChR2- Venus and CMV-GLuc-mCherry constructs. Through FACS analysis, clones will be isolated that express ChR2/GLuc at high, medium, and low levels. These three lines (and wild-type PC 12 cells, as well as PC12-ChR2 cells and PC12-GLuc cells as controls) will be analyzed in detail for biophysical parameters (as above).
  • Example 4 Production of transgenic mouse lines which express the light-generating and the light-transducing proteins in a subset of neurons involved in the central reward circuit.
  • mice After having gained information on critical basic parameters through the experiments described above, the information gained will be applied to generate transgenic mouse lines expressing ChR2 and GLuc in a genetically defined striatal pathway of the central reward circuit. These mice will be used to interrogate the role of a specific set of neurons in relapse behavior.
  • the 'switch' from voluntary drug use to habitual and progressively compulsive drug use may represent a transition at the neural level from prefrontal cortical to striatal control over drug-seeking and drug-taking behaviors, as well as a progression from ventral to more dorsal domains of the striatum, mediated at least in part by its stratified dopaminergic innervation.
  • Adaptive changes in the striatal dopaminergic system may be central to alterations in the experience-dependent plasticity that underlies drug-induced behavior.
  • mice In order to selectively activate the striatonigral pathway, transgenic lines will be generated in which the light-generating and light-transducing components of the system are expressed from promoters which specifically intersect in neurons of the direct pathway. Specifically, mice in which expression of ChR2 is regulated by dopamine receptor 1 (Drdl), and expression of GLuc is regulated by Dynorphin (Pdyn) will be generated.
  • Drdl dopamine receptor 1
  • Dynorphin Dynorphin
  • the projection pattern of MSNs from dorsal striatum in Drdl -EGFP or Drd2-EGFP BAC transgenic mice has been characterized through analysis of GFP expression (Gensat).
  • Single-cell RT-PCR profiling confirmed that the BAC Dl-EGFP striatonigral neurons had detectable levels of Pdyn and Dl receptor mRNAs, but not Penk or D2 receptor mRNA; conversely, labeled striatal neurons from BAC D2-EGFP mice had detectable levels of Penk and D2 receptor mRNA, but not Pdyn or Dl receptor mRNAs.
  • Acute midbrain slices from double transgenic, single transgenic and wild-type mice will be used for patch clamp recordings. Resting properties, currents, and action potentials will be measured without substrate and with different concentrations of substrate in the different genotypes. Current elicited by substrate will be compared to currents elicited by a physical light source. These experiments will validate that substrate-induced currents can be elicited and cause depolarization sufficient to generate action potentials and evoke synaptic responses. And they will validate that cells expressing ChR2 and/or GLuc are indistinguishable from their wild-type counterparts in their electrophysiological properties.
  • the transgenic lines generated in experimental paradigms of relapse behavior will be employed.
  • Relapse to drug-seeking and drug-taking behaviors following prolonged periods of abstinence constitutes one of the most significant problems for the long-term treatment of drug-dependent individuals.
  • a number of factors are known to contribute to craving and relapse, including exposure to environmental stimuli previously paired with drug use (that is, conditioned drug cues), negative mood states or stress.
  • These trigger factors have been used in animal models of relapse to drug-seeking behavior, particularly in the extinction-reinstatement model following withdrawal from chronic drug self-administration.
  • animals are trained to self-administer a drug (for example, ***e) for prolonged periods of time.
  • mice from the double and single transgenic lines as well as wild-type littermates will undergo self-administration training followed by extinction training. It will then be asked if activation of the striatonigral pathway induces or prevents relapse behavior, or is without effect.
  • substrate will be administered, i.e. to activate the striatonigral pathway: a) instead of a trigger, and b) together with a trigger. If activation of this pathway re -instates drug-seeking behavior, a similar effect will be seen with either the substrate or a trigger. If activation of this pathway prevents re-instating drug- seeking behavior, an increase in lever pressing will be seen only when the trigger is supplied, but not when substrate and trigger are applied together. Alternatively, there could be no effect of activating the pathway, in which case we will test different pathways.
  • Example 5 Gaussia Luciferase - Channelrhodopsin2 (GLuc-ChR2) fusion protein.
  • a fusion gene of the light generating Gaussia luciferase (GLuc) and the light responsive Channelrhodopsin-2 (ChR2) were generated such that in the protein encoded by this fusion, the light produced by the GLuc activated ChR2.
  • ChR2 is a transmembrane channel protein and GLuc is a naturally secreted protein.
  • the GLuc was positioned upstream of the ChR2; specifically, a codon-optimized GLuc gene was inserted into the pcDNA3.1/hChR2(H134R)-EYFP (Dr.
  • pcDNA3.1/GLuc-hChR2(H134R)-EYFP SEQ ID NO: 1; see Figures 3 and 5.
  • the CMV promoter in pcDNA3.1/hChR2(H134R)-EYFP drives a mammalian codon-optimized channelrhodopsin 2 gene with the histidine-134 to arginine mutation, which increases peak and steady state photo currents relative to wildtype ChR2.
  • a yellow fluorescent protein gene was fused to the carboxy terminus of ChR2 gene.
  • PC12 cells (ATCC; CRL-1721) were cultured in DMEM. For transient transfections, 3-6 x 10 4 cells per well were seeded onto poly-D-lysine (Sigma P6407) coated glass coverslips in 24-well plates. Cells were transfected with 0.2 ⁇ g/well plasmid pcDNA3.1/GLuc- hChR2(H134R)-EYFP DNA (SEQ ID NO: 1) using Effectene Transfection Reagent (Qiagen). Cells were analyzed for luminescence or in electrophysiology experiments 36 hours after transfection. For electrophysiology experiments involving ChR2, all-trans-retinal (1 ⁇ M final concentration) was added to the cultures several hours before the experiments.
  • transfected PC 12 cells grown on coverslips were examined on an upright epifluorescence microscope (Eclipse E600-FN; Nikon) equipped with a mercury arc lamp.
  • the YFP fused to ChR2 was excited (465-495 nm) and emitted fluorescence (515-555 nm) was detected with a cooled CCD camera (CoolSNAP-fx; Photometries, Arlington, AZ).
  • ChR2 was excited with bandpass filtered light pulses (430-450 nm) from the same arc lamp, and pulse duration was controlled by an electronic shutter (Uniblitz VS25S; Vincent, Rochester, NY).
  • the extracellular solution consisted of 150 mM NaCl, 3 mM KCl, 2 mM CaCl 2 , 2 mM MgCl 2 , 20 mM D-glucose, and 10 mM HEPES, (pH 7.35 titrated with NaOH). Experiments were performed at room temperature (21-24°C).
  • FIG 7A is a wide-field fluorescent image showing YFP -tagged ChR2 expression in a trans fected PC 12 cell
  • Figure 7B is a transmittant light image of the same field. The cell was whole-cell voltage-clamped by a glass pipette (left).
  • Figure 7C shows a family of photocurrents activated by various intensity of light in a PC 12 cell expressing ChR2.
  • FIG. 7D shows the relationship between photocurrent amplitude and light intensity obtained from the same cell depicted in Figures 7A-C. Fitting of the Hill equation yielded the maximum current of -220 pA, the Hill coefficient of 1.1, and the half-maximum light intensity of 400 ⁇ W/mm 2 .
  • Figures 7E-H are the same as Figures 7A-D except the cell was expressing GLuc- ChR2. Fitting of the Hill equation yielded the maximum current of -350 pA, the Hill coefficient of 1.3, and the half-maximum light intensity of 240 ⁇ W/mm 2 .
  • Native Gaussia luciferase is a secreted protein. It was shown that when GLuc was connected to ChR2 through a flexible linker, as in the GLuc-ChR2 fusion protein (pcDNA3.1/GLuc-hChR2(H134R)-EYFP; SEQ ID NO: 1), it functioned as the native enzyme GLuc did.
  • PC 12 cells were trans fected with both a plasmid carrying the fusion protein GLuc-ChR2 (pcDNA3.1/GLuc-hChR2(H134R)-EYFP; SEQ ID NO: 1), and with a pair of plasmids in equal amounts, one plasmid expressing native GLuc (pCMV-GLuc-1; SEQ ID NO: 3) and the other plasmid expressing ChR2 alone (pcDNA3.1/hChR2(H134R)-EYFP; SEQ ID NO: 2).
  • Cells transfected with pcDNA3.1/hChR2(H134R)-EYFP (SEQ ID NO: 2) alone were included as controls.
  • the luciferase substrate coelenterazine (CTZ) was added to cells expressing GLuc-ChR2 or cells expressing secreted GLuc. Luminescence was immediately measured in a luminometer (Veritas Microplate Luminometer, Turner Biosystems). As shown in Figure 8, cells transfected with plasmids wherein GLuc was linked to the channel ChR2 did not interfere with GLuc activity. For the results shown in Figure 8A and 8B, 1 x 10 4 cells were seeded in each well; for Figure 8C and 8D dilutions of cell were plated as indicated. 24 hours after transfection medium was collected and the cells were washed with PBS.
  • CTZ luciferase substrate coelenterazine
  • Substrate (coelenterazine, CTZ) was added at different concentrations or at 100 ⁇ M concentration.
  • CTZ was added to the medium to determine concentration of secreted luciferase. Only cells which were transfected with native GLuc secreted luciferase into the culture medium.
  • substrate was added to the cells, transferred to a reading plate 3 minutes later, and luminescence was measured.
  • the fusion protein GLuc-ChR2 oxidized its substrate when anchored to the cell membrane; ChR2 alone had no activity on CTZ ( Figure 8B).
  • GLuc-ChR2 membrane-bound GLuc
  • GLuc + ChR2 native GLuc
  • Figure 8D Note different scales for the relative luminescence when measuring medium (24 hour accumulation of secreted luciferase) versus cells (3 minute exposure to substrate).
  • PC12 cells transfected with pcDNA3.1/GLuc-hChR2(H134R)-EYFP (SEQ ID NO: 1) and expressing GLuc-ChR2 (as determined by YFP expression) were voltage-clamped as described above.
  • Coelenterazine (CTZ) was purchased from Nanolight, Inc.
  • CTZ was dissolved to 5 mg/mL (11.8 mM) in acidified ethanol (60 mM HCl). Substrate was applied at a 200 ⁇ M concentration in extracellular solution.
  • CTZ was applied by gravity from a glass pipette (tip diameter: -30 ⁇ m); start and stop of flow were controlled by moving a reservoir containing the substrate up or down, respectively.
  • the pipette was placed 200-300 ⁇ m away from the cell being recorded; this method insured that the entire cell being recorded was bathed in substrate for the time intended. Electrical responses were recorded as described above. Simultaneously, emitted bioluminescence was long-pass-filtered (>460 nm) and then collected by a cooled CCD camera every 10 sec.
  • HEK293 cells (Invitrogen) were cultured in DMEM. For transient transfections, 3-4 x 10 4 cells per well were seeded onto poly-D-lysine coated glass coverslips in 24-well plates. Cells were transfected with 0.2 ⁇ g/well plasmid DNA using Effectene Transfection Reagent (Qiagen). The transfection efficiency with the plasmid encoding the GLuc-ChR2 fusion protein was significantly higher in HEK cells compared to PC12 cells (50-80% versus 20%). Cells were tested for luminescence and in electrophysiology experiments 36 hours after transfection. For electrophysiology experiments involving ChR2, all-trans-retinal (1 ⁇ M final concentration) was added to the cultures several hours before the experiments.
  • the ChR2 response to light generated by GLuc in the trans fected HEK cells was tested.
  • HEK cells expressing GLuc-ChR2, as determined by YFP expression, were voltage-clamped as described in detail above.
  • the substrate coelenterazine (CTZ) was applied by a theta tube in 100 ms puffs at a 200 ⁇ M concentration in extracellular solution. Electrical responses were recorded and, simultaneously, emitted bioluminescence was measured (sampled every second). As shown in Figure 12, application of substrate resulted in a strong bioluminescence signal and a concomitant robust inward current.
  • Results shown in Figure 14 identified 200 ⁇ M CTZ as the optimal concentration for eliciting the highest luminescence in these in vitro experiments.
  • the substrate CTZ was applied to HEK cells at different concentrations (100 ⁇ M, 200 ⁇ M, and 500 ⁇ M) and bioluminescence was measured individually for 5 or more cells. The highest magnitude of luminescence was achieved with 200 ⁇ M CTZ. (Note: different scales for luminescence in Figures 14 and 15 are due to the use of different cameras.)
  • HEK cells were transiently transfected using the Effectene reagents with the GLuc-ChR2 construct (pcDNA3.1/GLuc-hChR2(H134R)-EYFP; SEQ ID NO: 1). Thirty-six hours after transfection, cells were washed, scraped off the tissue culture plates (rather than trypsinized), centrifuged, and resuspended in PBS.
  • GLuc-ChR2 construct pcDNA3.1/GLuc-hChR2(H134R)-EYFP; SEQ ID NO: 1).
  • CTZ coelenterazine
  • concentrations of substrate CTL
  • substrate CTL
  • Conventional white-light surface images were obtained immediately before each photon counting session to provide an anatomical outline of the animal.
  • post-processing and visualization were performed using the IVIS-associated software (Living Image, Xenogen). Images were recorded as pseudo-color photon count images and superimposed on a gray-scale anatomic white-light image. Regions of interest were defined using an automatic intensity contour procedure to identify bioluminescent signals with intensities significantly greater than background. The sum of the photon counts in these regions was then calculated.
  • HEK cells (1-2 million) were transiently transfected with the GLuc-ChR2 fusion construct and implanted into both flanks of an ICR mouse ( Figure 15A). Different amounts of coelenterazine (50 and 5 micrograms left and right side, respectively) were injected into the animals' flanks and the photon count was acquired for 1 second immediately after substrate application. As a control, an animal received 2 million HEK cells alone in each flank ( Figure 15B), and photon counts were zero. Total photon counts per second were calculated for a constant number of HEK cells exposed to varying concentrations of CTZ ( Figure 15C).
  • Implanted HEK cells were exposed to 50 and 5 micrograms of coelenterazine; mice were imaged for 1 second before (time point 0), immediately after (time point 1), and 20 minutes after application of CTZ (time point 20) (Figure 15D).
  • the signal intensity produced by a constant number of HEK cells in a mouse expressing GLuc-ChR2 was compared.
  • Fifty and 5 micrograms of coelenterazine resulted in the maximal achievable signal with the number of cells provided.
  • Further 10-fold dilutions of substrate resulted in approximately 4-fold decreases in signal generated. No signal was detected at all with either substrate alone or cells alone.
  • signal intensity was highest immediately after substrate application, but was still significant after 20 minutes (see Figure 15D).
  • Lentiviral particles (10 11 cfu/mL) will be stereotactically injected into the right and left motor cortex of wildtype ICR mice. One side will receive Lenti-ChR2, the other side will receive Lenti-GLuc-ChR2.
  • mice Four weeks after lentiviral injections, mice will receive the substrate CTZ intravenously at different concentrations, and photons will be acquired over the entire animal using the Xenogen IVIS system. Emission of blue light will occur on the side which received Lenti-GLuc-ChR2, and not the control side which received Lenti-ChR2.
  • these animals will be used for behavioral studies, specifically for correlating the degree of circling behavior induced by application of CTZ with the concentration of iv- administered substrate.
  • Example 6 Gaussia Luciferase - Volvox carteri Channelrhodopsinl (GLuc-VChRl) fusion protein.
  • VChRl had slower opening kinetics than ChR2, it was sensitive to dim light.
  • a fusion of GLuc and VChRl was engineered analogous to the GLuc-ChR2 fusion protein described above.
  • the plasmid pcDNA3.1/VChRl -EYFP (SEQ ID NO: 7; see Figure 18) was kindly provided by Dr. Karl Deisseroth (Stanford University, Stanford, CA).
  • the CMV promoter drove a mammalian codon-optimized Volvox channelrhodopsin 1 gene. Yellow fluorescent protein was fused to the carboxy terminus of ChR2.
  • the codon-optimized GLuc (pCMV-GLuc-1) was obtained from Nano light, Inc.
  • the GLuc sequence was inserted upstream of and in frame with the VChRl sequence.
  • the end of the GLuc sequence was separated from the start of the VChRl sequence by a 15 amino acid linker.
  • the resulting plasmid was pcDNAS.l/GLuc-VChRl-EYFP (SEQ ID NO: 6; Figure 18).
  • HEK cells were transiently transfected with the GLuc-VChRl plasmid (PCDNA3.1/GLUC- VChRl -EYFP; SEQ ID NO: 6) as described above. Recordings were done 36 hours after transfection.
  • Figure 19 shows an example of a robust current elicited by application of the substrate CTZ.
  • GLuc-generated blue light blue trace
  • evokes a strong inward current black trace
  • Figure 20 shows an example of voltage clamp and current clamp experiments using primary neurons expressing the Gluc-VChRl fusion protein. As shown in Figure 20, application of substrate (CTZ) resulted in a transient bioluminescence signal and a concomitant robust current and voltage, respectively.
  • CTZ substrate

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Abstract

L'invention concerne des procédés de suivi et/ou de modulation de la signalisation cellulaire, et spécifiquement, des polynucléotides, des polypeptides, des cellules, des tests, des animaux non humains génétiquement modifiés, et des procédés qui impliquent une protéine produisant de la lumière et une protéine transduisant la lumière, qui peuvent être utilisés pour manipuler, suivre et étudier la signalisation cellulaire dans divers types de cellules.
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