WO2017060510A1 - Methods and pharmaceutical compositions for the treatment of alzheimer's disease - Google Patents

Abstract

The present invention relates to methods and pharmaceutical compositions for the treatment of Alzheimer's disease. In particular, the present invention relates to a method of treating Alzheimer's disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated viral (AAV) vector comprising a polynucleotide encoding for an interleukin 2 (IL-2) polypeptide.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT

OF ALZHEIMER'S DISEASE

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of Alzheimer's disease.

BACKGROUND OF THE INVENTION:

Alzheimer's disease (AD) is the most prevalent form of dementia worldwide (Selkoe,

1999) for which no therapy is available, yet. It has been shown that the incidence of dementia in people over the age of 65 years doubles for every 5-year age interlude (Galimberti and Scarpini, 2012). Accumulating evidence suggests that activated glial cells have an important role in the pathophysiology of several psychiatric and neurological disorders, including AD (Skaper, 2007). The accumulation of Amyloid-β peptide (Αβ), processed from the β-amyloid precursor protein (APP) is a major hallmark of AD; however, the neuropathological mechanisms underlying its age-related deposition remain poorly understood. Insufficient clearance of Αβ from the brain is pondered to play a central role in amyloid accumulation (Sambamurti et al., 2011). This accumulation of amyloid in senile plaques prompts the recruitment of both microglia and astrocytes that generate proinflammatory elements, such as cytokines and chemokines, leading to neuroinflammation (Farfara et al., 2008). Glial cells maintain brain homeostasis and brain repair, as well as afford functional rescue from disturbing injuries (Streit, 2000). Though, chronic inflammation or glial cells dysfunction may induce synaptic loss and decreased neurogenesis, cognitive/motor impairment and finally neurodegeneration (Monje et al., 2003). Thus, inflammation has been considered a feature of AD pathogenesis and believed to pave the way to neurodegeneration (Rogers et al., 1988). Hence, anticipation of injurious neuroinflammation, by using particular cytokines (IL-4 and IL- 10) stimulating anti-inflammatory cascades, has been reported in AD transgenic mice (Kiyota et al., 2010; Kiyota et al., 2012), as an attempt to treat AD symptoms. In the other hand, it has been reported that sustained hippocampal IL-Ιβ overexpression mediated inflammation ameliorating AD plaque pathology (Shaftel et al., 2007).

Interleukin-2 (IL-2), a cytokine with pleotropic immune function, also has effects on the CNS. IL-2 can contact the CNS through the blood-brain-barrier (BBB) by a non-saturable transport mechanism (Waguespack et al., 1994). IL-2 has numerous effects on hippocampal neurons where its receptors are enriched, modifying cognitive performances in rodents (Hanisch et al., 1997; Lacosta et al., 1999). IL-2 shifts cellular and molecular substrates of learning and memory, like long-term potentiation (LTP) (Tancredi et al., 1990) and release of acetylcholine (Seto et al., 1997), the later playing a key role in cognition. Furthermore, IL-2 can afford trophic support to both neurons and glia (Awatsuji et al., 1993), affecting the morphology of neurite branching of rat hippocampal cultures (Sarder et al., 1996), enhancing dendritic development and spinogenesis (Shen et al., 2010), thus playing a role in neuronal development (Sarder et al., 1993). Importantly, IL-2 knockout (KO) mice display cytoarchitectural hippocampal modifications (Beck et al., 2005), impaired learning and memory ability and altered hippocampal development (Petitto et al., 1999).

In addition to these effects that might be interesting in the setting of AD, IL-2 is the key cytokine controlling regulatory T cells (Tregs) survival and function (reviewed in (Klatzmann and Abbas, 2015). Tregs represent a subset of T cells which main role is to control inflammation and (likewise) autoimmunity. The anti-inflammatory effects of Tregs have been observed in various models of inflammatory diseases in mice. Little is known about Tregs and neuroinflammation. However, peripheral blood Tregs numbers have been described as the best biomarkers predicting prognostic in ALS, with fewer Tregs correlating with worse clinical outcome. Recent clinical trials of IL-2 at low dose showed that it is safe and improves autoimmune and alloimmune inflammatory conditions in human.

SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of Alzheimer's disease. In particular, the present invention is defined by the claims. DETAILED DESCRIPTION OF THE INVENTION:

Interleukin-2 (IL-2) knockout mice have impaired learning and memory ability. Furthermore, IL-2 at low dose stimulates regulatory T cells (Tregs) which main role is to control inflammation. As neuroinflammation contributes to neurodegeneration in Alzheimer's disease (AD), the inventors investigated IL-2 in AD. They first showed that IL-2 is decreased in hippocampal biopsies from of AD patients. The inventors then treated with IL-2, APP/PS1AE9 mice having established AD. IL-2 induced systemic Treg expansion and activation, all along the 5-months follow-up. In the hippocampus, IL-2 induced astrocytic activation and recruitment around amyloid plaques, a decrease of amyloid plaques load and of the Αβ (42/40) ratio, and a restoration of the N-methyl-D-aspartate receptor subunit NR2A. Noteworthy, this tissue remodeling was associated with the recovery of memory deficits. Thus, IL-2 can alleviate AD in APP/PS1AE9 AD mice and this should prompt the investigation of low-dose IL-2 in AD.

Accordingly, an object of the present invention relates to a method of treating Alzheimer's disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated viral (AAV) vector comprising a polynucleotide encoding for an interleukin 2 (IL-2) polypeptide.

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably a subject according to the invention is a human. In the context of the present invention, a "subject in need thereof denotes a subject, preferably a human suffering from Alzheimer's disease.

As used herein, the term "Alzheimer's disease" denotes chronic neurodegenerative disease that usually starts slowly and gets worse over time. Alzheimer's disease (AD) is characterized by amyloid deposits, intracellular neurofibrillary tangles, neuronal loss and a decline in cognitive function (Hardy and Allsop, 1991, Selkoe, 2001). The most common early symptom is difficulty in remembering recent events (short-term memory loss). As the disease advances, symptoms can include: problems with language, disorientation (including easily getting lost), mood swings, loss of motivation, not managing self-care, and behavioral issues.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

In particular, the method of the present invention is particularly suitable for activating astrocytes, improving clearance of clearance of Αβ from the brain, improving amyloid pathology, and/or improving subject's memory learning and/or cognition.

As used herein, the term "IL-2" has its general meaning in the art and refers to the interleukin-2. The term "IL-2" polypeptide designates any source of IL-2, including mammalian sources such as e.g., human, mouse, rat, primate, and pig... IL-2 may be or comprise the native polypeptide sequence, or can be an active variant of the native IL-2 polypeptide. Preferably the IL-2 polypeptide is derived from a human source. Typically, the IL-2 polypeptide of the present invention comprises a amino acid sequence having at least 90% of identity with SEQ ID NO: l. According to the invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar are the two sequences. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A., 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5: 151-153, 1989; Corpet et al. Nuc. Acids Res., 16: 10881-10890, 1988; Huang et al., Comp. Appls Biosci., 8: 155-165, 1992; and Pearson et al., Meth. Mol. Biol., 24:307-31, 1994). Altschul et al., Nat. Genet., 6: 119-129, 1994, presents a detailed consideration of sequence alignment methods and homology calculations. By way of example, the alignment tools ALIGN (Myers and Miller, CABIOS 4: 11-17, 1989) or LFASTA (Pearson and Lipman, 1988) may be used to perform sequence comparisons (Internet Program® 1996, W. R. Pearson and the University of Virginia, fasta20u63 version 2.0u63, release date December 1996). ALIGN compares entire sequences against one another, while LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available on the Internet at the NCSA Website, for instance. Alternatively, for comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function can be employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). The BLAST sequence comparison system is available, for instance, from the NCBI web site; see also Altschul et al., J. Mol. Biol., 215:403-410, 1990; Gish. & States, Nature Genet., 3:266-272, 1993; Madden et al. Meth. Enzymol., 266: 131-141, 1996; Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997; and Zhang & Madden, Genome Res., 7:649-656, 1997.

SEQ ID NO: l (IL-2; Homo Sapiens):

MYRMQLLSCI ALSLALVTNS APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WIT FCQSIIS TLT

As used herein, the term "polynucleotide encoding for a IL-2 polypeptide" refers to any nucleic acid molecule encoding for the IL-2 polypeptide as defined above. As used herein, the term "nucleic acid molecule" has its general meaning in the art and refers to a DNA molecule. However, the term captures sequences that include any of the known base analogues of DNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fiuorouracil, 5- bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl- aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 - methylpseudouracil, 1 -methyl guanine, 1- methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-methyladenine, 7- methylguanine, 5-methylaminomethyluracil, 5- methoxyamino-methyl-2-thiouracil, beta-D- mannosylqueosine, 5'- methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil- 5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4- thiouracil, 5-methyluracil, -uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

In some embodiments, the polynucleotide of the present invention comprises a sequence having at least 80% (i.e. 80; 81, 82, 83, 84, 85, 86, 87, 88, 89 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100%) of identity with SEQ ID NO:2.

SEQ ID NO:2 (IL-2, Homo Sapiens):

AGTTCCCTAT CACTCTCTTT AATCACTACT CACAGTAACC TCAACTCCTG CCACAATGTA CAGGATGCAA CTCCTGTCTT GCATTGCACT AAGTCTTGCA CTTGTCACAA ACAGTGCACC TACTTCAAGT TCTACAAAGA AAACACAGCT ACAACTGGAG CATTTACTGC TGGATTTACA GATGATTTTG AATGGAATTA ATAATTACAA GAATCCCAAA CTCACCAGGA

TGCTCACATT TAAGTTTTAC ATGCCCAAGA AGGCCACAGA

ACTGAAACAT CTTCAGTGTC TAGAAGAAGA ACTCAAACCT

CTGGAGGAAG TGCTAAATTT AGCTCAAAGC AAAAACTTTC

ACTTAAGACC CAGGGACTTA ATCAGCAATA TCAACGTAAT

AGTTCTGGAA CTAAAGGGAT CTGAAACAAC ATTCATGTGT

GAATATGCTG ATGAGACAGC AACCATTGTA GAATTTCTGA

ACAGATGGAT TACCTTTTGT CAAAGCATCA TCTCAACACT GACTTGATAA TTAAGTGCTT CCCACTTAAA ACATATCAGG CCTTCTATTT ATTTAAATAT TTAAATTTTA TATTTATTGT TGAATGTATG GTTTGCTACC TATTGTAACT ATTATTCTTA ATCTTAAAAC TATAAATATG GATCTTTTAT GATTCTTTTT GTAAGCCCTA GGGGCTCTAA AATGGTTTCA CTTATTTATC CCAAAATATT TATTATTATG TTGAATGTTA AATATAGTAT CTATGTAGAT TGGTTAGTAA AACTATTTAA TAAATTTGAT AAATATAAAA AAAAAAAAAA AAAAAAAAAA AA

As used herein the term "AAV" has its general meaning in the art and is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all serotypes and variants both naturally occurring and engineered forms. According to the invention the term "AAV" refers to AAV type 1 (AAV-1), AAV type 2 (AAV- 2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV- 6), AAV type 7 (AAV-7), and AAV type 8 (AAV-8) and AAV type 9 (AAV9). The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC_001401 (AAV-2), AF043303 (AAV2), and NC_006152 (AAV-5). As used herein, a "rAAV vector" refers to an AAV vector comprising the polynucleotide of interest (i.e the polynucleotide encoding for the IL-2 polypeptide). The rAAV vectors contain 5' and 3' adeno- associated virus inverted terminal repeats (ITRs), and the polynucleotide of interest operatively linked to sequences, which regulate its expression in a target cell.

The AAV vector of the present invention typically comprises regulatory sequences allowing expression and, secretion of the encoded polypeptide (i.e. the IL-2 polypeptide), such as e.g., a promoter, enhancer, polyadenylation signal, internal ribosome entry sites (IRES), sequences encoding protein transduction domains (PTD), and the like. In this regard, the vector comprises a promoter region, operably linked to the polynucleotide of interest, to cause or improve expression of the protein in infected cells. Such a promoter may be ubiquitous, tissue- specific, strong, weak, regulated, chimeric, inducible, etc., to allow efficient and suitable production of the protein in the infected tissue. The promoter may be homologous to the encoded protein, or heterologous, including cellular, viral, fungal, plant or synthetic promoters. Examples of such regulated promoters include, without limitation, Tet on/off element- containing promoters, rapamycin-inducible promoters and metallothionein promoters. Examples of ubiquitous promoters include viral promoters, particularly the CMV promoter, CAG promoter (chicken beta actin promoter with CMV enhancer), the RSV promoter, the SV40 promoter, etc. and cellular promoters such as the PGK (phosphoglycerate kinase) promoter. The promoters may also be neurospecific promoters such as the Synapsin or the NSE (Neuron Specific Enolase) promoters (or NRSE (Neuron restrictive silencer element) sequences placed upstream from the ubiquitous PGK promoter), or promoters specific for RPE cell types such as the RPE65, the BEST1, the Rhodopsin or the cone arrestin promoters. The vector may also comprise target sequences for miRNAs achieving suppression of transgene expression in non- desired cells. In some embodiments, the vector comprises a leader sequence allowing secretion of the encoded protein. Fusion of the polynucleotide of interest with a sequence encoding a secretion signal peptide (usually located at the N-terminal end of secreted polypeptides) will allow the production of the therapeutic protein in a form that can be secreted from the transduced cells. Examples of such signal peptides include the albumin, the β-glucuronidase, the alkaline protease or the fibronectin secretory signal peptides.

The recombinant AAV vector of the present invention is produced using methods well known in the art. In short, the methods generally involve (a) the introduction of the rAAV vector into a host cell, (b) the introduction of an AAV helper construct into the host cell, wherein the helper construct comprises the viral functions missing from the rAAV vector and (c) introducing a helper virus into the host cell. All functions for rAAV virion replication and packaging need to be present, to achieve replication and packaging of the rAAV vector into rAAV virions. The introduction into the host cell can be carried out using standard virological techniques simultaneously or sequentially. Finally, the host cells are cultured to produce rAAV virions and are purified using standard techniques such as CsCl gradients. Residual helper virus activity can be inactivated using known methods, such as for example heat inactivation. The purified rAAV vector is then ready for use in the method of the present invention.

By a "therapeutically effective amount" of AAV vector as above described is meant a sufficient amount of the AAV vector for the treatment of Alzheimer's disease. It will be understood, however, that the total dosage of the AAV vector of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. Typically, from 10⁸ to 10¹⁰ viral genomes (vg) are administered per dose in mice. Typically, the doses of AAV vectors to be administered in humans may range from 10¹⁰ to 10¹² vg.

Administering the recombinant AAV vector of the present invention vector to the subject is preferably performed by sublingually, subcutaneously, intramuscularly, intravenously, transderaially delivery. In some embodiments, the recombinant AAV vector of the present invention is not administered to the subject by intraventricular or intracerebral injection. In some embodiments, the recombinant AAV vector of the present invention is administered to the subject by the intravenous injection. The recombinant AAV vector of the present invention is typically formulated into pharmaceutical compositions. For example, the AAV vector of the present invention is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The AAV vector of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifusoluble agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum- drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media, which can be employed, will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. The invention will be further illustrated by the following figures and examples.

However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Interleukin-2 protein levels are decreased in hippocampal biopsies from

AD patients.

Representative western-blot of hippocampal biopsies from Alzheimer's disease patients Braak 6/Thal5 (n=5) and healthy controls (n=5) show decreased levels of Interleukin-2 (IL-2) (Student's T test; p<0.05) (A); decrease levels of APP revealed with the 6E10 antibody (Student's T test; p=0.001) (B); and PSD-95 synaptic protein (Student's T test; p=0.004) (C).

ELISA analysis showing increased Αβ42/Αβ40 ratio in hippocampal biopsies from Alzheimer' s disease patients (n=5), compared to healthy age-matched controls (n=5) (Student's T test; p= 0.069) (D). Interleukin-2 protein levels are positively correlated with APP levels (6E10 antibody) (r2=0.72) (E) and PSD-95 protein levels (r2=0.68) (F) in the hippocampus of Alzheimer's disease patients (n=5).

Figure 2: Tregs are expanded and activated in blood and brain during Interleukin- 2 treatment.

Blood and Brain Interleukin-2 concentrations (respectively A and B), Tregs percentages (respectively C and D) and CD25 expression on Tregs (respectively E and F) in littermates and APP/PS1AE9 mice 5 months after AAV-LUC or AAV-IL-2 injection. The data shown are a pool from two different experiments: littermates LUC (n=5-8), APP/PS1AE9- LUC (n=5-9), littermates IL-2 (n=5-12) and APP/PS1AE9 IL-2 (n=5-8). Statistical significances are assessed using the Mann- Whitney test. Figure 3: Interleukin-2 treatment rescues spatial memory impairment in APP/PS1AE9 mice.

Transgenic APP/PS1AE9 mice (n=7/8 per group) or littermate controls (n=8 per group) were intraperitoneally injected with either rAAV8-IL-2 or rAAV8-LUC vectors at 8 months of age and tested 5 months later, i.e. at 13 months of age. Training phase consisted of daily sessions with three trials/day during 5 consecutive days. Four hours after the last training trial (day 5), the platform was removed and memory retention was assessed during a probe test. (A) Probe trial performance at 4h (short-term memory). rAAV8-LUC treated APP/PS1AE9 mice were impaired compared to rAAV8-LUC treated littermates, confirmed by no preference for the trained target quadrant (TQ) (% of time and % of distance). Strikingly, by contrast to APP/PS1AE9 mice injected with rAAV8-LUC, APP/PS1AE9 mice that had been injected with rAAV8-IL-2 showed a clear preference for the trained target quadrant (rAAV-LUC-treated vs IL-2-treated APP/PS1AE9 mice: % of time in TQ; p=0.0034 and % of distance in TQ; p<0.0001). (B and D) Probe trial performance at 72h. Long-term memory of APP/PS1AE9 mice was impaired in comparison to age-matched littermates after rAAV8-LUC injection (no preference for the trained target quadrant). Interleukin-2 treated APP/PS1AE9 mice displayed a noticeable preference towards the trained target quadrant, compared to APP/PS1AE9 mice injected with the control vector (% of time in TQ; Tukey Post-Hoc test: p=0.02 and % of distance in TQ: p=0.009) (Fig.3B and D); (TQ: Target Quadrant; OQ: Other Quadrants).

Figure 4: Interleukin-2 expression rescues structural and functional synaptic deficits in APP/PS1AE9 mice.

(A) Summary bar-graphs showing differences in mean values of LTP magnitude between genotypes. Long-term potentiation (LTP) was induced by delivering the theta-burst stimulation (TBS). Slices from transgenic APP/PS1AE9 mice injected with rAAV8-LUC exhibited significant deficit in LTP expression when compared with littermates injetced with rAAV8-LUC. LTP magnitude observed in Interleukin-2 treated APP/PS1AE9 mice was significantly lower than that in Interleukin-2 treated littermates; however, it was significantly improved in comparison to APP/PS1AE9 injected with the control rAAV8-LUC. (B) spine number was reduced in APP/PS1AE9 (1.12+ 0.03) compared to littermate controls injected with the control vector (1.42+ 0.05) or rAAV-IL-2 (1.35+ 0.04), a phenotype which could be rescued by injection with rAAV-IL-2 (1.29+ 0.04). N=3 animals per genotype, 8-10 dendrites per animal. Values represent means+SEM.

Figure 5: AAV-mediated Interleukin-2 administration decreases amyloid patology in the hippocampus of APP/PS1AE9 mice. Representative western-blot of hippocampal biopsies from APP/PS1AE9 mice and littermates injected with rAAV8-IL-2 or rAAV8-LUC vectors. The membrane probed with the 6E10 and 22C11 antibodies detects the presence of human APP or total APP, respectively. For optical densitometry quantification (A and B) signal intensities were normalized to GAPDH used as a loading control. (C) ELISA quantification of β-CTF in the hippocampus of APP/PS1AE9 mice: no major differences were detectable between rAAV8-IL-2 or rAAV8- LUC injected APP/PS1AE9 mice (Student's T test; p=0.08). (D-F) ELISA quantification (MSD immunoassay) of TBS soluble Αβ38, Αβ40 and Αβ42 in the hippocampus of APP/PS1AE9 mice (n=6/7 per group): a non-statistical significant decrease (-10%) in soluble Αβ42 levels in the hippocampus of mice injected with either rAAV8-IL-2 or rAAV8-LUC (Student T test; p=0.08) and a trend towards increased amounts of Αβ38 (-40%) (Student T test; p=0.27) and Αβ40 (-40%) (Student T test; p=0.28) were observed in the hippocampus of APP/PS1AE9 mice injected with rAAV8-IL-2, relatively to APP/PS1AE9 mice injected with rAAV8-LUC. Decreased Αβ42/Αβ40 ratio (-42%) in hippocampal biopsies from APP/PS1AE9 mice injected rAAV8-IL-2 relatively to APP/PS1AE9 mice injected with rAAV8-LUC (Student's T test; p=0.0013) (G). Diaminobenzidine (DAB)-staining using the 4G8 antibody in brain sections from 13.5-month aged APP/PS1AE9 mice treated with rAAV8-IL-2 or rAAV8-LUC vectors. A decrease in the hippocampal surface covered by plaques is observed in the hippocampus of 13.5 months old IL-2 treated APP/PS1AE9 mice relatively to APP/PS1AE9 mice injected with rAAV8-LUC (18%; Student's T test; p=0.023) mice (H).

Figure 6: Interleukin-2 promotes astrocytic recruitment around amyloid plaques and activates the JAK/STAT3 pathway in APP/PS1AE9 mice

Western-blot showing the expression of Ibal and GFAP in hippocampal biopsies from APP/PS1AE9 mice injected with the rAAV8-IL-2 or rAAV8-LUC vectors. (n=6-7/group). For optical densitometry quantification (A and B) signal intensities were normalized to GAPDH used as a loading control. rAAV8-IL-2 treatment increases the levels of the astrocytic marker GFAP, but not microglial Ibal. Laser confocal microscopy showing double staining using the 6E10 antibody and anti-Ibal or the anti-GFAP antibodies in coronal brain sections from APP/PS1AE9 mice. No major modifications were observed in Ibal immunoreactivity around amyloid plaques (6E10) between APP/PS1AE9 mice injected with rAAV8-IL-2 or rAAV8- LUC (C). (D) Increased GFAP immunoreactivity in APP/PS1AE9 mice injected with in rAAV8-IL-2, where hypertrophic astrocytes with thick proximal processes overlap the amyloid plaque as compared to APP/PS1AE9 mice treated with rAAV8-LUC. Representative western- blot showing a -2-fold increase in the levels of Stat3-a in APP/PS1AE9 mice treated with rAAV8-IL-2 as compared to APP/PS1AE9 mice treated with rAAV8-LUC (p<0.0001). (E) No statistical significant differences were found between APP/PS1AE9 mice treated with rAAV8- LUC and wild-type mice injected with either Interleukin-2 or LUC viral vectors (optical densitometry in E). The levels of pSTAT-3 are increased in APP/PS1AE9 mice injected with rAAV8-LUC compared to wild-type littermates receiving rAAV8-IL-2 or rAAV8-LUC (A). A ~2-fold increase of p-STAT3 was observed in APP/PS1AE9 mice treated with rAAV8-IL-2 relatively to rAAV8-LUC injected APP/PS1AE9 mice (B) (p<0.0001) (optical densitometry in F).

Figure 7: Western blot analysis showing increased levels of IL-2 in hippocampal biopsies from APP/PS1AE9 mice injected with rAAV8-IL-2, relatively to APP/PS1AE9 mice injected with rAAV8-LUC or littermates injected with both vectors. For quantification signal intensities were normalized to GAPDH, used as a loading control. Experiments were performed using 6-7 mice per group. Values represent means+SEM.

Figure 8: Ex vivo bioluminescence imaging in brain and liver at 2 weeks after intraperitoneal injection of 10E10 vg rAAV8-CAG-LUC vector in 8 months old APP/PS 1ΔΕ9 transgenic mice. A. Quantitative results of ex vivo bioluminescence imaging in rAAV8-CAG- LUC APP/PS 1ΔΕ9 mice (n = 2) and in non-injected APP/PS 1ΔΕ9 mice (n = 2). B. Quantitative PCR using primers for ITR2 detection in liver and brain samples from APP/PS 1ΔΕ9 transgenic mice injected with rAAV8-C AG-LUC or from non-injected APP/PS 1ΔΕ9 mice (controls) (n = 2 for non-injected and n=8 for rAAV8-CAG-LUC injected mice). ITR2 sequences were found in the liver of rAAV8-CAG-LUC injected mice relatively to the liver of non-injected mice and the brain of both non-injected or rAAV8-LUC injected mice. Statistical analysis: One-way ANOVA with Tukey post-hoc test.

Figure 9: (A, B and C) ELISA quantification (MSD immunoassay) showing increased levels of Αβ peptides (38, 40 and 42) and β-CTF (D) in hippocampal samples from 8 months aged APP/PS 1ΔΕ9 mice compared to age-matched wild- type littermates.

Figure 10: Western-blots showing no major differences in the levels of the microglial markers Arginase-1 (A), TGF-β (B), TREM-2 (C) and Insulin degrading enzyme (IDE) (D) in hippocampal biopsies of APP/PS 1ΔΕ9 mice injected with rAAV8-IL2 or the control vector rAAV8-LUC.

EXAMPLE:

Material & Methods

Human AD brain samples Post-mortem samples were obtained from brains collected as part of the Brain Donation Program of the GIE-Neuro-CEB Brain Bank, Pitie-Salpetriere Hospital (Paris). Autopsies were carried out by accredited pathologists, after informed consent had been obtained from the relatives, in accordance with French Bioethics laws. Five hippocampal samples from five patients with sporadic forms of Alzheimer's disease (male and female; Braak 6 Thai 5; aged between 69 and 89 years, with a post-mortem interval (PMI) of 30 to 59 h) and five hippocampal samples from five age-matched control subjects (male and female, aged between 69 and 92 years, PMI of 6 to 63 h) were used in this study.

Animals

Eight-month old APPs we/PS 1ΔΕ9 male mice (n=31) (hereafter referred as

APP/PS1AE9) and the wild-type littermates (n=32) (Jackson Laboratories, Bar Harbor, Maine, USA) were used in this study. APP/PS1AE9 mice overexpress the mutated human APP (Swedish mutation, K595N/M596L) gene as well as the Human PS1 gene deleted from its ex on 9 (Jankowsky et al., 2004). APP/PS1AE9 mice and wild-type littermates were bred and maintained in our animal facility under specific pathogen-free conditions. Mice were housed in a temperature-controlled room and maintained on a 12 h light/dark cycle. Food and water were available ad libitum. The experiments were carried out in accordance with the European Community Council directive (86/609/EEC) for the care and use of laboratory animals. All procedures were approved by the Regional Ethics Committee in Animal Experiment No. 5 of the Ile-de-France region (Ce5/2012/031).

rAA V generation and in vivo administration of AA Vs

Recombinant AAV8 vectors were generated by triple transfection of human embryonic kidney 293T cells, as described previously (Churlaud et al., 2014). Transgenes used were luciferase (LUC) and murine Interleukin-2 (IL-2) driven by the hybrid cytomegalovirus enhancer/chicken beta-actin constitutive promoter (CAG). Mice were injected once intraperitoneally with 1010 viral genomes (vg) of rAAVs (AAV8-CAG-IL2 or AAV8-CAG- LUC) diluted in 100 of 0.1M phosphate-buffered saline (PBS) (APP/PS 1ΔΕ9 AAV8- CAG- IL2, n=16; APP/PS 1ΔΕ9 AAV8-CAG-LUC, n=15; wild-type littermates AAV8-CAG-IL2, n=16; wild-type littermates AAV8-CAG-LUC, n=16.

Detection of Interleukin-2 in the serum

Sera were collected, frozen and kept at -80 °C until use. Levels of Interleukin-2 were measured using a mouse Interleukin-2 ELISA (eBioscience) according to the manufacturer's recommendations.

Analysis of cell surface markers and Foxp3 expression Direct ex vivo immuno staining was performed on 150 μΐ_^ of heparinized fresh whole blood from mice after red blood cells lysis, as described (Churlaud et al., 2014). Briefly, blood was stained with the following monoclonal antibodies for 20 min at 4 °C: CD3-PE, CD8- Alexa700, CD4-HorizonV500, CD25-PeCy7, NKp46-APC and B220-FITC (eBioscience). Intracellular detection of Foxp3 (Foxp3-E450, eBioscience) was performed on fixed and permeabilized cells using appropriate buffer (eBioscience). Cells were acquired on an LSR II (Becton Dickinson) and analyzed with FlowJo (Tree Star, Inc.) software. Dead cells were excluded by forward/side scatter gating. Tregs were defined as CD25+ Foxp3+ cells among CD4+ cells, and activated effector T cells as CD25+ cells among CD4+ Foxp3- cells. For the fluorescence-activated cell sorting (FACS) analysis of Tregs, CD25 Mean Fluorescence Intensity (MFI) in Tregs and Foxp3 MFI in Tregs were assessed.

Brain samples

APP/PS1AE9 mice and wild-type littermates were sacrificed 5 months post-injection (13 months old). The animals, given an overdose of sodium pentobarbital, were perfused transcardially with ice-cold PBS 0,1M before brain extraction. For flow cytometry analysis, freshly perfused brain was dissociated and digested in collagenase/DNase solution in RPMI medium (Churlaud et al., 2014). A Percoll (Sigma-Aldrich) gradient was used to isolate brain- infiltrating lymphocytes. Cells were then stained as described earlier for blood.

For histological processing, the left cerebral hemisphere was dissected and post-fixed in 4% paraformaldehyde (PFA) in 0.1M PBS for 1 week. Brains were cryoprotected by incubation in a 30% sucrose/0.1 M PBS solution. Coronal brain sections (40 μιη) were cut on a freezing microtome (Leica, Wetzlar, Germany), collected serially, and stored at -20°C until additional analysis. The right hemisphere was dissected to extract the hippocampus, used biochemistry analysis. Samples were then homogenized in a lysis buffer (TBS, NaCl 150mM and Triton 1%) containing phosphatase (Pierce) and protease (Roche) inhibitors. After centrifugation (20min, 13 000 rpm, 4°c), the supernatant was collected and the protein concentration was quantified (BCA Protein Assay, Thermo Fisher Scientific, Waltham, USA). Lysate aliquots (3mg of protein/ml) were stored at -80°C until use. The same procedure was conducted for human samples (GIE NeuroCEB Brain Bank).

Western Blot

Total protein concentrations were determined using the BCA kit (Pierce). Equal amounts of total protein extract (30 μg) were electrophoretically separated using SDS-PAGE in 4-12% Bis-Tris gels (NuPAGE® Novex Bis-tris midi gel 15 or 26 wells, Life Technologies, Carlsbad, USA) and transferred to nitrocellulose membranes. Blocked membranes (5% non-fat dry milk in TBS-0.1% Tween-20) were incubated with primary antibodies overnight at 4°C, and washed three times with TBS-0.1% Tween-20 (T-BST) for 10 min. Membranes were then labeled with secondary IgG-HRP antibodies raised against each corresponding primary antibody. After three washes with T-BST, the membranes were incubated with ECL chemiluminescent reagent (Clarity Western ECL substrate; GE Healthcare, Little Chalfont, UK) according to the instructions of the supplier. Peroxydase activity was detected with camera system Fusion TX7 (Fisher Scientific). Normalization was done by densitometry analysis with the Quantity One ID image analysis software (version 4.4; Biorad, Hercules, CA, USA). The optical densities were normalized with respect to a "standard protein" (GAPDH). A partition ratio was calculated and normalized with respect to the sample with the highest value defined as 1.

Primary antibodies

Antibodies used in western-blot (WB) and immunohistochemical (IHC) analyses:

Primary antibodies Source WB IHC rabbit anti-IL2 (mouse/human) Abeam 1:500 1:500 mouse anti-beta amyloid 1-16

(clone 6E10) Covance 1:4000 1:2000 rabbit anti-APP/ beta amyloid

(clone 22C11) LifeSpan Biosciences 1:2000

mouse anti-beta amyloid 17-24

(clone 4G8) Covance - 1:2000 rabbit anti-Glial Fibrillary Acidic

Protein (GFAP) Dako 1:5000 1:4000 rat monoclonal anti-mTREM-2B R&D systems 1:2000 - rabbit anti-ionized calcium

binding adapter molecule 1 (Ibal) Wako 1:500 1:3000 rabbit anti-Insulin degrading

enzyme (IDE) Abeam 1:1000 - rabbit anti-arginase Abeam 1:2000

rabbit anti-TGF-β Abeam 1:2000 - rabbit anti-pSTAT3 Cell signalling 1:1000

rabbit anti-STAT3 Cell signalling 1:1000 - mouse anti-GAPDH Abeam 1:4000 -

ELISA assay

Αβ-38, Αβ-40 and Αβ-42 were measured using the MSD Human Αβ42 V-PLEX Kit and the triplex Αβ Peptide Panel 1 (6E10) V-PLEX Kit (Mesoscale Discovery, Rockville, MD, USA). β-CTF was measured using the Human APP β-CTF Assay Kit (IBL, Hamburg, Germany). Interleukin-2 was measured using the MSD proinflammatory panel 1 (Mesoscale Discovery, Rockville, MD, USA). ELISA assays were performed following supplier instructions.

Immunostaining

The immunohistochemical procedure was initiated, by incubating slices in 88% formic acid solution for 15 min (antigen retrieval) and then by quenching endogenous peroxidase by incubating free-floating sections in hydrogen peroxide for 30 min at room temperature (RT). After three washes, slices were blocked in PBS/0.1% Triton X-100 containing 10% Normal Goat Serum (NGS, Gibco) for lh at RT. The sections were then incubated with the primary antibody (4G8), overnight at 4°C. After three washings, the sections were incubated with the corresponding biotinylated secondary antibody (1:250; Vector Laboratories Inc., CA, USA) diluted in PBS/0.1% Triton X-100 and 10% NGS for 2 h at RT. After three washes, bound antibodies were visualized by the ABC amplification system (Vectastain ABC kit, Vector Laboratories, West Grove, USA) and 3,3' -diaminobenzidine tetrahydrochloride (peroxidase substrate kit, DAB, Vector Laboratories, CA, USA) as the substrate. The sections were mounted, dehydrated by passing twice through ethanol and toluol solutions, and coverslipped with Eukitt® (O. Kindler GmbH & CO, Freiburg, Germany).

For immunofluorescence, slices were washed with PBS 0.1 M, permeabilized in PBS- Triton 0.1% before blocking in PBS-Triton 0.1% containing 5% normal goat serum (NGS) for 1 hour. Sections were then incubated with the respective primary antibodies, overnight at 4°C. After three successive washes, brain slices were incubated for 2 hours at room temperature with fluorescent secondary Alexa Fluor-conjugated antibodies (Invitrogen). Slices were stained with DAPI (1:5000; Sigma), mounted in Vectashield fluorescent mounting media (Vector laboratories) and conserved at 4°C.

Quantification of the microglia and astrocytes around plaques

Plaques, GFAP and Ibal immunoreactivity were quantified using Image J (NIH, Bethesda, USA) or Icy (Institut Pasteur, Paris, France). Laserpower, numeric gain and magnification were kept constant between animals to avoid potential technical artefacts. Images were first converted to 8-bit gray scale and binary thresholded to highlight a positive staining. At least 3 sections per mouse (between -1.7 mm to -2.3 mm caudal to bregma) were quantified. The average value per structure was calculated for each mouse. For quantification of Ibal and GFAP immunoreactivity around plaques, a region of interest (ROI) was drawn around the center of the plaque. The diameter of the circular ROI was set as 3 times the diameter of the plaque. Mean fluorescence intensity values were measured for either Ibal or GFAP immunoreactivity and were processed via Icy software (Institut Pasteur, Paris, France). Analysis of data was blind with respect to treatments and genotypes.

Image acquisition

Images of immunostained sections were acquired with a Z6 APO macroscope (Leica) and LAS V3.8 (Leica) software, at room temperature, with a brightfield Leica DM 5000B microscope equipped with a Leica DFC310FX digital camera. Confocal images were acquired with a Leica SP8 confocal microscope. Photographs for comparison were taken under identical conditions of image acquisition, and all adjustments of brightness and contrast were applied uniformly to all images.

Behavioral assessment-Morris Water Maze

Experiments were performed in a 120-cm diameter, 50 cm deep tank filled with opacified water kept at 21°C and equipped with a 10 cm diameter platform submerged 1 cm under the water surface. Visual clues were disposed around the pool as spatial landmarks for the mouse and luminosity was kept at 430 lux. Training consisted of daily sessions (three trials per session) during 5 consecutive days. Start positions varied pseudo-randomly among the four cardinal points. Mean inter- trial interval was 15 min. Each trial ended when the animal reached the platform. A 60- second cut-off was used, after which mice were gently guided to the platform. Once on the platform, animals were given a 30-second rest before being returned to their cage. Four hours (short-term memory) and 72 hours after the last training trial (day 8), retention was assessed during probe trial in which the platform was no longer present. Animals were video tracked using Ethovision software (Noldus, Wageningen, Netherlands) and behavioral parameters (swim speed, travelled distance, latency, percentage of time in each quadrant) were automatically calculated. Experiments and statistical evaluation of data were performed by an experimenter blind to genotype and treatment group.

Electrophysiology

Female 13.5 months-old APP/PSldE9 mice injected with the rAAV-IL-2 (n=5) or rAAV8-LUC (n=5) and their littermates administered with rAAV-IL-2 (n=4) or rAAV8-LUC (n=4) were used for electrophysiological examination of synaptic plasticity in hippocampus. Mice, deeply anesthetized by inhalation of high concentration of C02, were decapitated and brain was removed and divided into two parts. One hemisphere was transferred into the Golgi staining solution and processed accordingly to allow investigation of neuronal morphology of regions of interest. The second hemisphere was transferred into cold (4 °C) artificial cerebrospinal fluid (ACSF), containing the following (in mM): 124 NaCl, 4.9 KC1, 1.2 KH2P04, 2.0 MgS04, 2.0 CaC12, 24.6 NaHC03, 10 D-glucose, equilibrated with 95% 02 and 5% C02. The hippocampus was dissected from the second hemisphere and transverse hippocampal slices of 400 μιη were cut using tissue chopper. Hippocampal slices were incubated at 32 °C in an interface chamber with the constant flow of carbogenated (95% 02 and 5% C02) ACSF for 2 hours prior recording. Field excitatory postsynaptic potentials (fEPSPs) were recorded in stratum radiatum of the CA1 region of hippocampus. Synaptic responses were evoked by stimulation of the Schaffer collaterals. An input/output curve (dependence of fEPSP slope on stimulation intensity) was plotted prior to each experiment.

Data of electrophysiological recordings were collected, stored and analyzed with

LAB VIEW software (National Instruments, Austin, USA). The initial slope of fEPSPs elicited by stimulation of the Schaffer collaterals was measured over time, normalized to baseline and plotted as average + SEM. Analysis of the Paired-pulse facilitation (PPF) data was performed by calculating the ratio of the slope of the second fEPSP divided by the slope of the first one and multiplied by 100.

Dendrite and spine analysis in Golgi- Cox stained slices

Golgi- Cox staining

Golgi staining was performed using the Golgi Staining Kit (FD NeuroTechnologies, Columbia, USA) according to the manufacturer's instructions. All procedures were performed under dark conditions. Brains hemispheres used for Golgi cox staining were immersed in 2 ml mixtures of equal parts of kit solutions A and B and stored at RT for 2 weeks. Then, brain tissues were stored in solution C at 4°C for at least 48h and up to 7 days before sectioning. Solutions A, B and C were renewed within the first 24h. Coronal sections of 200 μιη were cut with a vibrating microtome (Leica, VT1200S) while embedded in 2% Agar in 0.1M PBS. Each section was mounted with Solution C on an adhesive microscope slide pre-coated with 1% gelatin/0.1% chromalaun on both slides and stained according to the manufacturer's protocol with the exception that AppliClear (AppliChem) was used instead of xylene. Finally, slices were covers lipped with Permount (Thermo Fisher Scientific).

Imaging and analysis of spine density in Golgi- Cox stained slices Imaging of dendritic branches of hippocampal pyramidal neurons was done with an Axioplan 2 imaging microscope (Zeiss) using a 63x oil objective (NA 1.3) and a z-stack thickness of 0.5 μιη under reflected light. The number of spines was determined per micrometer of dendritic length (in total 100 μιη) at apical compartments using ImageJ (1.48v, National Instruments of Health, USA). Three animals per genotype and 8-10 neurons per animal were analyzed blinded to genotype and injected AAV. Data were analyzed using Graphpad Prism (Version 5.01) software. Spine density is expressed as mean + SEM. Differences between genotypes were detected with one-way ANOVA followed by Bonferroni's post hoc test using IBM SPSS Statistics 21.

Statistical analysis

Statistical analyses were defined regarding the experimental design used. All data are presented as the mean + SEM. In most cases, data were analyzed using Student's T test, the Mann-Whitney test or one-way ANOVA with experimental group as factor. One-way ANOVA with repeated measures were carried out when required by the experimental plan to assess statistical effects. Correlations were generated using non-parametric Spearman rank correlation coefficient. For all analysis statistical significance was set to a p-value<0.05. All analyses were performed using Statistica (StatSoft Inc., Tulsa, USA) or GraphPad Prism (GraphPad Software, La Jolla, USA). Results:

Interleukin-2 expression is decreased in the hippocampus of AD patients

We first analyzed Interleukin-2 protein levels in frozen hippocampal biopsies from five severely affected Alzheimer's disease patients (Braak 6/Thal5) and five age-matched healthy controls. We observed a 2-fold decrease in Interleukin-2 levels (p<0.05) (Fig. 1A) paralleled by a ~ 80% reduction in APP levels (p=0.001) and synaptic protein PSD-95 levels (p=0.004) (Fig. 1B-C) in the hippocampus of AD patients relatively to controls. The Αβ42/Αβ40 ratio, an outcome measure for Alzheimer's disease severity (Ferrari et al., 2014), was found increased in Alzheimer's disease patients (p=0.07) (Fig. ID). Remarkably, there is a significant positive Spearman correlation between Interleukin-2 and APP levels (correlation coefficient 0.72, p = 0.0017) and between Interleukin-2 and PSD-95 levels (correlation coefficient 0.68, p = 0.003) (Fig. IE and F).

Peripheral Interleukin-2 delivery induces increased Interleukin-2 and Tregs levels in the brain of APP/PS1AE9 mice As Alzheimer's disease is a slow developing disease, we anticipated that long term Tregs stimulation could be necessary for obtaining therapeutic benefit. As Interleukin-2 has a short half-life in mice, maintaining an effect on Tregs would require frequent sub-cutaneous (sc) injections that could have an effect on mice behavior. We thus delivered Interleukin-2 by intraperitoneal (ip) injection of a recombinant adeno-associated virus (AAV) coding for murine Interleukin-2, which allows sustained and stable release of Interleukin-2 for at least 20 weeks (Churlaud et al., 2014; Wang et al., 2005). Eight-month old APP/PS1AE9 mice and littermates was injected with rAAV8-IL2 or luciferase-expressing control vectors (rAAV8-LUC). Four months after rAAV8 injections, serum Interleukin-2 was undetectable in mice receiving rAAV8-LUC, and was of 25.2 + 5.3 or 29.5 + 4.8 pg/mL (mean + SEM) in Interleukin-2 treated littermates and APP/PS1AE9, respectively (Fig.2A). These serum Interleukin-2 levels are those necessary for expanding and activating Tregs without effects on effector T cells, as previously described (Churlaud et al., 2014). Indeed, peripheral Interleukin-2 production expanded blood Tregs, significantly more in the APP/PS1AE9 mice than wild type littermates (Fig 2C). Tregs from Interleukin-2 treated mice were also more activated as assessed by increased CD25 cell surface expression (Rosenzwajg et al., 2015) (Fig 2E). Mice were sacrificed at 5.5 months post- injection (13.5 months of age). In hippocampal biopsies, there was a significant increase of Interleukin-2 levels only in Interleukin-2 treated APP/PS1AE9 mice (Figure 2B). These levels were approximately doubled compare to Interleukin-2 treated wild-type littermates and rAAV8-LUC controls, as detected both by western blot (Figure 7) and ELISA (Figure 2B). There was a concomitant 3-fold increase of brain Tregs in Interleukin-2 treated normal and APP/PS1AE9 mice (Fig 2D). These Tregs also showed a higher activation status as attested by an increased CD25 expression (Fig 2F). In order to evaluate whether the increased Interleukin- 2 levels observed in APP/PS1AE9 transgenic mice could be due to the transduction of brain cells by AAV vectors, we analyzed the biodistribution of AAV after injection of rAAV8-LUC in normal and in APP/PS1AE9 transgenic mice. We assessed luciferase bioluminescence in peripheral organs including the brain. As negative control, we used non-injected age-matched APP/PS1AE9 mice. Two weeks post-injection, luciferase expression could be detected in most of peripheral organs (liver, heart, kidney and spleen). In accordance with the known tropism of AAV8, most of the expression was detected in the liver. In contrast, no expression could be detected in the brain (Figure 8A). To further substantiate these data, we also used a sensitive qPCR targeting the inverted terminal repeat (ITR2) sequence from the AAV8 vector genome to probe its biodistribution. ITR2 sequences were readily detected in the liver, while no expression could be detected in the brain of APP/PS 1ΔΕ9 mice (Figure 8B). Thus, the increased brain Interleukin-2 levels in APP/PS1AE9 mice results from passage from the periphery and not from local production by rAAV8 transduced brain cells.

Interleukin-2 treatment rescues memory impairment in APP/PS1AE9 mice

To evaluate the therapeutic effects of increasing brain Interleukin-2 on spatial learning and memory, mice were tested in the Morris water- maze place navigation task (Fig. 3). Treated APP/PS1AE9 mice or wild-type littermates were tested 5 months post-injection. All mice learned platform position across time during learning session, as demonstrated by decreased latency (data not shown) or path length (data not shown) to reach the platform over the 5 days of training. Noteworthy, an overall significantly improved learning was detected in rAAV-IL- 2 treated littermates (p<0.05). Average swimming speed was comparable in all groups ruling out potential motor abilities differences (data not shown). The 4 hours probe trial that evaluates spatial reference memory (short-term memory) after the last training trial, revealed strong memory impairment in APP/PS1AE9 mice injected with rAAV-LUC compared to rAAV-LUC littermates (p=0.0002 and p=0.0002 using % of time and % of distance as readouts; Fig. 3A and C); wild-type littermates treated with either rAAV-LUC or rAAV-IL-2, showed similar preference. Strikingly, by contrast to APP/PS1AE9 mice injected with rAAV-LUC, Interleukin- 2 -treated APP/PS1AE9 mice showed a clear preference for the target quadrant (rAAV-LUC- treated vs Interleukin-2 -treated APP/PS1AE9 mice: % of time in TQ; p=0.0034 and % of distance in TQ; p<0.0001), and were statistically indistinguishable from control littermates. These results were confirmed at 72 hours (long-term memory; rAAV-LUC-treated vs IL-2- treated APP/PS1AE9 mice: % of time in TQ; p=0.0199 and % of distance in TQ: p=0.0092) (Fig. 3B and D) suggesting a beneficial effect of Interleukin-2 during memory consolidation phase. These data clearly demonstrate that Interleukin-2 rescued impairments in memory retention observed in APP/PS1AE9 mice.

Interleukin-2 rescues impaired synaptic plasticity and restores decreased spine density in APP/PS1AE9 mice

Αβ-induced damage of synaptic transmission is one probable mechanism inducing memory impairments in APPP/S1 mice (Snyder et al., 2005). We evaluated whether Interleukin-2 -based memory restoration in APP/PS1AE9 mice was reflected at the functional neuronal network level. We investigated synaptic plasticity at 13.5 months of age, which is considered to represent the basis of newly shaped declarative memory. Long-term potentiation (LTP) was induced at the Schaffer collateral to hippocampal CA1 pathway by theta-burst stimulation after baseline recording (data not shown). As expected, slices from transgenic APP/PS1AE9 mice exhibited significantly lower induction and maintenance of LTP compared with littermates after rAAV8-LUC administration (1.15 ± 0.001 (n; number of slices=12, p<0.05, F value 6.01, ANOVA) compared with 1.51 + 0.003 (n=9)). Interleukin-2 treated APP/PS1AE9 showed significantly improved LTP as evidenced by statistically significant increase of average potentiation (1.23 + 0.01, n=10, p<0.05, F value 5.92, ANOVA) compared with APP/PS1AE9 mice receiving rAAV8-LUC. However, LTP magnitude was lower relatively to Interleukin-2 treated littermates (1.45 + 0.001, n=8, p<0.05, F value 6.01) (Fig. 4A). The fEPSP slope was not different between different groups (data not shown), suggesting that basal synaptic transmission in all groups was not affected. In addition, we analyzed paired- pulse facilitation (PPF) of fEPSP to afferent stimulation, a form of short-term synaptic plasticity. Analysis of the EPSP2/EPSP1 ratio revealed significant (p<0.05, ANOVA) facilitation of second response in all interpulse intervals in APP/PS1AE9 mice administered with rAAV-IL-2 compared with their littermates. Paired-pulse facilitation in APP/PS1AE9 mice administered with rAAV-LUC was also significantly (p<0.05, ANOVA) higher compared with littermates. Paired-pulse facilitation was not different between APP/PS1AE9 mice administered with rAAV-IL-2 or rAAV-LUC (data not shown). Spine density was analyzed as a correlate of excitatory synapses in the same animals used for electrophysiological recordings. Spine density of mid- apical dendritic segments (between 100 and 400 μιη form soma) of the hippocampal CAl pyramidal layer was analyzed by the Golgi cox method (data not shown). We found an overall decrease in spine density in APP/PS 1ΔΕ9 mice treated with control rAAV- LUC at the CAl apical dendritic compartment relatively to littermates injected with either rAAV-LUC or rAAV-IL-2 (p<0.001, 1-way ANOVA followed by Bonferroni's post-hoc test). Importantly, rAAV-IL-2-treated APP/PS 1ΔΕ9 mice revealed a complete restoration of the spine deficit in apical dendrites of the CAl layer (p>0.1) (Fig. 4B). Altogether, these data indicate that Interleukin-2 strikingly ameliorates both structural and functional synaptic impairments in Alzheimer's disease mice.

Interleukin-2 peripheral administration alleviates hippocampal amyloid pathology in APP/PS1AE9 mice

APP/PS 1ΔΕ9 mice show highly abundant plaques from 6 months (Jankowsky et al., 2004). Interleukin-2 treatment was started at 8 months of age, a time at which we already evidence increased levels of Αβ peptides (Αβ38, Αβ40 and Αβ42) and β-CTF in hippocampus from APP/PS 1ΔΕ9 mice (Figure 9). Mice were sacrificed at 13.5 months of age. Hippocampal APP levels were slightly increased in mice receiving rAAV8-IL-2 compared to rAAV8-LUC injected mice (Fig. 5A-B). The production of Αβ peptides and β-CTF, known to induce hippocampal neurophysiological impairments, was quantified in the hippocampus of injected mice by ELISA. No differences in β-CTF levels were detectable (Fig. 5C). There was a trend towards increased amounts of Αβ38 and Αβ40 (Fig. 5D-E) and a trend to decrease in soluble Αβ42 levels (Fig. 5F). This translated into a significant reduction in the Αβ42/Αβ40 ratio (p=0.0013) (Fig. 5G). This correlated with a decrease in the surface covered by plaques in APP/PS1AE9 mice (p=0.023) (Fig. 5H). Altogether, these data indicate that Interleukin-2 reduces amyloid load and plaque deposition in the hippocampus of APP/PS1AE9 mice with established pathology.

Interleukin-2 administration induces widespread recruitment of astrocytes in the vicinity of amyloid plaques

Amyloid plaques in APP/PS1AE9 mice were surrounded by microglia and astrocytes at the time of the treatment (8 months) (data not shown).

The protein levels of microglial Ibal were not statistically different in the hippocampus of IL-2 treated APP/PS1AE9 mice compared to APP/PS1AE9 mice receiving rAAV8-LUC (Fig. 6A); no differences were detected in littermates receiving rAAV8-LUC or rAAV8-IL-2. No differences were observed in the expression of microglia markers, cytokine transforming growth factor-beta (TGF-β), Arginase-1 and Triggering Receptor Expressed on Myeloid cells (TREM2) or insulin-degrading enzyme (IDE), which contributes to Αβ clearance (Leissring et al., 2003) (Figure 10). No major difference in Ibal immunoreactivity around hippocampal amyloid plaques was observed (Fig. 6C).

Astrocytic markers analysis demonstrated a 3 to 4-fold increase of GFAP expression

(p<0.0001) in APP/PS1AE9 mice injected with rAAV8-IL-2 compared to APP/PS1AE9 mice treated with rAAV8-LUC (Fig. 6B). Immuno staining clearly showed that these GFAP immunoreactive astrocytes were hypertrophic, indicating their activation (data not shown). Moreover, a statistically significant increase of GFAP immunoreactivity around amyloid plaques was found in rAAV8-IL-2 treated APP/PS1AE9 mice (Fig. 6D). In addition, in regions surrounding plaques, astrocytes were hypertrophic with thick proximal processes overlapping with the plaque, suggesting process invasion within plaques in AAV-IL2-treated APP/PS1AE9 mice. In contrast, astrocytes from APP/PS1AE9 mice treated with the control vector exhibited lower hypertrophic processes (data not shown). Littermates treated with Interleukin-2 or Luciferase vectors did not exhibit hypertrophic process.

Interleukin-2 administration activates the JAK/STAT3 pathway in the hippocampus of APP/PS1AE9 mice

We next assessed whether this Interleukin-2 mediated astrocytic activation was correlated with stimulation of the JAK/STAT3 pathway. STAT3 is an important signaling molecule for many cytokines and growth factor receptors that prompts astrocyte reactivity (Chiba et al., 2009; Heim, 1999). Western-blot analysis demonstrated a 2-fold increase in the levels of Stat3-a in APP/PSl ΔΕ9 mice receiving rAAV8-IL-2 compared to APP/PSl ΔΕ9 mice treated with the control vector (Fig. 6E) (p<0.0001). The phosphorylated form of STAT3 [Phospho-STAT3 (Tyr705)] was increased in APP/PSl ΔΕ9 mice treated with the control rAAV8-LUC, relatively to littermates treated with rAAV8-IL-2 or rAAV8-LUC (Fig. 6F). Remarkably, we found a 2.2-fold increase of phospho-STAT3 in Interleukin-2 treated APP/PSl ΔΕ9 mice as compared to APP/PSl ΔΕ9 mice that received the control vector (Luciferase) (Fig. 6F) (p<0.0001). Taken together, these results reveal an increased recruitment of astrocytes around amyloid plaques and the activation of the JAK/STAT3 pathway in APP/PS1AE9 mice treated with Interleukin-2.

Discussion:

Gliosis and inflammation are hallmarks of Alzheimer's disease (Schwab and McGeer, 2008). It is still not clear whether inflammation has a direct or indirect influence on the buildup of Αβ pathology. It has long been considered that the increase of pro-inflammatory mediators would contribute to Alzheimer' s disease progression, thereby implying potential benefit of antiinflammatory immunotherapies (Birch et al., 2014). Likewise, inhibiting the signaling of the pro-inflammatory cytokines IL-12/IL-23 in APPPSl mice decreased glial activation, amyloid load and cognitive decline (Vom Berg et al., 2012). The hippocampal AAV-mediated overexpression of the anti-inflammatory cytokines Interleukin-10 or Interleukin-4 in AD mice enhanced neurogenesis and improved spatial learning and Αβ deposition in APP/PSl mice (Kiyota et al., 2010; Kiyota et al., 2012; Latta et al., 2015). Despite this, two recent studies support a detrimental impact of Interleukin-10 in Alzheimer's disease pathology (Chakrabarty et al., 2015; Guillot-Sestier et al., 2015). Interestingly, hippocampal expression of IL-Ιβ in Alzheimer's disease mice did not result in the expected exacerbation of the amyloid plaque deposition, but instead in plaque improvement (Shaftel et al., 2007). AAV-mediated expression of Interleukin-6 (Chakrabarty et al., 2010) and TNF-a (Chakrabarty et al., 2011) induced massive gliosis that suppressed Αβ deposition. Recently, immune checkpoint blockade directed against the programmed death- 1 (PD-1) pathway evoked an interferon (IFN)-y-dependent systemic immune response leading to clearance of plaques and improved cognitive performance (Baruch et al., 2016). Thus, and surprisingly, it appears that modulation of the immune system towards both effector and regulatory functions may counteract Alzheimer's disease. In this context, there is a strong rationale to investigate the therapeutic effects of Interleukin-2 in Alzheimer's disease in vivo: (i) Interleukin-2 KO mice exhibit impaired learning and memory formation and altered hippocampal development (Petitto et al., 1999); (ii) serum Interleukin-2 levels are low in Alzheimer's disease patients, compared with both elderly and middle-aged subjects (Beloosesky et al., 2002); (iii) Interleukin-2, at low dose, has an antiinflammatory effect (Saadoun et al., 2011). In this report, we show that Interleukin-2 prompts Treg expansion and activation in the brain of APP/PS1AE9 mice and improves Alzheimer's disease pathology. Increased Interleukin-2 concentrations were observed in the hippocampus of Interleukin-2 treated APP/PS1AE9 mice but not in Interleukin-2 treated littermates. Since AAV vectors do not transduce brain cells and Interleukin-2 is produced in the periphery, we may speculate that CNS penetration of peripherally produced Interleukin-2 could be favored in APP/PS1AE9 mice due to BBB leakage. Indeed, the BBB is relatively impermeable in healthy subjects, however compromised in Alzheimer's disease given disruption of the tightly packed endothelial cells that support brain vasculature. Importantly, BBB permeability has been reported in Alzheimer' s disease mouse models (Tanifum et al., 2014) and was shown to increase before plaque formation in Alzheimer's disease mice (Ujiie et al., 2003). This has important implications for the route of administration of therapeutic molecules such as Interleukin-2 (Waguespack et al., 1994).

Tregs have a controversial role in controlling or worsening Alzheimer's disease. Treg depletion was reported to improve Alzheimer's disease in 5XFAD mice (Baruch et al., 2015) or to accelerate the onset of cognitive deficits in APPPS1 mice (Dansokho et al., 2016). In any case, how transient Treg ablation (observed in the periphery but not asserted in the brain) can impact a disease that develops along months is unexplained. Actually, the brief and transient Tregs depletion reported as improving Alzheimer's disease (Baruch et al., 2015) resulted in a marked enrichment of Tregs in the brain three weeks after the last Treg depletion modality (53.4% vs 18.1%) (Baruch et al., 2015). The authors concluded that Tregs recruitment to cerebral sites of Alzheimer's disease pathology may have led to reduction of gliosis and Αβ plaque, with improvement of cognitive functions (Baruch et al., 2015). As efficient Treg depletion in mice leads to rapid (3 to 6 weeks) catastrophic autoimmunity, inflammation and death (Fontenot et al., 2003), this precludes the evaluation of the effects of long-term Treg ablation that might be important for slow-developing diseases. Thus, Treg depletion might not be an optimal method to assess Treg role in Alzheimer's disease. Other investigators (Dansokho et al., 2016) used an anti-CD25 monoclonal antibody to deplete Tregs in vivo in APPPS1 mice. We considered that this strategy was not suitable in our study, as Treg depletion is only transient and CD25 is also expressed by other immune cells such as NK cells, activated B cells, activated effector T cells, myeloid cells, which could induce non expected effects by depleting these populations (Baeyens et al., 2013). In this study, we used the reverse setting and evaluated whether Treg activation and expansion could improve Alzheimer's disease. We administered Interleukin-2 over 5 months and observed increased Treg numbers and activation in the brain, correlating with histological and clinical Alzheimer's disease improvement. Similar observations were also recently reported, although the authors did not assess Tregs in the brain (Dansokho et al., 2016). Thus, altogether, indirect evidences suggest a beneficial role of activated Tregs in Alzheimer's disease. We show that Interleukin-2 -induced Alzheimer's disease improvement is linked to an astrocytic activation. We observed a marked astrocytic activation that is considered as reflecting an attempt to recover from CNS injury (Wegiel et al., 2001 ) . Indeed, astrocytes were proposed to protect neurons by forming a physical barrier around plaques (Wegiel et al., 2001). Interestingly, the astrocytic phenotypic activation was correlated with stimulation of the JAK/STAT3 pathway that was shown to activate astrocytes in models of acute brain injury and is involved in cell growth, neuronal survival and differentiation (Bareyre et al., 2011; Lang et al., 2013). Astrocytes have been described as a potential source of brain Interleukin-2 (Eizenberg et al., 1995). Astrocytes make part of the BBB and may well be the primary brain parenchymal cell type encountered by peripheral Tregs. Indeed, previous reports have described astrocyte/T cells interactions (Barcia et al., 2013). More recently, a close interaction between Tregs and astrocytes has been reported, in which the activation of an Interleukin-2/STAT5 signaling pathway is implicated in an astrocyte-mediated maintenance of Tregs (Xie et al., 2015). Further studies are still needed to better understand the sequential crosstalk between astrocytes and Tregs during Interleukin-2-based therapy.

Increased astrocytic reactivity around amyloid plaques suggests that astrocytes may influence Alzheimer's disease-like pathogenesis through invasion of plaques as an attempt to clear Αβ and limit its extracellular deposition. In line with this finding, mouse astrocytes were reported to degrade amyloid-beta in vitro and in situ (Wyss-Coray et al., 2003), and exogenous astrocytes transplanted into the brain of plaque -bearing Alzheimer' s disease mice, were shown to migrate towards Αβ deposits, internalizing them (Pihlaja et al., 2008). Furthermore, it was shown that attenuating astrocyte activation accelerates plaque deposition in Alzheimer's disease mice (Kraft et al., 2013).

Consistent with these properties of astrocytes, Interleukin-2 treatment and consequent astrocytic activation were accompanied by a reduction of amyloid plaques and a decrease in the Αβ42/Αβ40 ratio (Murray et al., 2012). The strong Αβ40 increase in Interleukin-2 treated APP/PS1AE9 mice is in line with in vitro and in vivo data showing that Αβ40 protects neurons from Αβ42 induced damage in culture and in rat brain (Zou et al., 2003) and inhibits amyloid deposition in Alzheimer's disease mice, protecting them from premature death (Kim et al., 2007). In a different mouse model of Alzheimer's disease (APPPS1 mice), Interleukin-2 was proposed to work by a microglial activation (Dansokho et al., 2016). In our APP/PS1AE9 mice we did not observe such activation. Ibal, arginase-1, TGF-β, IDE and Trem2B detection by western blot were unchanged in the hippocampus. There was no increase of Ibal-positive cells by immunofluorescence around plaques.

We further report that the recovery of memory deficits observed in APP/PS1AE9 mice was supported by a remarkable Interleukin-2-mediated tissue remodeling in the brain characterized by increased synaptic plasticity and restoration of spine density. To our knowledge, this is the first demonstration that in vivo immunomodulatory treatment can actually induce such brain tissue remodeling. These findings are in accordance with previous reports showing that Interleukin-2 promotes survival and neurite extension of cultured neurons as well as enhances dendritic development and spinogenesis (Awatsuji et al., 1993; Sarder et al., 1993; Shen et al., 2010).

Interleukin-2 is an approved drug used for the stimulation of effector cells for the treatment of metastatic melanoma and renal cell carcinoma. In these indications it is given at very high doses (up to 160 MIU per day) and actually poorly used because of severe side effects (Klatzmann and Abbas, 2015). The demonstration that low-dose Interleukin-2 is safe and selectively activates and expands Tregs without activating effector T cells in humans has changed the paradigm for Interleukin-2 therapeutic use. Interleukin-2 is now intensively developed as a stimulant of Tregs at daily dose around 1 to 3 MIU. At these low doses, Interleukin-2 is well tolerated in humans with autoimmune diseases (Castela et al., 2014; Hartemann et al., 2013; He et al., 2016; Saadoun et al., 2011). Noteworthy, these doses lead to increased serum concentration of Interleukin-2 that are in the range of the long-term elevated concentrations observed during pregnancy (Curry et al., 2008). Finally, a pre-clinical study of the long-term effects of Interleukin-2 in mice showed that a yearlong treatment is well tolerated (Churlaud et al., 2014). Thus, long-term treatment with low-dose Interleukin-2 of Alzheimer's disease patients can be envisioned. In summary, our results demonstrate the therapeutic effects of Interleukin-2 in AD mice with established pathology. Although it remains to elucidate the direct and Treg-mediated contribution of Interleukin-2 to Alzheimer's disease improvements, these results warrant investigating the low dose Interleukin-2 for neuroinflammatory diseases. REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Awatsuji, H., et al., 1993. Interleukin-2 as a neurotrophic factor for supporting the survival of neurons cultured from various regions of fetal rat brain. J Neurosci Res. 35, 305-11.

Baeyens, A., et al., 2013. Limitations of IL-2 and rapamycin in immunotherapy of type 1 diabetes. Diabetes. 62, 3120-31.

Barcia, C, Sr., et al., 2013. Imaging the microanatomy of astrocyte-T-cell interactions in immune-mediated inflammation. Front Cell Neurosci. 7, 58.

Bareyre, F.M., et al., 2011. In vivo imaging reveals a phase-specific role of STAT3 during central and peripheral nervous system axon regeneration. Proc Natl Acad Sci U S A. 108, 6282-7.

Baruch, K., et al., 2015. Breaking immune tolerance by targeting Foxp3(+) regulatory T cells mitigates Alzheimer's disease pathology. Nat Commun. 6, 7967.

Baruch, K., et al., 2016. PD-1 immune checkpoint blockade reduces pathology and improves memory in mouse models of Alzheimer's disease. Nat Med.

Beck, R.D., Jr., et al., 2005. IL-2 deficiency results in altered septal and hippocampal cyto architecture: relation to development and neurotrophins. J Neuroimmunol. 160, 146-53.

Beloosesky, Y., et al., 2002. Cytokine levels and phagocytic activity in patients with

Alzheimer's disease. Gerontology. 48, 128-32.

Birch, A.M., Katsouri, L., Sastre, M., 2014. Modulation of inflammation in transgenic models of Alzheimer's disease. J Neuroinflammation. 11, 25.

Castela, E., et al., 2014. Effects of low-dose recombinant interleukin 2 to promote T- regulatory cells in alopecia areata. JAMA Dermatol. 150, 748-51.

Chakrabarty, P., et al., 2010. Massive gliosis induced by interleukin-6 suppresses Abeta deposition in vivo: evidence against inflammation as a driving force for amyloid deposition. FASEB J. 24, 548-59.

Chakrabarty, P., et al., 2011. Hippocampal expression of murine TNFalpha results in attenuation of amyloid deposition in vivo. Mol Neurodegener. 6, 16.

Chakrabarty, P., et al., 2015. IL-10 alters immunoproteostasis in APP mice, increasing plaque burden and worsening cognitive behavior. Neuron. 85, 519-33.

Chiba, T., et al., 2009. Amyloid-beta causes memory impairment by disturbing the JAK2/STAT3 axis in hippocampal neurons. Mol Psychiatry. 14, 206-22. Churlaud, G., et al., 2014. Sustained stimulation and expansion of Tregs by IL2 control autoimmunity without impairing immune responses to infection, vaccination and cancer. Clin Immunol. 151, 114-26.

Curry, A.E., et al., 2008. Maternal plasma cytokines in early- and mid-gestation of normal human pregnancy and their association with maternal factors. J Reprod Immunol. 77, 152-60.

Dansokho, C, et al., 2016. Regulatory T cells delay disease progression in Alzheimer- like pathology. Brain.

Eizenberg, O., et al., 1995. Interleukin-2 transcripts in human and rodent brains: possible expression by astrocytes. J Neurochem. 64, 1928-36.

Farfara D., V. Lifshitz, D. Frenkel, Neuroprotective and neurotoxic properties of glial cells in the pathogenesis of Alzheimer's disease. Journal of cellular and molecular medicine 12, 762-780 (2008).

Ferrari, R., et al., 2014. Frontotemporal dementia and its subtypes: a genome-wide association study. Lancet Neurol. 13, 686-99.

Fontenot, J.D., Gavin, M.A., Rudensky, A.Y., 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol. 4, 330-6.

Galimberti D., E. Scarpini, Progress in Alzheimer's disease. Journal of neurology 259, 201-211 (2012).

Guillot-Sestier, M.V., et al., 2015. 1110 deficiency rebalances innate immunity to mitigate Alzheimer-like pathology. Neuron. 85, 534-48.

Hanisch, U.K., et al., 1997. Neurotoxic consequences of central long-term administration of interleukin-2 in rats. Neuroscience. 79, 799-818.

Hartemann, A., et al., 2013. Low-dose interleukin 2 in patients with type 1 diabetes: a phase 1/2 randomised, double-blind, placebo-controlled trial. Lancet Diabetes Endocrinol. 1, 295-305.

He, J., et al., 2016. Low-dose interleukin-2 treatment selectively modulates CD4(+) T cell subsets in patients with systemic lupus erythematosus. Nat Med. 22, 991-3.

Heim, M.H., 1999. The Jak-STAT pathway: cytokine signalling from the receptor to the nucleus. J Recept Signal Transduct Res. 19, 75-120.

Jankowsky, J.L., et al., 2004. Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific gamma secretase. Hum Mol Genet. 13, 159-70. Kim, J., et al., 2007. Abeta40 inhibits amyloid deposition in vivo. J Neurosci. 27, 627-

33.

Kiyota, T., et al., 2010. CNS expression of anti-inflammatory cytokine interleukin-4 attenuates Alzheimer's disease-like pathogenesis in APP+PSl bigenic mice. FASEB J. 24, 3093-102.

Kiyota, T., et al., 2012. AAV serotype 2/1-mediated gene delivery of anti-inflammatory interleukin-10 enhances neurogenesis and cognitive function in APP+PSl mice. Gene Ther. 19, 724-33.

Klatzmann, D., Abbas, A.K., 2015. The promise of low-dose interleukin-2 therapy for autoimmune and inflammatory diseases. Nat Rev Immunol. 15, 283-94.

Kraft, A.W., et al., 2013. Attenuating astrocyte activation accelerates plaque pathogenesis in APP/PS1 mice. FASEB J. 27, 187-98.

Lacosta, S., Merali, Z., Anisman, H., 1999. Influence of acute and repeated interleukin- 2 administration on spatial learning, locomotor activity, exploratory behaviors, and anxiety. Behav Neurosci. 113, 1030-41.

Lang, C, et al., 2013. STAT3 promotes corticospinal remodelling and functional recovery after spinal cord injury. EMBO Rep. 14, 931-7.

Latta, C.H., et al., 2015. Determining the role of IL-4 induced neuroinflammation in microglial activity and amyloid-beta using BV2 microglial cells and APP/PS1 transgenic mice. J Neuroinflammation. 12, 41.

Leissring, M.A., et al., 2003. Enhanced proteolysis of beta- amyloid in APP transgenic mice prevents plaque formation, secondary pathology, and premature death. Neuron. 40, 1087- 93.

Monje M. L., H. Toda, T. D. Palmer, Inflammatory blockade restores adult hippocampal neurogenesis. Science 302, 1760-1765 (2003).

Murray, P.S., et al., 2012. beta-Amyloid 42/40 ratio and kalirin expression in Alzheimer disease with psychosis. Neurobiol Aging. 33, 2807-16.

Petitto, J.M., et al., 1999. Impaired learning and memory and altered hippocampal neurodevelopment resulting from interleukin-2 gene deletion. J Neurosci Res. 56, 441-6.

Pihlaja, R., et al., 2008. Transplanted astrocytes internalize deposited beta-amyloid peptides in a transgenic mouse model of Alzheimer's disease. Glia. 56, 154-63.

Rogers J., J. Luber-Narod, S. D. Styren, W. H. Civin, Expression of immune system- associated antigens by cells of the human central nervous system: relationship to the pathology of Alzheimer's disease. Neurobiology of aging 9, 339-349 (1988). Rosenzwajg, M., et al., 2015. Low-dose interleukin-2 fosters a dose-dependent regulatory T cell tuned milieu in T1D patients. J Autoimmun. 58, 48-58.

Saadoun, D., et al., 2011. Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis. N Engl J Med. 365, 2067-77.

Sambamurti K., N. H. Greig, T. Utsuki, E. L. Barnwell, E. Sharma, C. Mazell, N. R.

Bhat, M. S. Kindy, D. K. Lahiri, M. A. Pappolla, Targets for AD treatment: conflicting messages from gamma- secretase inhibitors. Journal of neurochemistry 117, 359-374 (2011).

Sarder, M., Saito, H., Abe, K., 1993. Interleukin-2 promotes survival and neurite extension of cultured neurons from fetal rat brain. Brain Res. 625, 347-50.

Sarder, M., et al., 1996. Comparative effect of IL-2 and IL-6 on morphology of cultured hippocampal neurons from fetal rat brain. Brain Res. 715, 9-16.

Schwab, C, McGeer, P.L., 2008. Inflammatory aspects of Alzheimer disease and other neurodegenerative disorders. J Alzheimers Dis. 13, 359-69.

Selkoe D. J., The molecular pathology of Alzheimer's disease. Neuron 6, 487-498 (1991).

Seto, S D.. Kar, R. Quirion, Evidence for direct and indirect mechanisms in the potent modulatory action of interleukin-2 on the release of acetylcholine in rat hippocampal slices. British journal of pharmacology 120, 1151-1157 (1997).

Shaftel, S.S., et al., 2007. Chronic interleukin-lbeta expression in mouse brain leads to leukocyte infiltration and neutrophil-independent blood brain barrier permeability without overt neurodegeneration. J Neurosci. 27, 9301-9.

Shen, Y., et al., 2010. Interleukin-2 enhances dendritic development and spinogenesis in cultured hippocampal neurons. Anat Rec (Hoboken). 293, 1017-23.

Skaper, S.D., 2007. The brain as a target for inflammatory processes and neuroprotective strategies. Ann N Y Acad Sci. 1122, 23-34.

Streit W. J., Microglial response to brain injury: a brief synopsis. Toxicologic pathology 28, 28-30 (2000).

Snyder, E.M., et al., 2005. Regulation of NMDA receptor trafficking by amyloid-beta. Nat Neurosci. 8, 1051-8.

Tancredi V., C. Zona, F. Velotti, F. Eusebi, A. Santoni, Interleukin-2 suppresses established long-term potentiation and inhibits its induction in the rat hippocampus. Brain research 525, 149-151 (1990).

Tanifum, E.A., et al., 2014. Cerebral vascular leak in a mouse model of amyloid neuropathology. J Cereb Blood Flow Metab. 34, 1646-54. Ujiie, M., et al., 2003. Blood-brain barrier permeability precedes senile plaque formation in an Alzheimer disease model. Microcirculation. 10, 463-70.

Vom Berg, J., et al., 2012. Inhibition of IL-12/IL-23 signaling reduces Alzheimer's disease-like pathology and cognitive decline. Nat Med. 18, 1812-9.

Waguespack, P.J., Banks, W.A., Kastin, A.J., 1994. Interleukin-2 does not cross the blood-brain barrier by a saturable transport system. Brain Res Bull. 34, 103-9.

Wang, Z., et al., 2005. Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart. Nat Biotechnol. 23, 321-8.

Wegiel, J., et al., 2001. The role of microglial cells and astrocytes in fibrillar plaque evolution in transgenic APP(SW) mice. Neurobiol Aging. 22, 49-61.

Wyss-Coray, T., et al., 2003. Adult mouse astrocytes degrade amyloid-beta in vitro and in situ. Nat Med. 9, 453-7.

Xie, L., et al., 2015. Cerebral regulatory T cells restrain microglia/macrophage- mediated inflammatory responses via IL-10. Eur J Immunol. 45, 180-91.

Zou, K., et al., 2003. Amyloid beta-protein (Abeta)l-40 protects neurons from damage induced by Abetal-42 in culture and in rat brain. J Neurochem. 87, 609-19.

Claims

CLAIMS:

1. A method of treating Alzheimer's disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a recombinant adeno- associated viral (AAV) vector comprising a polynucleotide encoding for an interleukin 2 (IL-2) polypeptide.

2. The method of claim 1 wherein the polynucleotide encodes for a IL-2 polypeptide comprising an amino acid sequence having at least 90% of identity with SEQ ID NO: l.

3. The method of claim 1 wherein the polynucleotide comprises a sequence having at least 80% of identity with SEQ ID NO:2.

4. The method of claim 1 wherein the administration of the recombinant AAV vector is performed by sublingually, subcutaneously, intramuscularly, intravenously, transdermally delivery.

5. The method of claim 4 wherein the recombinant AAV vector is not administered to the subject by intraventricular or intracerebral injection.

6. The method of claim 4 wherein the recombinant AAV vector is administered to the subject by the intravenous injection.