WO2016171549A1 - Alpha b-crystallin for use in treating glutamate excitotoxicity or tauopathies - Google Patents

Abstract

The present invention describes a compound for use in treating or preventing glutamate excitotoxicity and/or tauopathy in a subject, preferably a disorder selected from the group of epilepsy, spinal cord injury, stroke, traumatic brain injury, Alzheimer's disease, Pick's disease, amyotrophic lateral sclerosis, frontotemporal dementia, frontotemporal lobar degeneration, progressive supranuclear palsy, Parkinsonism linked to chromosome 17, cortical basal degeneration, hearing loss, and in alcoholism or alcohol withdrawal problems, wherein said compound comprises a small heat shock protein having an alpha-crystallin domain, and methods for treating or preventing glutamate excitotoxicity and/or tauopathy in a subject, using said compound.

Description

ALPHA B-CRYSTALLIN FOR USE IN TREATING GLUTAMATE EXCITOTOXICITY OR

TAUOPATHIES

FIELD OF THE INVENTION

The invention is in the field of medicine. In particular, the invention is in the field of medicaments used in the treatment of acute chronic neurodegenerative disorders.

BACKGROUND OF THE INVENTION

Glutamate excitotoxicity is a process by which neurons are damaged and/or killed as the result of excessive stimulation by high levels of extracellular glutamate in the central nervous system (CNS). Glutamate is the most abundant neurotransmitter in the CNS. It is released by a signaling neuron into the synaptic space, and subsequently binds to glutamate receptors of the opposing post-synaptic neuron which

consequently takes over the nerve impulse. In this manner, glutamate controls conduction of nerve impulses that are crucial to a wide range of CNS functions, including cognitive functions like learning and memory.

In order to adequately control these functions as an excitatory signal, levels of glutamate are under tight control in a healthy CNS. During acute or chronic damage to the CNS, however, excessive amounts of glutamate can be released by neurons into the synaptic space. As a result, over- activation of glutamate receptors occurs. Beyond a critical point, such over- activation results in an intracellular calcium overload in neurons which disrupts mitochondrial functions, over-activates intracellular enzyme systems including lipases, proteases and endonucleases, and ultimately leads to cell death. In addition this may cause release of excessive amounts of glutamate from the damaged neuron, which triggers damage to the next neuron.

In this manner, glutamate excitotoxicity is widely believed to propagate neuronal damage and death in a several different acute and chronic pathological conditions of the CNS, including for example epilepsy, spinal cord injury, stroke, traumatic brain injury, Alzheimer's disease, Pick's disease, amyotrophic lateral sclerosis, frontotemporal dementia, frontotemporal lobar degeneration, progressive supranuclear palsy,

Parkinsonism linked to chromosome 17, cortical basal degeneration, hearing loss, and in alcoholism or alcohol withdrawal problems. For this reason, glutamate-mediated excitotoxicity is considered a promising target for therapeutic strategies to ameliorate such disorders. One solution to the problem of glutamate excitotoxicity could be to inhibit production and release of glutamate by neurons into the extracellular synaptic space, or block the interaction of glutamate with its post-synaptic receptors. However, long-term blocking either of these processes is likely to result in serious side effects, since glutamate signaling has multiple excitatory functions in a healthy CNS. While a high level of glutamate causes neuronal death, a normal glutamate level is critical to survival of neurons and many

homeostatic signaling pathways in the CNS. Additionally applying the above blocking strategy is particularly difficult for treating acute trauma or ischemia, given that release of glutamate as the result of such events occurs already within minutes, after which any inhibitory intervention at the level of glutamate secretion or receptor signaling loses its protective value.

Instead, stimulating the removal of excess glutamate from the extracellular space is a more promising solution to control glutamate excitotoxicity. Being intimately associated with synapses, astrocytes play a central role in removing glutamate from synaptic spaces. Glutamate uptake by astrocytes has significance both in regulation of the potentially toxic accumulation of glutamate in the synaptic space, and in recycling glutamate for use as a neurotransmitter. Following intracellular uptake of glutamate by astrocytes, it is converted into glutamine and shuttled back to neurons via specialized transporters. Once in neurons, glutamine is enzymatically converted back to glutamate, which can be utilized for synaptic transmission again. Amongst the several different types of glutamate transporters that are expressed on the surface of human astrocytes for this purpose, the excitotory amino acid transporter 2 (EAAT2) is by far the most important one. In mice, the equivalent transporter is glutamate transporter 1 (GLT1). EAAT2/GLT1 on astrocytes is responsible for the removal of as much as 90% of extracellular glutamate from synapses. This key

contribution is reflected by the fact that disruption of EAAT2/GLT1 functions in laboratory animals rapidly causes excitotoxicity and leads to paralysis, increased susceptibility to acute cortical injury, and even lethal spontaneous seizures. Inducing enhanced expression of EAAT2/GLT1 on astrocytes in the face of glutamate excitotoxicity is therefore expected to exert neuroprotective effects, without compromising routine CNS functions. Indeed, targeted overexpression of GLT1 in mice has been found to exert neuroprotective effects against ischemic brain injury.

The experience with a number of candidate inducers of

EAAT2/GLT1 activity, particularly expression thereof, support the above notion. Several 6-lactam antibiotics have been identified as transcriptional activators of EAAT2/GLT1. Treatment of animals with one such antibiotic, viz. ceftriaxone, leads to an increase of GLT1 expression and glutamate transport activity in the brain. This inhibits neuronal cell death by preventing glutamate excitotoxicity, and exerts marked neuroprotective effects in vitro as well as in vivo. Similar effects have been reported for penicillin G, another β-lactam antibiotic, and for several other substances including cytidine-5'-diphosphocholine (citicoline or CDP-choline), thyroid hormone, 17P-estradiol, maslinic acid, a natural product from the Olea europaea plant, and the anti-breast cancer drugs tamoxifen and raloxifene, selective estrogen receptor modulators. Among their many other biological effects, these substances all induce enhanced expression of EAAT/GLTl, and mediate neuroprotection. More recently, the same has been reported for experimental small-molecule activators of EAAT2/GLT1 translation. Finally, EAAT2/GLT1 induction has also been implicated in the beneficial effects of mesenchymal stem cells in various experimental

neurodegenerative conditions.

Together, these findings demonstrate that enhanced activity of the astrocyte glutamate transporter EAAT2 in humans, and likewise enhanced activity of GLT1 in murine animals, is likely to offer

neuroprotection, and will be a useful strategy to control neuronal death during acute and chronic neurodegenerative conditions.

Therapeutic implementation of current pharmacological options to accomplish increased expression of EAAT2/GLT1 on astrocytes, however, has so far been hampered by the lack of biological selectivity of candidate substances such as the ones listed above. Apart from enhancing the activity of EAAT2/GLT1, the above substances have many more biological activities, which can readily lead to side effects. Secondly, entry into the CNS is another hurdle for therapeutic implementation of EAAT2/GLT1 inducers. In order to exert their desired local effect, therapeutic substances must first cross the blood-brain barrier (BBB) to reach CNS astrocytes. This is often problematic as the BBB excludes many substances from entering the CNS. The challenge thus remains to find a way to selectively induce enhanced astrocytic EAAT2/GLT1 activity, particularly expression thereof, without disrupting many other biological pathways that would cause unwanted side effects.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a compound for use in treating or preventing glutamate excitotoxicity and/or tauopathy in a subject, preferably a disorder selected from the group of epilepsy, spinal cord injury, stroke, traumatic brain injury, Alzheimer's disease, Pick's disease, amyotrophic lateral sclerosis, frontotemporal dementia, frontotemporal lobar degeneration, progressive supranuclear palsy, Parkinsonism linked to chromosome 17, cortical basal degeneration, hearing loss, and in alcoholism or alcohol withdrawal problems. wherein said compound comprises a small heat shock protein having an alpha-crystallin domain. The compound comprising a small heat shock protein having an alpha-crystallin domain in aspects of this invention may be a protein complex of small heat shock proteins comprising an alpha-crystallin domain. Preferably the small heat shock protein having an alpha-crystallin domain is alpha B-crystallin (HspB5). In a preferred embodiment of aspects of this invention the compound is a small heat shock protein comprising an alpha-crystallin domain. Still more preferably the compound is HspB5. The protein complex may for instance have a molecular weight of at least 200 kDa, more preferably at least 300 kDa, still more preferably at least 400 kDa, even more preferably at least 500 kDa, still more preferably approximately between 500 - 800 kDa, and most preferably approximately between 500-600 kDa.

In another aspect, the present invention provides a composition for use in the treatment or prevention of glutamate excitotoxicity and/or tauopathy in a subject, which composition comprises a compound according to the invention as described above and a pharmaceutically acceptable carrier or excipient. Preferably, the composition is in a pharmaceutical formulation suitable for systemic administration, in particular for

intravenous administration.

In another aspect, the present invention provides a method for producing a cell-secreted compound secreted by microglia, monocytes or macrophages that increases the activity of excitatory amino acid

transporter 2 (EAAT2) in human astrocyte cells, or of glutamate transporter 1 (GLT1) in murine astrocyte cells, the method comprising the steps of culturing microglia, monocytes or macrophage cells in culture medium in the presence of an effective amount of a compound comprising a small heat shock protein comprising an alpha-crystallin domain, followed by separating the cells from the secreted compound so as to isolate said compound, and optionally further purifying the secreted compound from the culture medium. In preferred embodiments, of this aspect, the cells are human adult microglial cells, monocytes or macrophages, and/or wherein the effective amount of said small heat shock protein is an amount of 0.1-1000 μg/mL, preferably about 50 μg/mL, in the initial cell culture medium.

In another aspect, the present invention provides a method for treating or preventing glutamate excitotoxicity and/or tauopathy in a subject, the method comprising culturing microglia, monocytes or

macrophages in culture medium in the presence of an effective amount of a compound comprising a small heat shock protein that comprises an alpha- crystallin domain, culturing the cells in the presence of said small heat shock protein for an effective amount of time so as to allow the secretion by said microglia, monocytes or macrophages of a compound, separating the cells from the secreted compound so as to isolate said secreted compound, optionally further purifying the secreted compound from the culture medium, and administering to the subject a therapeutically or

prophylactically effective amount of said secreted compound. In preferred embodiments of this aspect, the cells are human adult cells of the CD 14- expressing myelomonocytic lineage, including microglia, monocytes or macrophage cells, and/or wherein the effective amount of said small heat shock protein is an amount of 0.1-1000 μg/mL, preferably about 50 μg/mL, in the initial cell culture medium.

In another aspect, the present invention provides a method for treating or preventing glutamate excitotoxicity and/or tauopathy in a subject, comprising admininistering to a subject in need thereof a

therapeutically effective amount of a compound comprising a small heat shock protein that comprises an alpha-crystallin domain. Preferably, herein, the compound is alpha B-crystallin (HspB5). DESCRIPTION OF THE DRAWINGS

Figure 1 provides a diagram explaining the therapeutic strategy of methods using HspB5 to counteract glutamate excitotoxicity. By inducing increased expression of the most important astrocyte glutamate transporter EAAT2 in human astrocytes, removal of excess glutamate from the synaptic cleft between two communicating neurons in the CNS is enhanced.

Excessively high glutamate levels may occur during acute or chronic injury or damage to the CNS, and otherwise propagate damage by inducing neuronal injury or death.

Figure 2 illustrates the strong and selective increase in expression of EAAT2 in cultured human astrocytes, 24 h after these are exposed to culture medium harvested from HspB5-activated microglia. These data demonstrate that such induction of EAAT2 is achieved by soluble factors released by HspB5-activated microglia. Similar strong induction of EAAT2 can be obtained by using culture medium of HspB5-activated monocytes or macrophages instead of microglia. The selectivity of the effect is emphasized by the observation that purified TNF-oc, IFN-y or IL-Ιβ do not induce the same effect. Adding only HspB5 to astrocytes also does not cause any induction of EAAT2 since HspB5 is unable to directly activate CD 14- negative astrocytes.

Figure 3 illustrates by immunocytochemical staining the elevated levels of EAAT2 protein expression on the surface of human astrocytes, 24, 48 and 72 h after they have been exposed to culture medium from HspB5- activated microglia, as compared to control medium of cells not stimulated with HspB5.

Figure 4 shows how intracerebral infusion of HspB5 in transgenic P301 mice leads to significantly elevated levels of GLTl, which is selectively induced on astrocytes. Fig. 4E illustrates the quantitative and statistical evaluation of such GLTl induction by HspB5, confirming the statistical significance of this in vivo effect. Figure 5 illustrates how intracerebral infusion of HspB5 in transgenic P301S mice almost completely restores neuronal survival after 3 months. In 5A, it is shown that without HspB5 treatment, transgenic P301S mice that accumulate mutant tau protein develop dramatic neuronal loss especially in the outer cortical layers I and II over the course of 3 months. This is illustrated by comparing neuronal content in such mice with the healthy parent strain C57/BL6. This comparison shows an approximately 65% reduction in cortical areas with detectable signals of the neuronal marker NeuN in 3-month old P301S mice relative to normal mice. In Fig 5B, it is shown that intracerebral infusion of HspB5 into these mice between months 2 and 3 leads to the almost complete restoration of neuronal survival to normal levels.

Fig. 6 illustrates penetration of intravenously administered HspB5 across the BBB into the CNS parenchyma of normal healthy mice. A small covalent fluorescent label, viz. fluorescein isothiocyanate (FITC), was used to trace HspB5 in these experiments. In Figs 6A and B, HspB5-stained brain tissue samples illustrate the readily detectable extravasation of FITC- labeled HspB5 from cerebral blood vessels into brain tissues 90 minutes after intravenous dosing (compare the HspB5-stained sample of 6B with the control sample of 6A). In Fig. 6C-E, the dose-dependent accumulation of HspB5 in mouse brains 90 min after intravenous administration is illustrated by the fluorescent signal of FITC. In Fig. 6F, a more detailed image illustrates the localization of FITC-labeled HspB5 inside endosomal vesicles of microglia. Such localization is strikingly similar in morphology to what is seen after uptake of soluble HspB5 by human microglia in a cell culture model (Fig. 6G). This latter image shows the result of double fluorescent staining of HspB5-fed human microglia for both HspB5 and its signaling receptor TLR2. With a yellow fluorescent signal reflecting co- localization, Fig. 6G illustrates the extensive occurrence of such co- localization between HspB5 (carrying a red fluorescent label) and TLR2 (carrying a green fluorescent label) in endosomal vesicles (as described in more detail by Bsibsi et al, 2014). In Fig. 6 H and 61, results are shown of a quantitative and statistical analysis of the dose-dependent appearance of FITC-HspB5-containing microglial endosomes in mouse brains following intravenous administration of the labelled protein. Different settings in the Image J software were used to capture and quantify microglial endosomes of a certain morphology and pixel size, with very stable results. Data obtained with two of such settings for particle morphology and pixel size are shown. They confirm the statistically highly significant, dose-dependent

accumulation of HspB5 in microglial endosomes after intravenous

administration of the protein. Together, the data of Fig. 6 demonstrate the surprising ability of HspB5 to penetrate the CNS parenchyma after intravenous administration even to healthy mice with a fully functional BBB. They show that the originally free soluble protein is taken up by microglia, and ends up in endosomes where HspB5 meets its signaling receptor TLR1/2. The response thus triggered drives release of a variety of soluble microglial products, which ultimately leads to induction of GLT1/ EAAT2 on nearby astrocytes. DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term "small heat shock protein" as used herein refers to a proteinaceous entity having an amino acid sequence identity of at least about 50%, preferably at least 56%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 85%, still more preferably at least 90%, even more preferably at least 95%, and particularly preferably at least 97% more preferably 98% and most preferably at least 99%, with the alpha-crystallin domain of either one of the small heat shock proteins HspBl, HspB2, HspB3, HspB4, HspB5, HspB6, HspB7, HspB8, HspB9, or HspBlO. Preferably the amino acid sequence identity relates to a region of at least 40 contiguous amino acids, more preferably at least 50, more preferably at least 60, more preferably at least 70, more preferably at least 73, more preferably at least 74, more preferably at least 75, more preferably at least 77, most preferably at least 80 contiguous amino acids. Preferably, said small heat shock protein has an amino acid sequence having a sequence selected from the group of SEQ ID NOs: 2-11 (Table 2).

The term "tauopathy" as used herein refers to a class of

neurodegenerative diseases associated with the pathological aggregation of tau protein in the brain. The best-known of these illnesses is Alzheimer's disease (AD), wherein tau protein is deposited within neurons. Preferably, tauopathies include Alzheimer's disease, Pick's disease, amyotrophic lateral sclerosis, frontotemporal dementia, frontotemporal lobar degeneration, progressive supranuclear palsy, Parkinsonism linked to chromosome 17, and cortical basal degeneration. The neurodegeneration characteristic of tauopathies is associated with glutamate excitotoxicity resulting from an altered glutamate transporter function.

The term "glutamate excitotoxicity" refers to the pathological process by which nerve cells are damaged or killed by excessive stimulation by the neurotransmitter glutamate. This occurs when glutamate receptors such as the NMDA receptor and AMPA receptor are overactivated by a glutamatergic storm. The condition of glutamate excitotoxicity is considered herein a contributory factor to epilepsy, spinal cord injury, stroke, traumatic brain injury, Alzheimer's disease, Pick's disease, amyotrophic lateral sclerosis, frontotemporal dementia, frontotemporal lobar degeneration, progressive supranuclear palsy, Parkinsonism linked to chromosome 17, cortical basal degeneration , hearing loss, and in alcoholism or alcohol withdrawal problems. The term "amino acid sequence similarity" as used herein denotes the presence of similarity between two polypeptides or proteins over the entire length of the protein or molecule. Polypeptides have "similar" sequences if the sequence of amino acids in the two sequences is the same when aligned for maximum correspondence. Sequence comparison between two or more polypeptides is generally performed by comparing portions of the two sequences over a comparison window to identify and compare local regions of sequence similarity. The comparison window is typically from about 10 to 80 contiguous amino acids. The "percentage of sequence similarity" for polypeptides, such as 50, 60, 70, 80, 90, 95, 98, 99 or 100 percent sequence identity, may be determined by comparing two optimally ahgned sequences over a comparison window, wherein the portion of the polypeptide sequence in the comparison window may include amino acid deletions, modification or addition of single amino acids or groups of amino acids as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by: (a) determining the number of positions at which the identical amino acid occurs in both sequences to yield the number of matched positions; (b) dividing the number of matched positions by the total number of positions in the window of comparison; and (c) multiplying the result by 100 to yield the percentage of sequence similarity. The percentage sequence similarity is then determined over the entire length of the molecule. Optimal alignment of sequences for comparison may be conducted by computerized implementations of known algorithms, or by visual inspection. Sequence comparison and multiple sequence alignment algorithms are readily available on the internet, for instance William

Pearson's "LALIGN" program. The LALIGN program implements the algorithm of Huang and Miller, pubhshed in Adv. Appl. Math. (1991)

12:337-357. It can be found at http://www.ch.embnet.org/software/LALIGN form.html.

The term "pharmaceutically acceptable carrier" as used herein refers to a carrier for administration of the composition. The pharmaceutically acceptable carrier can comprise any substance or vehicle suitable for delivering said composition to a therapeutic target. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be one or more optional stabilizers, diluents, or excipients.

As used herein, the terms "therapeutically effective amount" and "pharmaceutically effective amount" as used herein refer to an amount of the compound, composition or substance as described herein effective to treat or preventing glutamate excitotoxicity associated with a tauopathy. Preferably, the compound, composition or substance induces EAAT2/LTR1 expression in astrocytes that results in increased astrocytic glutamate uptake and protects astrocytes and neurons against glutamate toxicity.

The term "therapeutically effective amount" as used herein refers to the quantity of the compound, composition or substance according to the invention necessary to prevent, cure or at least partially arrest the symptoms of the disorder and its complications. Amounts effective to achieve this goal will, of course, depend on the severity of the disease and the weight and general state of the patient.

The term "intravenous" is defined herein as a systemic administration by administering into a vein of the subject.

Embodiments

It has now been found by the present inventors that small heat shock proteins, particularly an alpha B-crystallin such as heat shock protein B5 (HspB5), may be used to enhance astrocytic EAAT2/GLT1 activity, particularly expression of EAAT2/GLT1 protein, both in vitro and in vivo. Small heat shock proteins for use in aspects of this invention include, preferably, small heat-shock proteins comprising an amino acid sequence having a sequence identity of at least 50% to any of the sequences listed as SEQ ID NOs: l and 12-26. Small heat shock proteins for use in aspects of this invention include, preferably, small heat-shock proteins having an amino acid sequence identity of at least 50% to any of the sequences listed as SEQ ID NOs: 2-11. Highly preferred is small heat shock protein B5 (HspB5).

The finding of the present inventors that certain small heat shock proteins may be used to enhance EAAT2/GLT1 activity in astrocytes is therefore surprising, since HspB5 is unable to directly activate astrocytes due to their lack of CD 14. Yet, the present inventors found that by first activating cells of the myelomonocytic lineage that do express CD 14, viz. microglia, monocytes or macrophages, HspB5 causes the release of soluble factors, which in turn induce significantly enhanced expression of both EAAT2 and GLT1 in astrocytes. The above finding is surprising since soluble products secreted by microglia, monocytes or macrophages have never before been implicated in the regulation of EAAT2/GLT1 in

astrocytes. The two growth factors previously suggested to induce

EAAT2/GLT1 as the result of their secretion by neurons, viz. EGF and TGF- a, are not secreted by HspB5-activated microglia, monocytes or

macrophages [supplementary data in Bsibsi et al, 2013 and van Noort et al, 2013]. The present inventors have uncovered this ability of HspB5-activated microglia, monocytes and macrophages to secrete products that stimulate astroglial EAAT2/GLT1 expression.

The present inventors have also found that HspB5 reaches brain or spinal cord microglia after systemic administration, even in fully healthy mammals without any prior disruption of the BBB. Although HspB5 is a relatively small 20-kDa protein, it exclusively exists as an oligomeric protein complex containing around 24 to 30 HspB5 molecules under physiological conditions. Such complexes have a molecular weight of approximately 500- 600 kDa and an estimated diameter of 14 nm [Jehle et al, 2010]. The current understanding of BBB permeability would suggest that under normal conditions, such a large protein complex is unhkely to cross the BBB and enter the CNS to reach the microglia. The present invention

demonstrates that in normal healthy mice, intravenously administered HspB5 nevertheless does enter the CNS in readily detectable quantities, and is subsequently taken up by brain microglia in endosomal

compartments that also contain CD 14 and TLR1/2, allowing HspB5 to trigger activation of microglia. Thus, the present invention surprisingly shows that activation of microglia in the CNS, followed by enhanced activation of EAAT2/GLT1, can be achieved by administering HspB5 systemically, greatly simplifying its therapeutic applicability.

Together, the present findings reveal that small heat shock proteins are suitable candidates to promote EAAT2/GLT1 expression on CNS astrocytes via activation of microgha, monocytes or macrophages. This counteracts the pathogenic effects of glutamate excitotoxicity during acute or chronic neurodegenerative conditions. Validating this notion is the presently disclosed observation that when administered to transgenic mice that accumulate mutant tau protein and otherwise develop dramatic neuronal loss in outer cortical layers, HspB5 almost fully restores neuronal survival.

Accordingly, in one aspect, the present invention provides a compound for use in treating or preventing glutamate excitotoxicity associated with a tauopathy, particularly a disorder selected from the group of Alzheimer's disease, Pick's disease, amyotrophic lateral sclerosis, frontotemporal dementia, frontotemporal lobar degeneration, progressive supranuclear palsy, Parkinsonism linked to chromosome 17, or cortical basal degeneration.in a subject. The compound is preferably a small heat shock protein comprising an alpha-crystallin domain.

Without wishing to be bound by any theory, it is considered that the compound comprising a small heat shock protein having an alpha- crystallin domain is not acting on the astrocytes directly, but that it induces the release from microglia, monocytes or macrophages of the subject of a factor or secretion product that acts on the astrocytes and stimulates the activity glutamate transporter in these astrocytes, thereby reducing the extracellular glutamate concentrations and preventing or treating

glutamate excitotoxicity.

The ability of a small heat shock protein to induce secretion of

EAAT2/GLT1 -inducing factors by microglia, monocytes or macrophages may be tested by using the methods as described in Example 1 herein below. While in the experimental part, the small heat shock protein HspB5 is used, it is envisioned herein that also other small heat shock proteins having the alpha-crystallin domain may be used in aspects of this invention. The soluble factors secreted by the microglia, monocytes or macrophage cells may be isolated and or purified form the cell culture medium following activation of the cells with HspB5.

In a preferred embodiments of aspects of this invention, the small heat shock protein is an alpha-crystallin, in particular alpha B-crystallin (HspB5).

The compound for use in aspects of this invention preferably comprises a protein complex of the small heat shock protein as indicated above, wherein, in preferred embodiments the protein complex has a molecular weight of at least 200 kDa, particularly at least 300 kDa, more particularly at least 400 kDa, more particularly at least 500 kDa, preferably approximately between 500 - 800 kDa, more preferably approximately between 500-600 kDa.

In another aspect, the present invention provides a composition for use in treating or preventing glutamate excitotoxicity in a subject, which composition comprises a compound comprising a small heat shock protein having an alpha-crystallin domain, said composition may further comprise a pharmaceutically acceptable carrier or excipient. In a preferred embodiment of this aspect, the composition for use according to the invention is in a pharmaceutical formulation suitable for systemic administration, in particular for intravenous administration.

The present invention also provides a method for producing a substance capable of increasing activity of excitatory amino acid transporter 2 (EAAT2) in human astrocyte cells or increasing activity of glutamate transporter 1 (GLT1) in murine astrocyte cells, wherein the method comprises culturing small heat shock protein-activated microglia, monocytes or macrophage cells in culture medium followed by harvesting the culture medium comprising the substance, and optionally isolating and purifying the substance from the culture medium.

The ability of a substance to increase activity of EAAT2/GLT1 may be tested as described in Example 1 herein below, e.g. by exposing the astrocytes to the substance in suitable culture medium followed by

harvesting of the astrocyte cells and examining those cells for expression levels of EAAT2/GLT1 gene transcripts.

In a preferred embodiment of this aspect, the method comprises activation of the microglia, monocytes or macrophages cells by culturing the cells in culture medium comprising a compound according to the first aspect.

In a further preferred embodiment of this aspect, the method produces a substance that is capable of increasing EAAT2/GLT1 expression in human/murine astrocyte cells. Although an increase in expression of EAAT2/GLT1 in astrocyte cells is preferred, an increased activity of the EAAT2/GLT1 receptors in uptake of glutamate, for instance via

posttranslational modifications of the EAAT2/GLT1 protein, is also contemplated in the present invention.

In a further preferred embodiment of this aspect, the method produces a substance that is capable of increasing EAAT2/GLT1 protein expression in human/murine astrocyte cells. In a another aspect, the present invention provides a method for increasing EEAT2/GLT1 protein expression in a cell or in a subject, comprising administering to the cell or subject a therapeutically or prophylactically effective amount of the compound comprising the small heat-shock protein as defined herein.

In a another aspect, the present invention provides a method for treating a neurodegenerative disease or condition, in particular a disease or condition selected from epilepsy, spinal cord injury, stroke, traumatic brain injury, Alzheimer's disease, Pick's disease, amyotrophic lateral sclerosis, frontotemporal dementia, frontotemporal lobar degeneration, progressive supranuclear palsy, Parkinsonism linked to chromosome 17, cortical basal degeneration , hearing loss, and in alcoholism or alcohol withdrawal problems, in a subject comprising administering to the subject a

therapeutically or prophylactically effective amount of the compound comprising the small heat-shock protein as defined herein.

In preferred embodiments of aspects of this invention the administration route is intravenous administration.

The present invention provides a compound, composition or substance for use in a method for treating or preventing glutamate excitotoxicity in a subject, wherein the compound, pharmaceutical composition or substance is administered at a dosage range that results in a serum concentration of the heat shock protein that induces microglia, monocytes or macrophages to secrete factors in effective amounts to enhance EAAT2/GLT1 expression in astrocytes.

The pharmaceutical composition may be in the form of a capsule, tablet, lozenge, dragee, pill, droplet, suppository, powder, spray, vaccine, ointment, paste, cream, inhalant, patch, aerosol, as a drip, an infusion, injection, and the like. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Yet in another alternative embodiment, the compound comprising a small heat shock protein having an alpha-crystallin domain or compositions of the invention may be administered from a controlled or sustained release matrix inserted in the body of the subject.

Further to the compound comprising a small heat shock protein having an alpha-crystallin domain, the pharmaceutical composition of the invention may comprise a suitable pharmaceutically acceptable carrier, and optionally additional pharmaceutically acceptable excipients.

For therapeutic treatment, the compound comprising a small heat shock protein having an alpha-crystallin domain may be administered to a subject by any suitable route, preferably in the form of a pharmaceutical composition adapted to such a route and in a dosage that is effective for the intended treatment. Therapeutically effective dosages of the compound comprising a small heat shock protein having an alpha-crystallin domain required for treating the disorder, for instance for prevention and/or treatment of tauopathy in the body of a human or animal subject, can easily be determined by the skilled person, for instance by using animal models. One suitable animal model is described in Example 2, below, wherein the transgenic P301S mouse model of tauopathy induced by the accumulation of mutant tau-protein is described, and wherein the compound comprising a small heat shock protein having an alpha-crystallin domain induced an almost completely restoration of neuronal survival in vivo.

A preferred dosage range in aspects of this invention results, in one embodiment, in a peak serum concentration of a compound comprising a small heat shock protein having an alpha-crystallin domain of between 0.1 and 100 μg/ml, more preferably between 1 and 10 μg/ml, more preferably around 3-5 μg/ml in the subject. In another embodiment, in a peak serum concentration of a compound comprising a small heat shock protein having an alpha-crystallin domain of between 10 and 500 μg/ml, more preferably between 10 and 100 μ£/ηύ, more preferably around 10-50 μ£/ιη1 in the subject.

The compound comprising a small heat shock protein having an alpha-crystallin domain may be administered in an amount of about 0.01-10 mg/kg body weight per day.

The compound comprising a small heat shock protein having an alpha-crystallin domain may be administered as a single unit dose of for instance 0.5-1000 mg, preferably 1-100 mg, more preferably 5-50 mg, still more preferably about 10-15 mg, of small heat shock protein once per day.

The current invention allows for the preferred intravenous delivery of the compound comprising a small heat shock protein having an alpha-crystallin domain, preferably in the form of a solubilized liquid formulation such as in the form of an injectable. When applied to humans, intravenous delivery has the advantage of allowing maximum dose control, circumventing the proteolytic environment of the stomach, and

circumventing any variations or complications that may be caused by the heterogeneous composition of the gut microflora amongst humans.

For the purpose of clarity and a concise description, features are described herein as part of the same or separate aspects and preferred embodiments thereof, however, it will be appreciated that the scope of the invention may include embodiments comprising combinations of all or some of the embodiments and features described unless explicitly otherwise specified.

The invention will now be illustrated by the following examples, which are provided by way of illustration and not of limitation. It will be understood that many variations in the methods described and the amounts indicated can be made without departing from the spirit of the invention and the scope of the appended claims.

EXAMPLES Example 1. HspB5-mediated activation of human microglia results in the release of soluble factors that induce strongly enhanced EAAT2 expression on human astrocytes. Methods

Post-mortem brain tissue samples were obtained in collaboration with the Netherlands Brain Bank in Amsterdam, with the written approval of the medical ethical committee of the VU University Medical Center in Amsterdam. All patients and control donors had given informed consent for autopsy and use of their brain tissue for research purposes. Tissue samples from subcortical white matter were obtained from control cases to isolate and culture microglia as well as astrocytes.

Human adult microglia were isolated as previously described [Bsibsi et al, 2013, 2014]. Briefly, tissue samples were dissected from subcortical white matter and visible blood vessels were removed. After a 20-min digestion in 0.25 % (w/v) trypsin (Sigma, St. Louis, MO), the cell suspension was gently triturated and washed with DMEM/HAM-FIO medium

containing 10 % (v/v) FCS and antibiotic supplements. After passage through a 100-μιη filter, myelin was removed by Percoll gradient

centrifugation. Erythrocytes were lysed by 15-min incubation on ice with 155 mM NH₄C1, 1 mM KHCO3 and 0.2 % (w/v) bovine serum albumin.

Microglia were cultured at 37 °C under 5% CO2 in a humidified stove.

Recombinant human granulocyte-macrophage colony stimulating factor (PeproTech Inc, Rocky Hill, NJ) was added to microglial cultures every 3 days at a final concentration of 20 μg/mL to promote proliferation and survival. The purity of the cultured microglia was verified by staining for CD68 before cell stimulation and RNA isolation. Generally, microglia cultures were found to be > 99 % pure. Activation of microglia by HspB5 at a final concentration of 50 μg/mL was performed using sterile clinical- grade recombinant human HspB5 (Delta Crystallon BV, Leiden, The Netherlands). Culture supernatants of microglia were harvested 24 h after the addition of HspB5.

Astrocytes were isolated from human brain material as previously described [Bsibsi et al., 2002]. Briefly, brain tissues dissected from corpus callosum or subcortical white matter was collected and blood vessels were removed. After a 20-min digestion in trypsin and DNAse, the cell suspension was gently triturated and washed with DMEM/HAM-F10 medium

containing 10% FCS and antibiotic supplements. After passage through a 100-μιη filter, myelin was removed by Percoll gradient centrifugation.

Erythrocytes were lysed by a 15-min incubation on ice with 155 mM NH₄C1, 1 mM KHCO3, 0.2% BSA. Primary cultures of astrocytes were set up in DMEM/HAM-F 10 medium containing 10% FCS and antibiotic supplements. Purity of the astrocyte cultures was verified by staining for glial fibrillary acidic protein (GFAP) as a positive marker for astrocytes and CD68 as a negative control marker for microglia. Astrocytes cultures used were at least 99% pure.

Purified astrocyte cultures were stimulated with either freshly prepared microglial culture medium, or supernatant from HspB5-stimulated microglia. Either non-conditioned or conditioned medium was diluted 1: 1 into astrocyte culture medium, and astrocytes were subsequently harvested and examined for expression levels of various gene transcripts including EAAT2 after 4, 24 or 48 h. As controls, parallel astrocyte cultures were stimulated with purified HspB5 alone (50 μg/mL), IL-10, IFN-γ, IL-Ιβ or TNF-oc. These individual cytokines were each added to a final concentration of 2 ng/mL, a levels that approximately reflects the maximum

concentrations of cytokines found in the microglia culture supernatants.

For real-time PCR analysis of astrocyte transcripts, total cellular RNA was isolated using TRizol® , reverse transcribed into cDNA and levels of different genes, including β-actin as a reference, were determined by real- time PCR (RT-PCR) using SYBR®green. The primers used (Biolegio, Nijmegen, The Netherlands) for b-actin were 5'-GGTCATCACCATTGGCAA- 3' (sense) and 5'-ACGTCACACTTCATGATG-3' (anti-sense), and for EAAT2 5'- TTCCCTGAAAACCTTGTCCA-3'(sense) and 5'- GGTGGTGCAACCAGGA CTTT-3'(anti-sense)

Quantitation of cytokines in microglial culture supernatants was performed using commercially available ELISA kits according to the manufacturer's instructions (eBioscience, San Diego, CA, and Sanquin, Amsterdam, the Netherlands).

Human astrocytes grown on chamber-slides were rinsed with phosphate-buffered saline (PBS) and fixed with 4 % formaldehyde, for 10 min at room temperature. To permeabihze the cells and to block non-specific protein-protein interactions, astrocytes were incubated in 1 % bovine serum albumin/10 % normal goat serum/0.3 M glycine in 0.1 % Tween in PBS for 1 h and then incubated overnight at 4 °C with 5 μg/ml anti-EAAT2 antibody (Abeam) diluted in 5 % normal goat serum, then washed in PBS and incubated for 1 h with Goat anti rabit Alexa fluor-594 (H+L) (Life

Technologies). After washing in PBS cells were mounted with vectashield and counterstained with 4',6-diamidino-2-phenylindole. Results

Fig. 2 illustrates how exposure of cultured human astrocytes to factors secreted by HspB5-activated human microglia leads to a dramatic, 80-fold increase in the levels of EAAT2- encoding mRNA after 24 h. In Fig. 3, it is illustrated by staining astrocytes for EAAT2 how surface protein levels of EAAT2 reflect this increase. No such induction of EAAT2 was observed when astrocytes were given non-conditioned fresh microglial culture medium, or several individual cytokines, some of which are known to be contained in this medium, such as IL-10, TNF-a, and IL-6. When supplied to astrocyte cultures, none of these factors induce similar levels of EAAT2 as found with the conditioned medium. Thus, the nature of microglial factors that are released by activation with HspB5 and are responsible for the EAAT2 -inducing effect on astrocytes remains to be clarified. Similar or even stronger induction of EAAT2 was obtained when astrocytes were stimulated with compounds secreted into the culture medium by purified human monocytes or macrophages activated with HspB5.

Example 2. Intrathecal administration of HspB5 to mutant tau P301S -expressing transgenic mice leads to selective induction of astroglial GLT1, and to almost complete restoration of neuronal survival.

Methods

For the experiment, transgenic mice were used that express the P301S mutant form of tau protein (Hampton et al, 2010). In these P301S mice, accumulation of mutant tau leads to progressive neuronal

degeneration. P301S mice 8 weeks of age were anesthetized with isofluorane combined with nitrous oxide, and positioned into a stereotaxic frame (David Kopf Instruments, Tujunga, CA). Once animals were anesthetized and the skull was exposed, a 1-mm burr hole was drilled 0.75 mm laterally from the midline and 1.2 mm forward from the bregma. By modifying the Alzet brain infusion kit 3 by cutting the cannula and using only one spacer, the infusion cannula was positioned approximately 0.5 mm deep, resting onto the surface of the cortex.

Alzet micro-osmotic pumps model 1004 (Durect Corporation,

Cupertino, CA) were filled with 100 μΐ of a 12.5 mg/mL solution of

recombinant human HspB5 in phosphate-buffered saline (PBS) (Delta Crystallon, Leiden, The Netherlands), or PBS only. Once pumps were filled, they were primed for 48 h in a saline solution at 37°C according to the manufacturer's instructions, before being surgically implanted under the skin along the animals hind flank. The tubing was connected to the cannula, resulting in infusion of about 2.5 μΐ_^ of the solution per day onto the surface of the cortex, for a period of 28 days. Animals were treated in two separate experiments, with a final group size of 8 HspB5-teated animals, 5 PBS control animals, and 3 'cannula alone' control animals.

Animals were euthanized 4 weeks post cannulation at 12 weeks of age by lethal intraperitoneal injection of sodium pentobarbitone (Euthatal) at 3 mL/g bodyweight, transcardially perfused and fixed using ice-cold PBS followed by 4% paraformaldehyde in PBS. Brains were removed, postfixed in 4% paraformaldehyde overnight, cryoprotected in 25% sucrose solution, and frozen in tissue-tec. Crysections were cut coronally (25 μηι), mounted onto superfrost-plus glass slides (VWR International, East Grinstead, UK) and stored at -80°C. For immunohistochemistry, cryosections were defrosted, air-dried and washed in PBS before being blocked with 0.2% Triton X100 in PBS (TX-PBS) containing using 3% normal goat or horse serum, depending on the secondary antibody used. Primary antibodies were applied overnight in PBS or TX-PBS containing 1% serum. They included biotinylated monoclonal mouse anti-neuronal nuclear antigen (NeuN) (1:400; Millipore Bioscience Research Reagents, Watford, UK), polyclonal rabbit anti-glial fibrillary acidic protein (GFAP) (1: 1,000; DAKO, Ely, UK), monoclonal mouse anti-GFAP (clone GA5-Cy3; 1:500; Sigma, Poole, UK), polyclonal rabbit anti-glutamate transporter-1 (GLT1) (1: 150; Alpha Diagnostics international, San Antonio, TX), and polyclonal rabbit anti-calbindin (1:50; AbCam, Cambridge, UK). After several washes in PBS, secondary

antibodies were added for 2 h in TX-PBS or PBS alone, containing 1% serum and bis-benzamide (Sigma Aldrich, 1:4,000). Secondary antibodies used were labelled with Alexa-488,-555 or -47 fluorescent probes (Molecular Probes/Invitrogen, UK, 1:1,000). Slides were washed in PBS followed by 2 final washes in Tris buffered non-saline before being mounted using fluorosave reagent (Calbiochem, Nottingham, UK). For analysis, Axiovision 4.8 (Zeiss microsystem, Cambridge, UK) was used to collect images on a Zeiss Axiovision upright microscope or Apotome system, or Zen 2009 software was used in conjunction with a Zeiss LSMZIO confocal microscope. Images were taken as z-stacks and max -projected prior to analysis. All images within a dataset were captured and handled identically for quantitative analysis. Zen blue or axiovision (Zeiss) or

SigmaScan Pro 5.0 (SPSS, Chicago, IL) software were used for subsequent quantitative density measurements and cell counts. GFAP-GLT1 double- positive cells were counted in a 155 x 155 μιη grid throughout the superficial cortex, using between 4 and 8 images per animal. Calbindin-positive cell counts were generated from a 327 x 150 μιη grid, and all counts were scaled up to generate mean numbers of cells per 1 mm² of tissue surface area.

Graphs were generated using graphing software (Sigma Plot 11), and statistical ANOVA tests were performed using a Tukey post-hoc test

(SigmaStat), using a threshold of p <0 .05 for statistical significance.

Results

The extent of neuronal loss observed in the present experiment in P301S mice was fully in line with previously published data (Hampton et al., 2010). After 3 months, untreated transgenic P301S mice had suffered marked neuronal loss in the outer cortical layers I and II. As compared to healthy control mice of the same C57/BL6 background, about 30% of all neurons in these areas had died, and the mean area in these cortical layers containing a readily detectable immunohistochemical signal for NeuN, a neuronal marker, had decreased by about 65% (see Fig. 5A). Clearly, 3 months of gradual accumulation of mutant tau protein in P301S mice results in dramatic neuronal death in the outer cortical layers.

Fig. 4 illustrates how intracerebral infusion of HspB5 into P301S mice results in significantly increased expression of the main astrocyte glutamate transporter GLTl, while Fig 5 illustrated how this results in the almost complete preservation of neurons. In Fig. 4, representative examples are shown of tissue samples taken from the outer cortical layers, after staining for GFAP and GLT1. Figs. 4A and B shows the result for control mice, while Fig. 4C and D shows the result for mice that had received an infusion of HspB5, highlighting enhanced expression of GLT1 and its astrocytic localization. In Fig. 4E, expression of astrocytic GLT1 in the outer cortical layers is quantified as GFAP/GLT1 -double positive astrocytes per surface area, illustrating the significant, more than 3-fold increase in mice that had received HspB5 relative to control mice.

Fig 5 summarizes immunohistochemical analyses that were

performed to evaluate neuronal survival, using the neuronal marker NeuN. Similar results were obtained using calbindin or gamma-aminobutyric acid as a neuronal marker (not shown). In Fig. 5A, it is illustrated how after 3 months, accumulation of mutant tau protein leads to a dramatic, 65% loss of cortical areas with readily detectable NeuN⁺ neurons in P301 mice as compared to C57/BL6 mice with the same genetic background. This loss is rather selectively observed in the outer cortical layers I and II. In Fig. 5B, it is illustrated that intracerebral administration of HspB5 over a period of 4 weeks, but not PBS, results in almost full restoration of normal neuronal counts in all cortical layers after 3 months.

Example 3. Intravenous administration of HspB5 to normal healthy mice allows its entry into the CNS, and subsequent HspB5-mediated activation of microglia in the CNS parenchyma.

Methods

C57/BL6 mice, 8-9 weeks of age (Janvier, France) were used for the experiment. They were housed and treated in compliance with all applicable local legal and ethical regulations throughout the study. Clinical grade recombinant human HspB5 (Delta Crystallon BV, The Netherlands) was labelled with fluorescein isothiocyanate (FITC) using the labelling kit of Thermo Scientific (Rockford, IL), according to the manufacturer's

instructions. Adequate labeling was verified by examining the absorbance spectrum of the resulting FITC-labelled HspB5 solution. The observed A495/A280 ratio of approximately 1 was taken as a sign of adequate labelling. Protein concentration in the resulting solution was determined using a bicinchoninic acid (BCA) assay, and adjusted to 1 mg/mL with PBS.

Mice (N=3) received a single intravenous dose of either 20 or 100 μg HspB5 in a final volume of 100 μΐ_^ phosphate-buffered saline, while control mice were given no injection at all. Given the half -life of intravenous HspB5 in serum of 1-2 h, all mice were terminated after 90 min, transcardially perfused with 2 mL ice-cold PBS, and brains were carefully dissected, snap- frozen in liquid nitrogen, embedded in optimal cutting temperature (OCT) compound, and frozen in a non-fixed state at -80°C. Frozen brain tissue sections (10 pm) were air dried, and immediately examined for fluorescence using a Leica DMI 6000B fluorescence microscope working station, equipped with a DFC 365FX camera (Leica, Germany). Of each animal, five regions of brain tissue were evaluated, randomly selected by the software controlling the microscope, and all acquired with the same setting. Images were processed by Image J to produce various numerical representations of the data. Results for different treatment groups, all obtained using the same Image J settings, were compared and tested for statistical significance by ANOVA followed by Dunnet's multiple comparison test. A p-value < 0.05 was considered significant. Several frozen and subsequently fixed brain sections were additionally subjected to immunohistochemical staining for HspB5 using a monoclonal antibody against human HspB5 (JAMOl; Delta Crystallon BV, The Netherlands).

Results Figs. 6A and 6B show representative brain samples of mice stained with a monoclonal antibody against HspB5. They clearly reveal direct passage of HspB5 from within small blood vessels into the CNS

parenchyma, 90 min following an intravenous dose of 100 μg of protein. Fig. 6C-E show a series of representative fluorescence micrographs of brain sections of mice, collected 90 min after receiving either no FITC-HspB5 (6C), or intravenous doses of 20 μg (6D) or 100 μg (6E) of FITC-HspB5. They illustrate the marked dose-dependent increase in fluorescence intensity in the brain parenchyma of mice receiving FITC-labelled HsopB5, most clearly visible in case of the highest i.v. dose of 100 μg. Especially in cortical areas, the largely vesicular localization of FITC-HspB5 signals is fully consistent with the expected rapid uptake of free soluble protein within the brain parenchyma by microglia. The detailed image of Fig. 6F illustrates this, and emphasizes the striking morphological similarity of fluorescent endosomal vesicles in mouse microglia with those found in cultured human microglia after pulsing them with free soluble HspB5. This latter condition is illustrated in Fig. 6G, which shows cultured human microglia that were fed soluble HspB5, and were subsequently double stained for HspB5 (red fluorescent signal) and TLR2 (green fluorescent signal) [Bsibsi et al, 2014]. By confocal laser scanning microscopy, co-localization of HspB5 and its signaling receptor TLR2 is reflected by a yellow signal. This signal is clearly present in cultured human microglia, and morphologically identical to the fluorescent signal in the mouse microglia of Fig. 6F.

Figure 6H and 61 provide a quantitation and statistical evaluation of microglial endosomes containing HspB5 in mice given an intravenous dose of the protein. Using Image J, a number of different threshold algorithms for signal strength were combined with lower and upper threshold values for pixel size to evaluated the present of FITC-positive endosomes in microglia. More particularly, the algorithms "Maximum Entropy", "YEN", "Triangle", "Moments", and "Renji Entropy" were combined with lower threshold values of 1, 3 or 5 pixels, and an upper threshold value of 5,000 pixels for particle size, and the outcome of each combination was evaluated. Confirming the robustness of the data, the above different modes of evaluating the result tended to produce very similar outcomes. The algorithms "Maximum

Entropy" and "YEN" combined with a pixel range of 3 to 5,000 for size demonstrated the greatest sensitivity. In Fig 6H and 61, the outcomes obtained using these two different modes of analyses are illustrated.

Importantly, no pre-selection was made of the brain regions used for these evaluations. Fig. 6H and I reflect average data collected from 5 randomly selected brain regions from each of the three animals in the different treatment groups, including cortical regions that contained many vesicles as well as white mater regions with much fewer positive vesicles. The result of this unbiased approach clearly demonstrating a highly significant dose- dependent increase in the number of microglial vesicles containing HspB5 following intravenous administration of the protein.

Apart from the number of FITC-HspB5-positive vesicles, also the mean integrated fluorescence signal density of FITC-HspB5 over randomly selected brain regions was calculated for each of the treatment groups (not shown here). This evaluation also revealed a highly significant increase in signal densities in animals that had received intravenous FITC-HspB5 relative to untreated control animals.

References

• Bsibsi M et al (2002) Broad expression of Toll -like receptors in the

human central nervous system. J Neuropathol Exp Neurol 61: 1013- 1021.

• Bsibsi M et al (2013) Alpha-B-crystallin induces an immune-regulatory and antiviral microglial response in preactive multiple sclerosis lesions. J Neuropathol Exp Neurol 72: 970-979. Bsibsi M et al (2014) Demyelination during multiple sclerosis is associated with combined activation of microglia/macrophages by IFN-γ and alpha B-crystallin. Acta Neuropathol 128: 215-229.

Hampton DW et al (2010) Cell-mediated neuroprotection in a mouse model of human tauopathy. J Neurosci 30: 9973-9983.

Jehle S et al (2010) Solid-state NMR and SAXS studies provide a structural basis for the activation of alphaB-crystallin oligomers. Nat

Struct Mol Biol 17: 1037-1042.

van Noort JM et al (2013) Activation of an immune-regulatory macrophage response and inhibition of lung inflammation in a mouse model of COPD using heat-shock protein alpha B-crystallin-loaded PLGA microp articles. Biomaterials 34: 831-840.

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<400> 11

Met Ala Ala Leu Ser Cys Leu Leu Asp Ser Val Arg Arg Asp lie Lys 1 5 10 15

Lys Val Asp Arg Glu Leu Arg Gin Leu Arg Cys lie Asp Glu Phe Ser

20 25 30

Thr Arg Cys Leu Cys Asp Leu Tyr Met His Pro Tyr Cys Cys Cys Asp

35 40 45

Leu His Pro Tyr Pro Tyr Cys Leu Cys Tyr Ser Lys Arg Ser Arg Ser 50 55 60

Cys Gly Leu Cys Asp Leu Tyr Pro Cys Cys Leu Cys Asp Tyr Lys Leu 65 70 75 80

Tyr Cys Leu Arg Pro Ser Leu Arg Ser Leu Glu Arg Lys Ala He Arg

85 90 95

Ala He Glu Asp Glu Lys Arg Glu Leu Ala Lys Leu Arg Arg Thr Thr

100 105 110

Asn Arg He Leu Ala Ser Ser Cys Cys Ser Ser Asn He Leu Gly Ser

115 120 125

Val Asn Val Cys Gly Phe Glu Pro Asp Gin Val Lys Val Arg Val Lys 130 135 140

Asp Gly Lys Val Cys Val Ser Ala Glu Arg Glu Asn Arg Tyr Asp Cys 145 150 155 160

Leu Gly Ser Lys Lys Tyr Ser Tyr Met Asn He Cys Lys Glu Phe Ser

165 170 175

Leu Pro Pro Cys Val Asp Glu Lys Asp Val Thr Tyr Ser Tyr Gly Leu

180 185 190

Gly Ser Cys Val Lys He Glu Ser Pro Cys Tyr Pro Cys Thr Ser Pro

195 200 205

Cys Ser Pro Cys Ser Pro Cys Ser Pro Cys Asn Pro Cys Ser Pro Cys 210 215 220

Asn Pro Cys Ser Pro Tyr Asp Pro Cys Asn Pro Cys Tyr Pro Cys Gly 225 230 235 240

Ser Arg Phe Ser Cys Arg Lys Met He Leu

245 250

<210> 12

<211> 81

<212> PRT

<213> Homo sapiens <220>

<221> MISC-FEATURE

<222> (1) .. (81) <223> HSPB1 a-Crystallin domain <400> 12 lie Arg His Thr Ala Asp Arg Trp Arg Val Ser Leu Asp Val Asn His 1 5 10 15

Phe Ala Pro Asp Glu Leu Thr Val Lys Thr Lys Asp Gly Val Val Glu

20 25 30 lie Thr Gly Lys His Glu Glu Arg Gin Asp Glu His Gly Tyr lie Ser

35 40 45

Arg Cys Phe Thr Arg Lys Tyr Thr Leu Pro Pro Gly Val Asp Pro Thr 50 55 60

Gin Val Ser Ser Ser Leu Ser Pro Glu Gly Thr Leu Thr Val Glu Ala 65 70 75 80

Pro

<210> 13

<211> 81

<212> PRT

<213> Homo sapiens

<220>

<221> MISC-FEATURE

<222> (1) .. (81)

<223> HSPB2 a-Crystallin domain <400> 13

Leu Arg Leu Ser Glu Gly Lys Phe Gin Ala Phe Leu Asp Val Ser His 1 5 10 15

Phe Thr Pro Asp Glu Val Thr Val Arg Thr Val Asp Asn Leu Leu Glu

20 25 30

Val Ser Ala Arg His Pro Gin Arg Leu Asp Arg His Gly Phe Val Ser 35 40 45

Arg Glu Phe Cys Arg Thr Tyr Val Leu Pro Ala Asp Val Asp Pro Trp 50 55 60

Arg Val Arg Ala Ala Leu Ser His Asp Gly lie Leu Asn Leu Glu Ala 65 70 75 80

Pro

<210> 14

<211> 77

<212> PRT

<213> Homo sapiens

<220>

<221> MISC-FEATURE

<222> (1) .. (77)

<223> HSPB5 a-Crystallin domain fragment

<400> 14

Arg Leu Glu Lys Asp Arg Phe Ser Val Asn Leu Asp Val Lys His Phe 1 5 10 15

Ser Pro Glu Glu Leu Lys Val Lys Val Leu Gly Asp Val lie Glu Val

20 25 30

His Gly Lys His Glu Glu Arg Gin Asp Glu His Gly Phe lie Ser Arg

35 40 45

Glu Phe His Arg Lys Tyr Arg lie Pro Ala Asp Val Asp Pro Leu Thr 50 55 60 lie Thr Ser Ser Leu Ser Ser Asp Gly Val Leu Thr Val

65 70 75

<210> 15

<211> 77

<212> PRT

<213> Homo sapiens <220>

<221> MISC-FEATURE

<222> (1) .. (77)

<223> HSPB3 a-Crystallin domain fragment <400> 15

Arg Glu Gly Lys Ser His Phe Gin lie Leu Leu Asp Val Val Gin Phe 1 5 10 15

Leu Pro Glu Asp lie lie lie Gin Thr Phe Glu Gly Trp Leu Leu lie

20 25 30

Lys Ala Gin His Gly Thr Arg Met Asp Glu His Gly Phe lie Ser Arg

35 40 45

Ser Phe Thr Arg Gin Tyr Lys Leu Pro Asp Gly Val Glu lie Lys Asp

50 55 60

Leu Ser Ala Val Leu Cys His Asp Gly lie Leu Val Val

65 70 75

<210> 16

<211> 81

<212> PRT

<213> Homo sapiens

<220>

<221> MISC-FEATURE

<222> (1) .. (81)

<223> HSPB4 a-Crystallin domain <400> 16

Val Arg Ser Asp Arg Asp Lys Phe Val lie Phe Leu Asp Val Lys His 1 5 10 15

Phe Ser Pro Glu Asp Leu Thr Val Lys Val Gin Asp Asp Phe Val Glu

20 25 30 lie His Gly Lys His Asn Glu Arg Gin Asp Asp His Gly Tyr lie Ser

35 40 45 Arg Glu Phe His Arg Arg Tyr Arg Leu Pro Ser Asn Val Asp Gin Ser 50 55 60

Ala Leu Ser Cys Ser Leu Ser Ala Asp Gly Met Leu Thr Phe Cys Gly 65 70 75 80

Pro

<210> 17

<211> 75

<212> PRT

<213> Homo sapiens

<220>

<221> MISC-FEATURE

<222> (1) .. (75)

<223> HSPB5 a-Crystallin domain fragment <400> 17

Glu Lys Asp Arg Phe Ser Val Asn Leu Asp Val Lys His Phe Ser Pro 1 5 10 15

Glu Glu Leu Lys Val Lys Val Leu Gly Asp Val He Glu Val His Gly

20 25 30

Lys His Glu Glu Arg Gin Asp Glu His Gly Phe He Ser Arg Glu Phe

35 40 45

His Arg Lys Tyr Arg He Pro Ala Asp Val Asp Pro Leu Thr He Thr 50 55 60

Ser Leu Ser Ser Asp Gly Val Leu Thr Val

70 75

<210> 18

<211> 75

<212> PRT

<213> Homo sapiens

<220>

<221> MISC-FEATURE <222> (1) .. (75)

<223> HSPB6 a-Crystallin domain fragment <400> 18

Asp Pro Gly His Phe Ser Val Leu Leu Asp Val Lys His Phe Ser Pro 1 5 10 15

Glu Glu lie Ala Val Lys Val Val Gly Glu His Val Glu Val His Ala

20 25 30

Arg His Glu Glu Arg Pro Asp Glu His Gly Phe Val Ala Arg Glu Phe

35 40 45

His Arg Arg Tyr Arg Leu Pro Pro Gly Val Asp Pro Ala Ala Val Thr 50 55 60

Ser Ala Leu Ser Pro Glu Gly Val Leu Ser lie

65 70 75

<210> 19

<211> 73

<212> PRT

<213> Homo sapiens

<220>

<221> MISC-FEATURE

<222> (1) .. (73)

<223> HSPB5 a-Crystallin domain fragment <400> 19

Asp Arg Phe Ser Val Asn Leu Asp Val Lys His Phe Ser Pro Glu Glu 1 5 10 15

Leu Lys Val Lys Val Leu Gly Asp Val lie Glu Val His Gly Lys His

20 25 30

Glu Glu Arg Gin Asp Glu His Gly Phe lie Ser Arg Glu Phe His Arg

35 40 45

Lys Tyr Arg lie Pro Ala Asp Val Asp Pro Leu Thr lie Thr Ser Ser 50 55 60

Leu Ser Ser Asp Gly Val Leu Thr Val

65 70

<210> 20

<211> 70

<212> PRT

<213> Homo sapiens

<220>

<221> MISC-FEATURE

<222> (1) .. (70)

<223> HSPB7 a-Crystallin domain fragment <400> 20

Asp Ala Tyr Glu Phe Ala Val Asp Val Arg Asp Phe Ser Pro Glu Asp 1 5 10 15 lie lie Val Thr Thr Ser Asn Asn His lie Glu Val Arg Ala Glu Lys

20 25 30

Leu Ala Ala Asp Gly Thr Val Met Asn Thr Phe Ala His Lys Cys Gin

35 40 45

Leu Pro Glu Asp Val Asp Pro Thr Ser Val Thr Ser Ala Leu Arg Glu 50 55 60

Asp Gly Ser Leu Thr lie

65 70

<210> 21

<211> 74

<212> PRT

<213> Homo sapiens

<220>

<221> MISC-FEATURE

<222> (1) .. (74)

<223> HSPB5 a-Crystallin domain fragment <400> 21

Phe Ser Val Asn Leu Asp Val Lys His Phe Ser Pro Glu Glu Leu Lys 1 5 10 15

Val Lys Val Leu Gly Asp Val lie Glu Val His Gly Lys His Glu Glu

20 25 30

Arg Gin Asp Glu His Gly Phe lie Ser Arg Glu Phe His Arg Lys Tyr

35 40 45

Arg lie Pro Ala Asp Val Asp Pro Leu Thr lie Thr Ser Ser Leu Ser 50 55 60

Ser Asp Gly Val Leu Thr Val Asn Gly Pro

65 70

<210> 22

<211> 74

<212> PRT

<213> Homo sapiens

<220>

<221> MISC-FEATURE

<222> (1) .. (74)

<223> HSPB8 a-Crystallin domain fragment <400> 22

Trp Lys Val Cys Val Asn Val His Ser Phe Lys Pro Glu Glu Leu Met 1 5 10 15

Val Lys Thr Lys Asp Gly Tyr Val Glu Val Ser Gly Lys His Glu Glu

20 25 30

Lys Gin Gin Glu Gly Gly lie Val Ser Lys Asn Phe Thr Lys Lys lie

35 40 45

Gin Leu Pro Ala Glu Val Asp Pro Val Thr Val Phe Ala Ser Leu Ser 50 55 60

Pro Glu Gly Leu Leu lie lie Glu Ala Pro

65 70 <210> 23

<211> 76

<212> PRT

<213> Homo sapiens

<220>

<221> MISC-FEATURE

<222> (1) .. (76)

<223> HSPB5 OC-Crystallin domain fragment <400> 23

Lys Asp Arg Phe Ser Val Asn Leu Asp Val Lys His Phe Ser Pro Glu 1 5 10 15

Glu Leu Lys Val Lys Val Leu Gly Asp Val He Glu Val His Gly Lys

20 25 30

His Glu Glu Arg Gin Asp Glu His Gly Phe He Ser Arg Glu Phe His

35 40 45

Arg Lys Tyr Arg He Pro Ala Asp Val Asp Pro Leu Thr He Thr Ser 50 55 60

Ser Leu Ser Ser Asp Gly Val Leu Thr Val Asn Gly

65 70 75

<210> 24

<211> 79

<212> PRT

<213> Homo sapiens

<220>

<221> MISC-FEATURE

<222> (1) .. (79)

<223> HSPB9 a-Crystallin domain fragment

<400> 24

Arg Asp Gly Phe Gin Met Lys Leu Asp Ala His Gly Phe Ala Pro Glu 1 5 10 15 Glu Leu Val Val Gin Val Asp Gly Gin Trp Leu Met Val Thr Gly Gin 20 25 30

Gin Gin Leu Asp Val Arg Asp Pro Glu Arg Val Ser Tyr Arg Met Ser

35 40 45

Gin Lys Val His Arg Lys Met Leu Pro Ser Asn Leu Ser Pro Thr Ala 50 55 60

Met Thr Cys Cys Leu Thr Pro Ser Gly Gin Leu Trp Val Arg Gly 65 70 75

<210> 25

<211> 71

<212> PRT

<213> Homo sapiens

<220>

<221> MISC-FEATURE

<222> (1) .. (73)

<223> HSPB5 a-Crystallin domain fragment <400> 25

Asn Leu Asp Val Lys His Phe Ser Pro Glu Glu Leu Lys Val Lys Val 1 5 10 15

Leu Gly Asp Val He Glu Val His Gly Lys His Glu Glu Arg Gin Asp

20 25 30

Glu His Gly Phe He Ser Arg Glu Phe His Arg Lys Tyr Arg He Pro

35 40 45

Ala Asp Val Asp Pro Leu Thr He Thr Ser Ser Leu Ser Ser Asp Gly 50 55 60

Val Leu Thr Val Asn Gly Pro

65 70

<210> 26 <211> 74

<212> PRT

<213> Homo sapiens

<220>

<221> MISC-FEATURE

<222> (1) .. (73)

<223> HSPBIO a-Crystallin domain fragment <400> 26

Ser Val Asn Val Cys Gly Phe Glu Pro Asp Gin Val Lys Val Arg Val 1 5 10 15

Lys Asp Gly Lys Val Cys Val Ser Ala Glu Arg Glu Asn Arg Tyr Asp

20 25 30

Cys Leu Gly Ser Lys Lys Tyr Ser Tyr Met Asn lie Cys Lys Glu Phe

35 40 45

Ser Leu Pro Pro Cys Val Asp Glu Lys Asp Val Thr Tyr Ser Tyr Gly 50 55 60

Gly Ser Cys Val Lys lie Glu Ser Pro

70

Claims

Claims

1. A compound for use in treating or preventing glutamate excitotoxicity and/or tauopathy in a subject, preferably a disorder selected from the group of epilepsy, spinal cord injury, stroke, traumatic brain injury, Alzheimer's disease, Pick's disease, amyotrophic lateral sclerosis, frontotemporal dementia, frontotemporal lobar degeneration, progressive supranuclear palsy, Parkinsonism linked to chromosome 17, cortical basal degeneration , hearing loss, and in alcoholism or alcohol withdrawal problems, wherein said compound comprises a small heat shock protein having an alpha-crystalhn domain.

2. Compound for use according to claim 1, wherein the compound comprises a protein complex of the small heat shock protein having an alpha-crystallin domain.

3. Compound for use according to claim 1 or 2, wherein the small heat shock protein having an alpha-crystallin domain is alpha B-crystallin (HspB5).

4. Compound for use according to claim 1 or 2, wherein the compound is a small heat shock protein having an alpha-crystalhn domain.

5. Compound for use according to any one of claims 1-4, wherein the compound is alpha B-crystallin (HspB5).

6. Compound for use according to claim 2 or 3, wherein the protein complex has a molecular weight of at least 200 kDa, particularly at least 300 kDa, more particularly at least 400 kDa, more particularly at least 500 kDa, preferably approximately between 500 - 800 kDa, more preferably approximately between 500-600 kDa.

7. A composition for use in the treatment or prevention glutamate excitotoxicity and/or tauopathy in a subject, which composition comprises a compound according to any one of claims 1-6 and a pharmaceutically acceptable carrier or excipient.

8. Composition for use according to claim 7, wherein the composition is in a pharmaceutical formulation suitable for systemic administration, in particular for intravenous administration.

9. A method for producing a cell-secreted compound that increases the activity of excitatory amino acid transporter 2 (EAAT2) in human astrocyte cells or of glutamate transporter 1 (GLT1) in murine astrocyte cells, the method comprising the steps of culturing microglial, monocyte or macrophage cells in culture medium in the presence of an effective amount of a compound comprising a small heat shock protein having an alpha- crystallin domain, followed by separating the cells from the cell-secreted compound so as to isolate said compound, and optionally further purifying the cell-secreted compound from the culture medium.

10. Method according to claim 9, wherein the cells are human adult microglia, monocytes or macrophages cells, and/or wherein the effective amount of said small heat shock protein is an amount of 0.1-1000 μg/mL, preferably about 50 μg/mL, in the initial cell culture medium.

11. Method according to claim 9 or 10, wherein the effective amount of said small heat shock protein is an amount that increases EAAT2/GLT1 gene expression or protein expression in human and/or murine astrocyte cells.

12. A method for treating or preventing glutamate excitotoxicity and/or tauopathy in a subject, the method comprising culturing microglia, monocytes or macrophagesin culture medium in the presence of an effective amount of a compound comprising a small heat shock protein having an alpha-crystallin domain, culturing the cells in the presence of said small heat shock protein for an effective amount of time so as to allow the secretion by said cells of a secreted compound, separating the cells from the secreted compound so as to isolate said secreted compound, optionally further purifying the secreted compound from the culture medium, and administering to the subject a therapeutically or prophylactically effective amount of said secreted compound.

13. Method according to claim 12, wherein the cells are human adult microglia, monocytes or macrophages cells, and/or wherein the effective amount of said compound comprising a small heat shock protein having an alpha-crystallin domain is an amount of 0.1-1000 μg/mL, preferably about 50 μg/mL, in the initial cell culture medium, preferably said amount is the amount of the small heat shock protein having an alpha-crystallin domain.

14. A method for treating or preventing glutamate excitotoxicity and/or tauopathy in a subject, comprising admininistering to a subject in need thereof a therapeutically effective amount of a compound comprising a small heat shock protein having an alpha-crystallin domain.

15. Method according to claim 14, wherein the compound is alpha B- crystallin (HspB5).