METHYLTHIONINIUM FOR USE IN THE TREATMENT OF SYNAPTOPATHIES

Abstract

The present invention relates generally to methods and materials for treating synaptopathies, based on the use of Leu-co-methylthioninium acid salts, which are disclosed herein to increase synaptophysin levels in various brain regions at therapeutically relevant doses both in animal models of neurodegenerative disease, and in normal animals.

Claims

1. A method of increasing the level of synaptophysin in the brain of a mammalian subject, which method comprises orally administering to said subject a methylthioninium (MT)-containing compound, wherein the MT-containing compound is an LMTX compound of the following formula: ##STR00010## wherein each of H.sub.nA and H.sub.nB (where present) are protic acids which may be the same or different, and wherein p=1 or 2; q=0 or 1; n=1 or 2; (p+q)×n=2, or a hydrate or solvate thereof.

2. A method of therapeutic treatment a synaptopathy disorder in a subject which disorder is selected from the list consisting of: schizophrenia; cerebral ischemia; Multiple sclerosis (MS); depression; epilepsy; Startle syndrome; Tourette's syndrome; Autism spectrum disorders (ASD); Focal hand dystonia; Experimental allergic encephalitis (EAE); Glaucoma; late onset Alzheimer's disease synaptic dysfunction type; a Lysozomal storage disease not associated with tau pathology which method comprises orally administering to said subject a methylthioninium (MT)-containing compound, wherein the MT-containing compound is an LMTX compound of the following formula: ##STR00011## wherein each of H.sub.nA and H.sub.nB (where present) are protic acids which may be the same or different, and wherein p=1 or 2; q=0 or 1; n=1 or 2; (p+q)×n=2, or a hydrate or solvate thereof.

3. A method as claimed in claim 1 wherein the treatment is combined with a further therapeutic agent for that disorder.

4. A method as claimed in claim 1 or claim 2 wherein the total daily dose is between 2 and 100 mg of MT, optionally 10-60 mg, to the subject per day, optionally split into 2 or more doses.

5. A method as claimed in claim 4 wherein the total daily dose is from around any of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mg to around any of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 mg.

6. A method as claimed in claim 4 wherein the total daily dose is between 20 and 40 mg.

7. A method as claimed in claim 4 wherein the total daily dose is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 mg.

8. A method as claimed in any one of claims 1 to 7 wherein the total daily dose of the LMTX compound is administered as a split dose twice a day or three times a day.

9. A method as claimed in any one of claims 1 to 8 wherein the subject has not historically received treatment with a neurotransmission modifying compound which is a modifier of the activity of acetylcholine or glutamate neurotransmitters.

10. A method as claimed in any one of claims 1 to 8 wherein the subject has historically received treatment with the neurotransmission modifying compound which is a modifier of the activity of acetylcholine or glutamate neurotransmitters, but ceased that treatment at least 1, 2, 3, 4, 5, 6, 7 days, or 2, 3, 4, 5, 6, 7, 8 weeks prior to treatment with the LMTX compound.

11. A method as claimed in any one of claims 1 to 8 wherein the subject is selected as one who is receiving treatment with the neurotransmission modifying compound which is a modifier of the activity of acetylcholine or glutamate neurotransmitters, wherein said treatment is discontinued prior to treatment with the LMTX compound.

12. A method as claimed in any one of claims 1 to 11 wherein the therapeutic treatment is not combined with a neurotransmission modifying compound which is a modifier of the activity of acetylcholine or glutamate neurotransmitters.

13. A method as claimed in any one of claims 9 to 12, wherein the neurotransmission modifying compound is an acetylcholinesterase inhibitor.

14. A method as claimed in any one of claims 9 to 13, wherein the neurotransmission modifying compound is selected from donepezil; rivastigmine; and galantamine.

15. A method as claimed in any one of claims 9 to 12, wherein the neurotransmission modifying compound is an N-methyl-D-aspartate receptor (NMDA) receptor antagonist.

16. A method as claimed in any one of claims 9 to 12 or claim 15, wherein the neurotransmission modifying compound is memantine.

17. A method as claimed in any one of claims 1 to 16 wherein the subject is a human who has been diagnosed as having said synaptopathy disorder, or wherein said method comprises making said diagnosis.

18. A method of prophylactic treatment of a synaptopathy disorder in a subject, which disorder is selected from the list consisting of: schizophrenia; cerebral ischemia; Multiple sclerosis (MS); depression; epilepsy; Startle syndrome; Tourette's syndrome; Autism spectrum disorders (ASD); Focal hand dystonia; Experimental allergic encephalitis (EAE); Glaucoma; late onset Alzheimer's disease synaptic dysfunction type; a Lysozomal storage disease not associated with tau pathology, which method comprises orally administering to said subject a methylthioninium (MT)-containing compound, wherein the MT-containing compound is an LMTX compound of the following formula: ##STR00012## wherein each of H.sub.nA and H.sub.nB (where present) are protic acids which may be the same or different, and wherein p=1 or 2; q=0 or 1; n=1 or 2; (p+q)×n=2, or a hydrate or solvate thereof.

19. A method as claimed in claim 18 wherein the treatment is combined with a further prophylactic agent for that disorder.

20. A method as claimed in claim 18 wherein the dose is as defined in any one of claims 4 to 8 and/or the subject is as defined in any one of claims 9 to 11.

21. A method as claimed in any one of claims 18 to 20 wherein the subject is a human who has been assessed as being susceptible to, or at risk of, the disorder, optionally based on familial or genetic or other data.

22. A method as claimed in any one of claims 1 to 21 wherein the LTMX compound has the following formula, where HA and HB are different mono-protic acids: ##STR00013##

23. A method as claimed in claim 22 wherein the LTMX compound has the following formula: ##STR00014## wherein each of H.sub.nX is a protic acid.

24. A method as claimed in claim 22 wherein the LTMX compound has the following formula and H.sub.2A is a di-protic acid: ##STR00015##

25. A method as claimed in claim 23 wherein the LTMX compound has the following formula and is a bis-monoprotic acid: ##STR00016##

26. A method as claimed in any one of claims 22 to 25 wherein the or each protic acid is an inorganic acid.

27. A method as claimed in claim 26 wherein each protic acid is a hydrohalide acid.

28. A method as claimed in claim 26 wherein the or each protic acid is selected from HCl; HBr; HNO.sub.3; H.sub.2SO.sub.4.

29. A method as claimed in any one of claims 22 to 25 wherein the or each protic acid is an organic acid.

30. A method as claimed in claim 29 wherein the or each protic acid is selected from H.sub.2CO.sub.3; CH.sub.3COOH; methanesulfonic acid, 1,2-ethanedisulfonic acid, ethansulfonic acid, naphthalenedisulfonic acid, p-toluenesulfonic acid.

31. A method as claimed in any one of claims 1 to 30 wherein the LTMX compound is LMTM: ##STR00017##

32. A method as claimed in claim 31 wherein the total daily dose of LMTM is around 34 to 67, 34 to 100, 34 to 134, or 34 to 167 mg/day.

33. A method as claimed in claim 32 wherein the dose of LMTM is about 34, 38, 67, or 100 mg/once per day.

34. A method as claimed in any one of claims 1 to 21 wherein the LTMX compound is selected from the list consisting of: ##STR00018## ##STR00019##

35. A method as claimed in any one of claims 1 to 34 wherein the LTMX compound is provided as a pharmaceutical composition comprising the LMTX compound and a pharmaceutically acceptable carrier or diluent in the form of a dosage unit.

36. A method as claimed in claim 35 wherein the amount of MT in the unit is about 4, 5, 6, 7, 8, 9, 10, 20, or 30 to about 40, 50 or 60 mg.

37. A method as claimed in claim 35 wherein the dosage unit comprises about 34 to 67 mg, 34 to 100, 34 to 134, or 34 to 167 LMTM.

38. A method as claimed in any one of claims 35 to 37 wherein the composition is a tablet or capsule.

39. A container comprising: (i) a plurality of dosage units as defined in any one of claims 35 to 38; (ii) a label and/or instructions for their use according to a method of treatment as defined in any one of claims 1 to 34.

40. A container as claimed in claim 39, wherein the container comprises dosage units, and the dosage units are present in a blister pack which is substantially moisture-impervious.

41. A container as claimed in claim 39 or claim 40 wherein the label or instructions provide information regarding the disorder for which the composition is intended.

42. A container as claimed in any one of claims 39 to 41 wherein the label or instructions provide information regarding the maximum permitted daily dosage of the dosage units.

43. A container as claimed in any one of claims 39 to 42 wherein the label or instructions provide information regarding the suggested duration of the treatment.

44. An LTMX compound or composition as defined in any one of claims 1 to 34, for use in a method of treatment as defined in any one of claims 1 to 38.

45. Use of an LTMX compound or composition as defined in any one of claims 1 to 34, in the manufacture of a medicament for use in a method of treatment as defined in any one of claims 1 to 38.

Description

FIGURES

[0153] FIG. 1. Treatment effects of LMTM alone or following chronic pretreatment with rivastigmine in wild-type mice on hippocampal levels of acetylcholine (A) or synaptophysin levels measured immunohistochemically as the mean in hippocampus, visual cortex, diagonal band and septum (B). (**, p<0.01; ***, p<0.001).

[0154] FIG. 2. Treatment effects of LMTM alone or following chronic pretreatment with rivastigmine in tau transgenic L1 mice on levels of (A) SNARE complex proteins (SNAP25, syntaxin and VAMP2) and (B) α-synuclein measured immunohistochemically as the mean in hippocampus, visual cortex, diagonal band and septum. (*, p<0.05; ***, p<0.001; ****, p<0.0001).

EXAMPLES

Example 1—Provision of MT-Containing Compounds

[0155] Methods for the chemical synthesis of the MT-containing compounds described herein are known in the art. For example:

[0156] Synthesis of compounds 1 to 7 can be performed according to the methods described in WO2012/107706, or methods analogous to those. Synthesis of compound 8 can be performed according to the methods described in WO2007/110627, or a method analogous to those.

Example 2—Features of the Tau Transgenic Mouse Model Used for Interference Studies

[0157] In the L1 mouse model which was used in some of the present studies, there is over-expression of a three-repeat tau fragment encompassing residues 296-390 of the 2N4R tau isoform under the control of the Thy 1 promotor in an NMRI mouse strain (WO2002/059150). This fragment corresponds to the segment of tau first identified within the proteolytically stable core of the PHF (Wischik et al., 1988a; Wischik et al., 1988b) and recently confirmed by cryo-electronmicroscopy of PHFs in AD and tau filaments in Pick's disease (Fitzpatrick et al., 2017; Falcon et al., 2018).

[0158] Further features of the L1 mouse model include a prominent loss of neuronal immunoreactivity for choline acetyltransferase in the basal forebrain region, and a corresponding reduction in acetylcholinesterase in neocortex and hippocampus, indicative of reduction in acetylcholine. There is also an approximate 50% reduction in glutamate release for brain synaptosomal preparations from L1 mice compared with those from wild-type mice. In these respects, therefore, L1 mice also model the neurochemical impairments in cholinergic (Mesulam, 2013; Pepeu and Grazia Giovannini, 2017) and glutamatergic (Revett et al., 2013) function that are characteristic of AD and also in other synucleinopathies.

[0159] Underlying these impairments in neurotransmitter function, the L1 mouse model shows a disturbance in integration of synaptic proteins. Quantitative immunohistochemistry for multiple synaptic proteins in the basal forebrain (vertical diagonal band) shows that there is normally a high degree of correlation in levels of proteins comprising the SNARE complex (e.g. SNAP-25, syntaxin, VAMP2; reviewed in Li and Kavalali, 2017), and the vesicular glycoprotein synaptophysin and α-synuclein in wild-type mice. These correlations are largely lost in L1 mice (Table 1). The only correlations that remain are between synaptophysin, syntaxin and VAMP2. Therefore, synaptic vesicular protein levels are no longer linked quantitatively to the proteins of the SNARE complex or α-synuclein. This suggests that the tau oligomer pathology of the L1 mice interferes with the functional integration between vesicular and membrane-docking proteins in the synapse.

Example 3—Experimental Paradigms, Results and Discussion

Experimental Paradigms

[0160] The treatment schedule used to study the negative interaction between symptomatic treatments and LMTM was designed to model the clinical situation in which subjects are first treated chronically with a cholinesterase inhibitor or memantine before receiving LMTM. In what follows, we summarise some of the key results obtained for the AChEI, rivastigmine.

[0161] Wild-type and L1 mice (n=7-16 for each group) were pre-treated with rivastigmine (0.1 or 0.5 mg/kg/day) or memantine (2 or 20 mg/kg/day) or vehicle for 5 weeks by gavage. For the following 6 weeks, LMTM (5 and 15 mg/kg) or vehicle were added to this daily treatment regime, also by gavage. Animals were tested behaviourly during weeks 10 and 11 using a problem solving task in the open field water maze and then sacrificed for immunohistochemical and other tissue analyses.

[0162] Translating doses from mice to humans requires consideration of a number of factors. Although 5 mg/kg/day in mice corresponds approximately to 8 mg/day in humans in terms of C.sub.max levels of parent MT in plasma, this dose is at the threshold for effects on pathology and behaviour. The higher dose of 15 mg/kg/day is generally required for LMTM to be fully effective in the L1 mouse model (Melis et al., 2015a). This may relate to the much shorter half-life of MT in mice (4 hours) compared to humans (37 hours in elderly humans). Tissue sectioned for immunohistochemistry was labelled with antibody and processed using Image J to determine protein expression densitometrically. Data are presented as Z-score transformations without units.

[0163] For measurement of acetylcholine (ACh) levels in hippocampus, animals (wild-type or L1) were treated with LMTM (5 mg/kg/day for 2 weeks) after prior treatment for 2 weeks with or without rivastigmine (0.5 mg/kg/day). Rivastigmine was administered subcutaneously with an Alzet minipump whereas LMTM was administered by oral gavage. Levels of ACh were measured in hippocampus using an implanted microdialysis probe and HPLC analysis of the extracellular fluid.

[0164] Data are presented as group averages and standard errors of mean and were analysed using parametric statistics, with alpha set to 0.05.

[0165] Experiments on animals were carried out in accordance with the European Communities Council Directive (63/2010/EU) with local ethical approval, a project license under the UK Scientific Procedures Act (1986), and in accordance with the German Law for Animal Protection (Tierschutzgesetz) and the Polish Law on the Protection of Animals.

Results

[0166] Effects of Treatment with LMTM and Rivastigmine in Wild-Type Mice

[0167] The effects of treatment with LMTM alone or on a chronic rivastigmine background are summarised in Table 2.

[0168] In wild-type mice, there was a significant, 2-fold increase in basal ACh levels in hippocampus following LMTM treatment, and a 30% reduction when mice received LMTM after prior treatment with rivastigmine (FIG. 1A).

[0169] There was also a 3-fold increase in mean synaptophysin levels measured in hippocampus, visual cortex, diagonal band and septum following LMTM treatment alone and a statistically significant reduction of the same magnitude when LMTM was given against a background of prior treatment with rivastigmine (FIG. 1B).

TABLE-US-00002 TABLE 2 Summary of treatment effects of LMTM given alone (5 or 15 mg/kg/day) or following chronic pretreatment with rivastigmine (0.1 or 0.5 mg/kg/day) in wild-type mice, given as approximate rounded percentages to indicate scale and direction of change. Numbers in black signify treatment effects which reached statistical significance, those in grey were directional, ‘—’ indicates no effect. Rivastigmine + Effects in wild-type mice LMTM alone LMTM ACh release ↑ × 200% ↓ × 30%  SNARE complex — — Synaptophysin ↑ × 300% ↓ × 300% α-Synuclein — — Mitochondrial complex IV — — Behaviour — —
Effects of Treatment with LMTM and Rivastigmine in Tau Transgenic L1 Mice

[0170] The activating effects of LMTM alone and the inhibitory effects of the combination with rivastigmine are larger and more generalised in the tau transgenic L1 mice than in the wild-type mice (see Table 3). LMTM alone produces significant increases in ACh release in the hippocampus, in glutamate release from brain synaptosomal preparations, in synaptophysin levels, in mitochondrial complex IV activity and in behavioural changes. None of these effects were seen when LMTM was preceded by chronic rivastigmine. Indeed, in the case of SNARE complex proteins (FIG. 2A) and synuclein (FIG. 2B), the reduction produced by the combination was to levels below those seen in the absence of LMTM treatment.

TABLE-US-00003 TABLE 3 Summary of treatment effects of LMTM given alone (5 or 15 mg/kg/day) or following chronic pretreatment with rivastigmine (0.1 or 0.5 mg/kg/day) in L1 mice, given as approximate rounded percentages to indicate scale and direction of change. Numbers in black signify treatment effects that reached statistical significance, those in grey were directional and n/a signifies that results are not yet available. Rivastigmine + Effects in L1 mice LMTM alone LMTM ACh release ↑ × 200% ↓ × 30%  Glutamate release ↑ × 200% n/a SNARE complex — ↓ × 300% Synaptophysin ↑ × 400% ↓ × 300% α-Synuclein — ↓ × 200% Mitochondrial complex IV ↑ × 50%  ↓ × 30%  Behaviour ↑ × 30%  ↓ × 20% 

Discussion of Example 3

[0171] The results presented here demonstrate that the reduction in efficacy of LMTM when given as an add-on to a symptomatic treatment in humans can be reproduced both in wild-type mice and in a tau transgenic mouse model.

[0172] The results we now report demonstrate that there are two classes of effect produced by LMTM treatment in wild-type and tau transgenic mice: those that are subject to dynamic modulation by prior exposure to cholinesterase inhibitor and those which are not. In tau transgenic mice, the treatment effects that can be modulated include increase in ACh release in the hippocampus, changes in synaptic proteins, increase in mitochondrial complex IV activity and reversal of behavioural impairment. The only treatment effects that are not subject to pharmacological modulation are the primary effect on tau aggregation pathology and its immediate effect on neuronal function, as measured for example by restoration of choline acetyltransferase expression in the basal forebrain.

[0173] Effects that are subject to pharmacological modulation are themselves of two types: those which are augmented by the effect on tau aggregation pathology and those which are also seen in wild-type mice. Of the outcomes we have measured, positive treatment effects of LMTM given alone in wild-type mice included an increase in ACh levels in hippocampus, and an increase in synaptophysin levels in multiple brain regions. Therefore, LMTM treatment is able to activate neuronal function at therapeutically relevant doses in wild-type mice lacking tau aggregation pathology.

[0174] An increase in synaptophysin signals an increase in number or size of the synaptic vesicles that are required for release of neurotransmitters from the presynapse following activation via an action potential. Therefore, an increase in synaptophysin levels appears to be associated with an increase in a number of neurotransmitters needed to support cognitive and other mental functions.

[0175] Although it has been reported that the MT moiety is a weak cholinesterase inhibitor (Pfaffendorf et al., 1997; Deiana et al., 2009), this is unlikely to be the mechanism responsible for the increase in ACh levels.

[0176] Specifically, further experiments using scopolamine to increase ACh levels (by blocking M2/M4 negative feedback receptors) showed that the increase produced by LMTM was less than that seen with rivastigmine alone, and that the combination was again inhibitory in wild type mice. Under the condition of cholinesterase inhibition used in these experiments (a very small amount of a cholinesterase inhibitor, 100 nanomolar rivastigmine, added to the perfusion fluid), ACh levels in the hippocampus rise, and when they rise strongly enough, they limit additional ACh release by activating pre-synaptic muscarinic receptors of the M2/M4 subtype (so-called negative feedback receptors).

[0177] In this situation, adding scopolamine (1 μM) to the perfusion fluid blocks these presynaptic receptors, and as a consequence, ACh levels rise by 3-5 fold. The fact that LMTM is not additive with rivastigmine in these experiments supports the conclusion that LMTM has a different mechanism of action from rivastigmine. In other words, although LMTM has been described as being a weak inhibitor of cholinesterases in high concentrations, the present effects seem to be unrelated to cholinesterase inhibition, because there is no additive effect with small quantities of rivastigmine.

[0178] The increase in ACh and synaptophysin levels might theoretically be explained by an increase in presynaptic mitochondrial activity, since the MT moiety is known to enhance mitochondrial complex IV activity (Atamna et al., 2012), and mitochondria have an important role in homeostatic regulation of presynaptic function (Devine and Kittler, 2018). In particular, The MT moiety is thought to enhance oxidative phosphorylation by acting as an electron shuttle between complex I and complex IV (Atamna et al., 2012). The MT moiety has a redox potential of approximately 0 mV, midway between the redox potential of complex I (−0.4 mV) and complex IV (+0.4 mV).

[0179] However, direct measurement of complex IV activity in wild type mice did not show any increase following LMTM treatment. The activating effects of LMTM were also not associated with improvement in spatial recognition memory in wild-type mice.

[0180] Although qualitatively similar, the effects of LMTM given alone are much more prominent and more broad-ranging in tau transgenic L1 mice. The most likely explanation for this is that LMTM combines an inhibitory effect on tau oligomers together with inherent activating effects which are not tau-dependent. The reduction in tau oligomer levels following LMTM treatment facilitates a more pronounced activation of synaptic function and release of neurotransmitters such as ACh and glutamate. Likewise, LMTM reverses the spatial memory deficit seen in tau transgenic L1 mice (Melis et al., 2015a). Alternatively, LMTM may act via a different mechanism that does not depend on tau, as seen for example in wild-type mice lacking tau pathology. The negative effects seen when LMTM is introduced on a chronic rivastigmine background appears simply to reflect the reversal of the activation seen with LMTM alone.

[0181] A deleterious effect of tau oligomers on functioning of synaptic proteins is readily understandable as being the result of direct interference with docking of synaptic vesicles, membrane fusion and release of neurotransmitter. In tau transgenic L1 mice for example, synaptic vesicular protein levels are no longer linked quantitatively to either the proteins of the SNARE complex or α-synuclein, implying a loss of functional integration between vesicular and membrane-docking proteins at the synapse. The consequence of this can be seen directly as an impairment in glutamate release from synaptosomal preparations from tau transgenic mice, and a restoration of normal glutamate release following treatment with LMTM.

[0182] A further consideration is whether the homeostatic downregulation that we have demonstrated would operate in the same way if LMTM treatment were primary and symptomatic treatment were added at a later date. The experiments we have conducted to date were originally designed to mimic the clinical situation in which LMTM is added in patients already receiving symptomatic treatments. If homeostatic downregulation is determined by the treatment that comes first, it is logical that the treatment effects of LMTM would dominate, albeit that the response to add-on symptomatic treatment could be reduced to some extent.

Example 4—Synaptopathies

[0183] As disclosed herein LMTX compounds are capable of increasing mean levels of synaptic proteins in various brain regions at therapeutically relevant doses both in the impaired and wild-type mice. This increase in synaptic proteins may be used to compensate for loss of integration of synaptic proteins in diseases such as synaptopathies i.e. brain disorders that have arisen from synaptic dysfunction, or in which such synaptic dysfunction contributes to the aetiology or symptoms of the disorder. A non-limiting list of such diseases includes the following:

[0184] Schizophrenia is a devastating mental disorder with a complex etiology that arises as an interaction between genetic and environmental factors. Schizophrenia is a neurodevelopmental disorder, and synaptic disturbances play a critical role in developing the disease. In 1982, Feinberg proposed that the schizophrenia might arise as a result of abnormal synaptic pruning. Synaptic disturbances cannot be studied and understood as an independent disease hallmark, but only as a part of a complex network of homeostatic events. Development, glial-neural interaction, changes in energy homeostasis, diverse genetic predisposition, neuroimmune processes and environmental influences all can tip the delicate homeostatic balance of the synaptic morphology and connectivity in a uniquely individual fashion, thus contributing to the emergence of the various symptoms of this devastating disorder. Faludi and Mirnics (2011) have broadly sub-stratified schizophrenia into “synaptic” “oligodendroglial”, “metabolic” and “inflammatory” subclasses.

[0185] The level of SNAP-25 is significantly depleted in the schizophrenic cerebellum (Mukaetova-Ladinska et al., 2002). Tau and MAP2 and synaptic proteins other than SNAP25, such as synaptophysin and syntaxin, are not affected. This provides evidence that alterations of the cerebellar synaptic network occur in schizophrenia. These changes may influence cerebellar-forebrain connections, especially those with the frontal lobes, and give rise to the cognitive dysmetria that is characteristic of the clinical phenotype in schizophrenia.

[0186] Pregulated formation of SNARE complexes and the abnormal expression of SNARE proteins and accessory molecules in a specific region (orbitofrontal cortex) of the human brain are associated with schizophrenia (Katrancha et al., 2015)

[0187] Depression. Atrophy of neurons and the loss of glutamatergic synaptic connections caused by stress are key contributors to the symptoms of depression. In addition to the HPA axis, synaptic number and function are altered by other factors (notably neurotrophic factors) that have been implicated in depression (Duman et al., 2016).

[0188] Autism spectrum disorders are a complex group of disorders associated with aberrant synaptic transmission and plasticity (Giovedi et al., 2014). Levels of both postsynaptic homer1 and presynaptic synaptophysin were significantly reduced in the adult brain of a shank3b-deficient zebrafish model of ASD (Liu et al., 2018).

[0189] Epilepsy: several synaptic proteins are implicated in epilepsy (Giovedi et al., 2014). Electrical kindling increases synaptophysin immunoreactivity in both the hippocampal formation and the piriform cortex in rats (Li et al., 2002).

[0190] Startle disease (hyperekplexia) is a rare non-epileptic disorder characterised by an exaggerated persistent startle reaction to unexpected auditory, somatosensory and visual stimuli, generalised muscular rigidity, and nocturnal myoclonus. The major form has a genetic basis: mutations in the al subunit of the glycine receptor gene, GLRA1, or related genes (Bakker et al., 2006). Related syndromes include Tourette's syndrome and anxiety disorders.

[0191] Focal hand dystonia, is a syndrome characterized by muscle spasms giving rise to involuntary movements and abnormal postures. Significant alterations in synaptic plasticity have been described in dystonic animal models as well as in patients (Quartarone and Pisani, 2011).

[0192] Cerebral ischemia causes synaptic alterations that are consistent with ischemic long-term potentiation (LTP) and represent a new model to characterize aberrant forms of synaptic plasticity. (Orfila et al., 2018). Although immunoreactivity for synaptophysin is transiently increased in ischemic lesions from 3 to 7 days after cerebral ischemia, synaptophysin immunostaining in the damaged areas gradually decreased and finally almost disappeared one month after transient cerebral ischemia in rats (Korematsu et al., 1993).

[0193] The inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1β (IL-1β) play important physiological roles in LTP and synaptic scaling. However, actions of these cytokines on synaptic plasticity can be altered under conditions of neuroinflammation. Altered synaptic plasticity occurs under either physiological or inflammatory conditions, in particular for experimental allergic encephalitis (EAE) and multiple sclerosis (MS) (Rizzo et al. 2018). Synaptophysin, synapsin I, and PSD-95 immunoreactivities were reduced in both the grey and white matter of both chronic and acute models of EAE (Zhu et al., 2013).

[0194] Glaucoma and AD share several features. They both affect the elderly, are neurodegenerative, chronic and progressive, leading to irreversible cell death. AD and glaucoma also share some common features such as the Aβ accumulation/aggregation, tau aggregation and hyperphosphorylation. Both diseases are characterized by early changes of neuronal circuitry and phosphorylation of mitogen-activated protein kinases (MAPK) followed by inflammatory process, glial reaction, reactive oxygen species production, oxidative stress and mitochondrial abnormalities, propagation of neurodegenerative processes leading to cell death. Both diseases are characterized by common features such as synaptic dysfunction and neuronal cell death at the level of the inner retina. Glaucoma is recognized as a disease frequently associated with AD and aging (Criscuolo et al., 2017).

REFERENCES FOR EXAMPLE 4

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