Methods of diagnosing or treating neurological disorders using P75ECD and / or P75

Abstract

The present invention relates to methods for diagnosing or treating neurological disorders using p75ECD and / or p75. Specifically, a method for treating and / or preventing neurological disorders is described herein, comprising administering a therapeutically effective amount of a p75 extracellular domain (p75ECD) or a functional fragment, variant, analog or derivative thereof to a subject in need. Also described herein is a method for diagnosing and / or prognosing Alzheimer's disease (AD) in a test subject, the method comprising comparing the p75ECD and / or p75FL concentration and / or the p75ECD: p75FL ratio in a biological sample of the test subject with the p75ECD and / or p75FL concentration and / or the p75ECD: p75FL ratio in a comparable biological sample obtained from a non-dementia control subject.

Description

[0001] This application is a divisional application of the application with international application date of March 25, 2016, international application number PCT / CN2016 / 077412, which entered the Chinese national phase on September 9, 2016, application number 201680000847.7, and invention name "Methods for diagnosing or treating neurological disorders using P75ECD and / or P75". Technical Field

[0002] The present disclosure relates to methods for diagnosis, detection and / or prognosis of neurological disorders and / or methods for treatment of neurological disorders. In a specific form, the present disclosure relates to the use of neurotrophin receptor p75 and / or its extracellular domain. Background Art

[0003] Neurotrophins are a small class of secreted neurotrophic factors that act through complex signal transduction pathways, which cause a wide range of effects in the nervous system, including involvement in neurogenesis, neuronal survival, proliferation, differentiation, myelination, axonal growth and synaptic plasticity, and conversely, in apoptosis and cell death at all stages of neuronal development, adult life, neuronal injury and disease. The neurotrophin family includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and neurotrophin-4 / 5 (NT4 / 5). Neurotrophins mediate their effects by binding to at least three different types of receptors, namely tropomyosin-related kinase (Trk) receptors (TrkA, TrkB, and TrkC), sortilin, and p75 neurotrophin receptor (p75; synonyms include p75NTR, p75(NTR), Gp80-LNGFR, low affinity nerve growth factor receptor, low affinity neurotrophin receptor, NGF receptor, and tumor necrosis factor receptor superfamily member 16 precursor (TNFRSF16). High affinity binding of neurotrophins often involves two of these receptor types interacting as co-receptors.

[0004] Depending on the relative expression level of the high-affinity neurotrophic factor Trk receptor, p75 mediates the survival or apoptosis of neurons. p75 undergoes extracellular domain (or extracellular domain) shedding, wherein the extracellular domain (p75ECD) of p75 is removed from the full-length protein. p75ECD shedding is physiologically regulated by tumor necrosis factor-α converting enzyme (TACE), followed by the regulation of intramembrane proteolysis regulated by γ-secretase. However, the physiological and pathological significance of p75 shedding is unknown. The regulation of p75 shedding and the function of p75ECD that can diffuse after shedding in vivo have not yet been determined.

[0005] Alterations in neurotrophin levels and signaling pathways have been implicated in neuronal survival and pathophysiology in a wide variety of neurodegenerative and psychiatric disorders, including Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS) and tauopathies, as well as in neuronal injury, such as spinal cord injury; however, no human disease has been shown to be directly caused by defects in neurotrophins or their receptors.

[0006] AD is the most common form of dementia, with about 24 million people suffering from the disease worldwide. AD is characterized by progressive cognitive deterioration, including memory loss, reduced ability to perform daily living activities, and neuropsychiatric symptoms or behavioral changes. Pathologically, the brain of AD patients is characterized by "neurofibrillary tangles" (NFTs) and "amyloid plaques" that are not seen in the normal brain. Amyloid plaques in AD are formed by the accumulation of misfolded and toxic amyloid peptide-β (Aβ) proteins in the cerebral cortex and hippocampus. Aβ is a metabolite of amyloid precursor protein (APP), and in the normal brain, the steady-state level of Aβ is maintained by the balance between Aβ production and removal. This balance is lost in AD patients. Interestingly, it has been found that the Aβ peptide is a ligand of p75, and it can activate signal transduction cascades. It has been proposed that p75 mediates Aβ-induced neurotoxicity.

[0007] Cerebral amyloid angiopathy (CAA) is increasingly recognized as an important cause of spontaneous intracerebral hemorrhage (ICH) and cognitive impairment in the elderly. It is characterized by progressive deposition of Aβ in the walls of small to medium-sized blood vessels in the cerebral cortex and pia mater, causing vascular dementia. It has a preference for the posterior cortical areas, followed by the frontotemporal and parietal lobes. CAA can also affect cerebellar vessels, but only rarely vessels in the brainstem or basal ganglia. CAA and AD are different conditions. However, a systematic review of four population-based autopsy studies indicated that the prevalence of CAA was 28–38% in non-demented patients and 55–69% in patients with dementia (Keage et al., 2009). CAA is present in 82–98% of AD cases, although severe CAA is seen in only 25% of AD cases (Charidimou et al., 2012; Jellinger, 2002).

[0008] Tauopathies are a class of diverse neurodegenerative diseases characterized by pathological aggregation of tau protein in the brain. Tau protein is a microtubule-binding protein (MAP) that interacts with tubulin in normal individuals to stabilize axonal microtubules and promote tubulin assembly into microtubules. Tau protein is abundant in neurons of the central nervous system and relatively rare elsewhere, but is also expressed at very low levels in CNS astrocytes and oligodendrocytes. When tau protein becomes defective and no longer correctly stabilizes microtubules, tau protein disease can be caused. This can cause intracellular accumulation and formation of neurofibrillary tangles (NFTs). Tau protein is a phosphoprotein with up to 79 potential serine (Ser) and threonine (Thr) phosphorylation sites on the longest tau protein isoform. It has been reported that in normal tau protein, phosphorylation occurs at about 30 of these sites; however, hyperphosphorylation is accompanied by the self-assembly of NFTs.

[0009] NFTs can be observed in a variety of tauopathies, including AD, progressive supranuclear palsy, dementia pugilistica (chronic traumatic encephalopathy), frontotemporal dementia and Parkinson's disease linked to chromosome 17, Lytico-Bodig disease (Guam Parkinson-dementia comorbidity), tangle-dominant dementia (which has NFTs similar to AD, but without plaques), gangliogliomas and ganglioneuromas, meningioangiomatosis, subacute sclerosing panencephalitis, Huntington's disease, etc. AD patients may have varying degrees of NFT involvement; however, NFTs may be present in patients who do not suffer from AD. Therefore, tauopathy is a different component of AD that is not accompanied by amyloid plaques.

[0010] As disclosed herein, p75ECD has been advantageously recognized as a neuroprotective factor that can be used to diagnose, prevent or treat neurological disorders and / or protect neurons from apoptosis and neurodegeneration. In addition, advantageously, the effects of full-length p75 (p75FL) can additionally or alternatively be used to diagnose neurological disorders, and / or this factor can be targeted to prevent or treat neurological disorders and / or protect neurons from apoptosis and neurodegeneration. Summary of the invention

[0011] According to one aspect of the present invention, there is provided a method for diagnosing and / or prognosing Alzheimer's disease (AD) in a test subject, the method comprising:

[0012] (a) obtaining a biological sample from the test subject,

[0013] (b) measuring the concentration of p75 extracellular domain (p75ECD) and / or full-length p75 (P75FL) in the biological sample of the test subject, and

[0014] (c) comparing the concentration of p75ECD and / or p75FL and / or the ratio of p75ECD:p75FL in said biological sample of said test subject with the concentration of p75ECD and / or p75FL and / or the ratio of p75ECD:p75FL in a comparable biological sample obtained from a non-demented control subject or a group of non-demented control subjects,

[0015] Wherein Alzheimer's disease is diagnosed and / or prognosed when the concentration of p75ECD and / or p75FL and / or the ratio of p75ECD:p75FL in the test subject is altered compared to the concentration of p75ECD and / or p75FL and / or the ratio of p75ECD:p75FL in the non-dementia control subject or a group of non-dementia control subjects.

[0016] In one embodiment, the method comprises:

[0017] (a) obtaining a biological sample from the test subject,

[0018] (b) measuring the concentration of p75 extracellular domain (p75ECD) in the biological sample of the test subject, and

[0019] (c) comparing the concentration of p75ECD in said biological sample from said test subject to the concentration of p75ECD in a comparable biological sample obtained from a non-dementia control subject or a group of non-dementia control subjects,

[0020] Wherein Alzheimer's disease is diagnosed and / or prognosed when the concentration of p75ECD in the test subject is reduced compared to the non-dementia control subject or a group of non-dementia control subjects.

[0021] In one embodiment, the method comprises:

[0022] (a) obtaining a biological sample from the test subject,

[0023] (b) measuring the concentrations of p75ECD and p75FL in the biological sample of the test subject, and

[0024] (c) comparing the concentration of p75ECD and p75FL in said biological sample from said test subject with the concentration of p75ECD and p75FL in a comparable biological sample obtained from a non-dementia control subject or a group of non-dementia control subjects,

[0025] Wherein Alzheimer's disease is diagnosed and / or prognosed when the ratio of p75ECD:p75FL is reduced in said test subject compared to said non-demented control subject or a group of non-demented control subjects.

[0026] In one embodiment, the method comprises:

[0027] (a) obtaining a biological sample from the test subject,

[0028] (b) measuring the concentration of p75FL in the biological sample of the test subject, and

[0029] (c) comparing the concentration of p75FL in said biological sample from said test subject to the concentration of p75FL in a comparable biological sample obtained from a non-dementia control subject or a group of non-dementia control subjects,

[0030] Wherein Alzheimer's disease is diagnosed and / or prognosed when the concentration of p75FL in the test subject is increased compared to the non-dementia control subject or a group of non-dementia control subjects.

[0031] According to another aspect of the present disclosure, a method for treating and / or preventing cerebral amyloid angiopathy (CAA) or tauopathy is provided, the method comprising administering to a subject in need thereof a therapeutically effective amount of a p75 extracellular domain (p75ECD) or a functional fragment, variant, analog or derivative thereof.

[0032] In another aspect of the present disclosure, a method for protecting neurons against apoptosis and / or neurodegeneration caused by neurotoxic factors such as Aβ is provided, the method comprising administering to a subject in need thereof a therapeutically effective amount of p75ECD or a functional fragment, variant, analog or derivative thereof.

[0033] In one embodiment, the p75ECD or a functional fragment, variant, analog or derivative thereof is p75ECD. In one embodiment, the p75ECD has an amino acid sequence provided by SEQ ID NO: 4. In one embodiment, the p75ECD is a p75ECD-Fc fusion. In one embodiment, the p75ECD-Fc has an amino acid sequence provided by SEQ ID NO: 6. In one embodiment, the p75ECD-Fc fusion is administered as a p75ECD-Fc fusion adeno-associated virus (AAV) vector. In one embodiment, the p75ECD-Fc fusion is a recombinant peptide. In one embodiment, the p75ECD is administered by intraperitoneal, intracerebroventricular, intracranial or intramuscular injection.

[0034] In one embodiment, the subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1AAV-p75ECD-Fc vector construction and characterization are provided, wherein: (a) shows a plasmid map of the AAV-GFP vector design, (b) shows SDS-PAGE analysis of the purity and homogeneity of AAV-GFP and AAV-p75ECD-Fc preparations, (c) shows a plasmid map of the AAV-p75ECD-Fc vector design;

[0036] Figure 2 Evidence for physiological levels of p75ECD in the central nervous system is provided, specifically: (a) graphic representation of ELISA analysis of p75ECD in TBS fractions of whole brain homogenates of Wt mice at different ages (mon=month; n=7), (b) p75ECD levels in different brain regions of 1-month-old and 12-month-old Wt mice (n=5, mean±sem, Student's t-test, **P<0.01), (c) Western blot analysis of p75FL and p75ECD in different brain regions of 12-month-old Wt mice;

[0037] Figure 3Provided are (a) a graphical representation of the levels of p75ECD in human cerebrospinal fluid (CSF) obtained by ELISA in Alzheimer's disease (AD) patients and age-matched non-demented controls (ND) (n=29 for AD, n=27 for ND, mean±sem, Student's t-test, **P<0.01), (b) Western blot analysis of full-length p75 (p75FL) and p75ECD in human AD and ND brain tissues, (c) quantitative analysis of the Western blot band densities of p75FL and p75ECD shown in (b), (d) the ratio of the p75ECD / p75FL Western blot band densities shown in (b) (n=12 for AD, n=12 for ND, mean±sem, Student's t-test, *P<0.05, **P<0.01); (e) Graphical representation of the levels of p75ECD obtained by ELISA in Tris-buffered saline (TBS) fractions of brain homogenates of 12-month-old AD mice (Tg) and wild-type littermates (Wt) (n=13 for Tg, n=14 for Wt, mean±sem, Student's t-test, **P<0.01), (f) Western blot analysis of full-length p75 (p75FL) in brain tissues from 12-month-old Tg and Wt mice, (g) Quantitative analysis of Western blot band densities of p75FL and p75ECD shown in (f), and (h) p75ECD / p75FL shown in (f). Ratio of Western blot band density (n=13 for Tg, n=14 for Wt, mean±sem, Student's t-test, *P<0.05, **P<0.01);

[0038] Figure 4The following graphical representations are provided: (a) the expression levels of ECD-FC analyzed by ELISA analysis of p75ECD in TBS fractions of AD mouse brain homogenates at 1 week (1w), 2 weeks (2w), 3 weeks (3w) and 9 months (9m) after intracerebroventricular injection of AAV-ECD-Fc or AAV-GFP in the left lateral ventricle of 3-month-old AD mice (n=5 per group for 1w, 2w and 3w, n=8 for 9m group), and (b) the expression levels of p75ECD in TBS fractions of 12-month-old AD mice analyzed by ELISA analysis of p75ECD after intracerebroventricular injection of AAV-ECD-Fc or AAV-GFP in the left lateral ventricle at 3 months of age compared with 12-month-old Wt mice (n=8 for 12-month-old Wt mice injected with AAV-GFP or AAV-ECD-Fc). n = 8 for month-old Tg mice, n = 13 for wt mice); mean ± sem, ANOVA, Tukey test, **P < 0.01);

[0039] Figure 5Provided are graphical representations of behavioral analysis of 12 month old mice in the Morris water maze assay, including Wt littermates (Wt, n=16), untreated AD mice (Tg, n=15), AD mice injected with AAV-GFP at 3 or 9 months of age as controls (Con, n=14), AD mice treated with AAV-ECD-Fc injection at 9 months of age (Tre, n=12), and AD mice prevented with AAV-ECD-Fc injection at 3 months of age (Pre, n=11), showing (a) latency (seconds) during the platform test, (b) distance traveled (cm) on the last day of training, (mean ± sem, ANOVA, Tukey's test, *P < 0.05, **P < 0.01, compared with Con), and (c) representative locomotor activity tracings; exploration test performed on day 5 shows (d) the number of crossings through the removed platform annulus, and (e) the distance traveled in the quadrant where the platform was located (mean ± sem; one-way ANOVA, Tukey's test, *P < 0.05, **P < 0.01, compared with Con), and (f) representative locomotor activity tracings; Y-maze analysis shows (g) arm alternations and (h) number of entries in the spontaneous alternation test, and (i) number of entries and (j) time in the novel arm test (mean ± sem; one-way ANOVA, Tukey's test Test, *P < 0.05, **P < 0.01, compared with Con; #P < 0.05, ##P < 0.01, compared with Wt); open field analysis shows (k) number of rearing times, (l) distance traveled (cm), and (m) representative locomotor activity training graphs (mean ± sem; one-way ANOVA, Tukey's test, *P < 0.05, compared with Con);

[0040] Figure 6Provided are analyses of the area fraction, density, and size of Aβ plaques in the hippocampus (HC) and neocortex (NC) of each animal in 5 equally spaced sections across the entire brain quantified by ImageJ software in 12-month-old AD mice injected with AAV-GFP at 3 or 9 months of age as controls (Con, n=7-9), AD mice treated with AAV-ECD-Fc injection at 9 months of age (Tre, n=8-9), and AD mice prevented with AAV-ECD-Fc injection at 3 months of age (Pre), showing (a) Aβ plaque deposition visualized using Congo red staining (scale bar=1 mm); comparison of (b, left panel) area fraction, (b, middle panel) plaque density, and (b, right panel) size of Congo red-stained plaques between groups; (c) Aβ plaques immunohistochemically stained with antibody 6E10 (scale bar = 1 mm); comparison of (d, left) area fraction, (d, middle) plaque density, and (d, right) size of 6E10-stained plaques between groups (for (a)-(d), n = 11 for Con, n = 9 for Tre, n = 8 for Pre, mean ± sem, one-way ANOVA, Tukey's test, *P < 0.05, **P < 0.01, compared with Con); Aβ40, Aβ42 in TBS, SDS, and formic acid (FA) fractions of (ei) brain homogenates and (e-ii) serum and ELISA analysis of total Aβ (for brain homogenates, n = 11 for Con, n = 9 for Tre, n = 8 for Pre; for serum, n = 14 for Con, n = 12 for Tre, n = 11 for Pre, mean ± sem, one-way ANOVA, Tukey's test, *P < 0.05, **P < 0.01, compared with Con); (f) micrographs showing cerebral amyloid angiopathy (CAA) visualized by Congo red staining (scale bar = 1 mm), (g) comparison of CAA characteristics between Con, Tre and Pre groups (n = 8, mean ± sem, one-way ANOVA, Tukey's test, **P < 0.01, compared with Con (h) Comparison of the number of cerebral microbleeds between Con, Tre and Pre groups (n=8, mean±sem, one-way ANOVA, Tukey's test, **P<0.01, compared with Con); (i) Micrographs showing the colocalization of Aβ plaques and ECD-Fc in APP / PS1 transgenic AD mice (scale bars: 200 μm (black), 40 μm (white), * indicates colocalization of ECD-Fc with amyloid peptide-β plaques; # indicates ECD-Fc expression in neurons);

[0041] Figure 7Results from AD mice injected with AAV-GFP at 9 months of age as control (Con), AD mice treated with AAV-ECD-Fc injection at 9 months of age (Tre), and AD mice prevented with AAV-ECD-Fc injection at 3 months of age (Pre) are provided, wherein: (a) micrographs showing the grossly preserved structure of neurons and dendrites detected using (upper) anti-MAP-2 antibody and anti-NeuN antibody immunofluorescence or (lower) anti-choline acetyltransferase (ChAT) antibody immunohistochemistry (n=11 for Con, n=9 for Tre, n=8 for Pre, mean±sem, one-way ANOVA, Tukey's test, *P<0.05, **P<0.01, compared with Con, scale bar = 100 μm), which were quantified (ai, a-ii, a-iii); (b) micrographs showing neuronal apoptosis detected by immunofluorescence using activated caspase-3 (cas-3) (scale bar = 50 μm, upper panel) or using TUNEL staining (scale bar = 250 μm, lower panel), which were quantified (bi, b-ii, b-iii, b-iv) (n = 11 for Con, n = 9 for Tre, n = 8 for Pre, mean ± sem, one-way ANOVA, Tukey's test, *P<0.05, **P<0.01, compared with Con); (ci) micrographs showing Golgi staining analysis of spines using images of representative Golgi staining of CA1 pyramidal neurons (left) and different dendritic spines (right) (scale bar = 10 μm, n = 3, mean ± sem, one-way ANOVA, Tukey's test, *P<0.05, compared with Con), (c-ii) quantitative analysis of spines per μm; (c-iii) Western blots of synaptic-related proteins (including SNAP25 and PSD95), (c-iv) quantitative analysis of the data in (c-iii), (cv) Western blots of synaptophysin I (Syn I), VAMP1, synaptic vesicle protein (SYP), and corresponding quantification of the data in (c-vi) (cv) (n = 8, mean ± sem, one-way ANOVA, Tukey's test, *P<0.05, **P<0.01, compared with Con); (di) Micrographs of Tau phosphorylation using pSer396 immunostaining (scale bar = 200 μm), (d-ii) Western blot analysis of Tau phosphorylation using anti-pS396, pT231, pS262, and pS199 antibodies (n = 8, mean ± sem, one-way ANOVA, Tukey's test, *P < 0.05, **P < 0.01, compared with Con), the results of which were quantified (d-iii), and the relative binding intensity of each (d-iv) anti-ps396 antibody, (dv) anti-pT231 antibody, (d-vi) anti-pS262 antibody, (d-vii) anti-ps199 antibody in each group; (e) micrographs of neurite outgrowth analysis of cultured primary mouse cortical neurons stained with anti-MAP-2 antibody and DAPI immunofluorescence (n=35, mean±sem, ANOVA, Tukey's test, **P<0.01, compared with Aβ42scr., #P<0.05, compared with Aβ42, scale bar=50 μm), which were quantified (f);.

[0042] Figure 8 Provided are (a) (left) Western blot results and from SHSY5Y cells differentiated in retinoic acid for three days and then treated with 5uM Aβ and the indicated agents, (right) quantification of the ratio of pTau262:total Tau protein, showing that ECD-Fc reversed Tau protein hyperphosphorylation induced by Aβ (n=3, mean±sem, one-way ANOVA, Tukey's test, **P<0.01, compared with control; ##P<0.01, compared with Aβ42), (b) (left) Western blot analysis and (right) quantification of the concentration-dependent inhibitory effect of recombinant p75ECD-Fc on Tau protein hyperphosphorylation induced by Aβ (n=5, mean±sem), and (c) (left) Western blot analysis and showing GSK3β phosphorylation at Serine 9, (right) Ser9 was upregulated with increasing p75ECD-Fc concentration. Quantification of the ratio of GSK3β phosphorylation / GSK3β at (n=5, mean±sem);

[0043] Fig. 9Results from peripheral injection of p75ECD or GFP in AD (APP / PS1 transgenic) or wild-type mice are provided, wherein (a) shows the escape latency (seconds) during the platform test in the Morris water maze, (b) shows the distance traveled to escape to the platform during the platform test, (c) shows the number of ring crossings in the exploration test, (d) shows the percentage of alternations and (e) novel arm entries in the Y-maze test, and (f) shows the distance traveled in the open field test; wherein p75ECD-Tg represents APP / PS1 mice injected intramuscularly with AAV-p75ECD-Fc, GFP-Tg represents APP / PS1 mice injected intramuscularly with AAV-GFP, Con-Tg represents APP / PS1 mice that did not receive injection, and Con-Wt represents wild-type mice that did not receive injection; number of animals = 8 per group, mean ± sem, Tukey test; *P < 0.05, **P < 0.01, compared with GFP;

[0044] Fig.10 Results from peripheral injection of p75ECD or GFP in AD mice are provided, wherein (a) shows Aβ deposition using 6E10 immunochemical staining, scale bar = 1 mm, (b) Aβ deposition using Congo red staining, scale bar = 1 mm, (c) quantification of the area fraction of Aβ deposition using 6E10 immunochemical staining, (d) quantification of the area fraction of Aβ deposition using Congo red staining, (e) ELISA quantification of Aβ levels in TBS, SDS fractions, FA extracts and serum, wherein NC indicates neocortex and HC indicates hippocampus, (f) 24 hours after peritoneal injection of p75ECD peptide (50 μg / 50 μl ip), serum Aβ40 and Aβ42 levels were significantly increased, pre-bleeding means before blood sampling, number of animals = 8, mean ± sem, T test; *P < 0.05, **P < 0.01;

[0045] Fig.11 Results from peripheral injection of p75ECD or GFP in AD mice are provided, wherein (a) shows astrocytes in the neocortex of AD mice immunohistochemically stained with anti-GFAP antibody, (b) quantification of the positive area fraction of GFAP staining, scale bar = 1 mm, (c) immunohistochemical staining of microglia in the neocortex of AD mice using anti-CD45 antibody, (d) quantification of the positive area fraction of CD45 staining, wherein NC indicates neocortex, HC indicates hippocampus, number of animals = 8, mean ± sem, T test, *P < 0.05, **P < 0.01;

[0046] Fig.12Results from peripheral injection of p75ECD or GFP in AD mice are provided, wherein (a) shows immunohistological staining of pS396 in the cortex, scale bar = 100 μm, (b) quantification of pS396-positive area fraction in the cortex, (c) immunohistological staining of pS396 in the hippocampus, scale bar = 100 μm, (d) quantification of pS396-positive area fraction in the hippocampus, relative intensities in (a) and (c) were normalized to Wt, (e) western blot analysis of pS396, pS199, pS262 and pT231, (f) quantification of relative intensities of bands of pS396, pS199, pS262 and pT231, number of animals = 8, mean ± sem, T test, **P < 0.01;

[0047] Fig.13 Provided are the results of pR5(Tau) mice after treatment with recombinant p75ECD-Fc or Fc during the Morris water maze (MWM) test, wherein (a) escape latency is shown, (b, c) distance traveled to find the platform in the progressive platform learning test, (d) time spent in the platform quadrant and (e) number of entries, (f) number of crossings of the hidden platform site in the exploration test between the different groups (*p<0.05 for p75ECD-Fc compared with the Fc group, #p<0.05 for wild type compared with the Fc group; $p<0.05 during the learning period compared with the first day of learning in the wild type; ￥p<0.05 during the learning period compared with the first day of learning in p75ECD-Fc, *p<0.05) (n=6 per group, mean±SEM, ANOVA, Tukey's post hoc test);

[0048] Fig.14 The following results are provided: A) Western blot showing the expression of BACE1, sAPPβ, full-length APP (APP.FL) and β-actin in the brain of PR5 mice in the p75ECD-Fc and Fc-treated groups, (B) the expression ratio of BACE1 / β-actin, (C) the expression ratio of sAPPβ / β-actin (n=5 per group, mean±SEM, Student's t-test; "Fc" was normalized to 100%); and

[0049] Fig.15The following results are presented: A) Western blot images showing the levels of Tau phosphorylated at Ser202 and Thr205 (using a single "AT8" antibody) and total Tau in the brains of p75ECD-Fc and Fc-treated PR5 mice, (B) statistical analysis of Tau phosphorylated at Ser202 and Thr205 (**p<0.01, compared with Fc) (n=5 per group, mean±SEM, Student's t-test; "Fc" was normalized to 100%). DETAILED DESCRIPTION

[0050] As disclosed herein, it has been recognized that the balance of full-length p75 and the extracellular domain of p75 (p75ECD) present in an individual may determine whether the individual suffers from neurodegeneration, which can cause a number of neurological diseases. Specifically, it has been recognized that p75ECD is a neuroprotective factor. Although not wishing to be bound by theory, it is believed that soluble p75ECD competes with cell membrane-bound p75 for ligands such as neurotrophic factors, thereby preventing p57 signaling that can cause neuronal degeneration and / or apoptosis.

[0051] As described herein, the level of p75ECD in the brain and cerebrospinal fluid (CSF) of AD patients and AD mouse models has been investigated. On the one hand, it has been recognized that AD is accompanied by low p75ECD levels, high full-length p75 (referred to herein as p75 or p75FL) levels and / or low p75ECD: p75FL ratios compared to normal non-demented control subjects. In addition, it has been recognized that the concentration of p75ECD, p75FL and / or the p75ECD: p75FL ratio can be used as a diagnostic or prognostic marker. In addition, in another aspect disclosed herein, it has been found that the restoration of p75ECD levels, such as by the administration of p75ECD, alleviates neurodegenerative diseases such as AD, cerebral amyloid angiopathy (CAA) and Tau protein disease, and improves learning and memory in both the early and late stages of AD in the APP / PS1 mouse model.

[0052] Therefore, in one aspect, the present invention provides a method for diagnosing and / or prognosing Alzheimer's disease (AD) in a test subject, the method comprising:

[0053] (a) obtaining a biological sample from the test subject,

[0054] (b) measuring the concentration of p75 extracellular domain (p75ECD) and / or full-length p75 (P75FL) in the biological sample of the test subject, and

[0055] (c) comparing the concentration of p75ECD and / or p75FL and / or the ratio of p75ECD:p75FL in said biological sample of said test subject with the concentration of p75ECD and / or p75FL and / or the ratio of p75ECD:p75FL in a comparable biological sample obtained from a non-demented control subject or a group of non-demented control subjects,

[0056] Wherein Alzheimer's disease is diagnosed and / or prognosed when the concentration of p75ECD and / or p75FL and / or the ratio of p75ECD:p75FL in the test subject is altered compared to the concentration of p75ECD and / or p75FL and / or the ratio of p75ECD:p75FL in the non-dementia control subject or a group of non-dementia control subjects.

[0057] The terms "p75FL," "full-length p75," "p75," and "p75NTR" are used interchangeably and, when used herein, refer to the entire (full-length) p75 neurotrophin receptor (also referred to as p75 or p75NTR).

[0058] The terms "extracellular domain of p75", "p75ECD", "extracellular domain of p75", "extracellular domain of p75" and "extracellular domain of p75" are used interchangeably and all refer to fragments that can be derived from the full-length p75 peptide after cleavage of p75 by tumor necrosis factor alpha converting enzyme (TACE) / ADAM17 at the juxtamembrane region, which releases or "falls off" the extracellular domain of p75FL (p75ECD) as a soluble peptide (Weskamp et al., 2004). In one embodiment, the p75ECD is shed from the p75FL peptide, and in such embodiments, the p75ECD is soluble. When used herein, the term "soluble" indicates that the protein, polypeptide or peptide is not bound to or does not become bound to a cell membrane and is therefore characterized by the absence or functional disruption of all or a substantial portion of a transmembrane (i.e., lipophilic) domain, such that the protein, polypeptide or peptide does not have any membrane anchoring function.

[0059] The p75 and / or p75ECD described herein can come from any suitable species. In one embodiment, the p75 and / or p75ECD are derived from humans. However, in one embodiment, the p75 and / or p75ECD can also be derived from animals with commercial, scientific, medical or personal significance, such as non-human primates, rats and / or mice and / or other mammals such as livestock, rare animals and companion animals (including race horses, sheep, cows, goats, pigs, lions, tigers, elephants, dogs, cats, rabbits, rats and mice, etc.). In one embodiment, the p75 and / or p75ECD can be derived from primates such as rhesus monkeys, chimpanzees, orangutans, chimpanzees, gorillas, etc.

[0060] The nucleotide sequence of human full-length p75 is provided at nucleotides 126-1409 of Genbank Accession No. NM_002507 and in SEQ ID NO:1.

[0061] The amino acid sequence of human full-length p75 is provided in SEQ ID NO:2.

[0062] Human p75ECD consists of amino acids 29 to 250 of SEQ ID NO: 2, and includes 28 amino acids as a single peptide, followed by four cysteine-rich domains, each containing 40 amino acid residues and each containing an N-glycosylation site (Schor, 2005). The nucleotide sequence of human p75ECD is provided in SEQ ID NO: 3.

[0063] The amino acid sequence of human p75ECD is provided in SEQ ID NO:4.

[0064] In one embodiment, the p75FL and / or p75ECD may be a genetic or peptide variant. "Variants" of p75 and / or p75ECD referred to herein should be understood to mean isoforms of p75FL encoded by, for example, allelic variants. Variants are further described in detail elsewhere herein.

[0065] As used herein, the term "test subject" refers to an individual who is being evaluated for diagnosis, detection, or prognosis of Alzheimer's disease. In one embodiment, the test subject can be a human. However, it is contemplated that the subject can also be an animal of commercial, scientific, medical, or personal interest, such as non-human primates and other mammals such as livestock, exotic animals, and companion animals (including race horses, sheep, cows, goats, pigs, lions, tigers, elephants, dogs, cats, rabbits, rats, and mice, etc.).

[0066] As used herein, the term "non-dementia control subject or a group of non-dementia control subjects" refers to an individual or a group of individuals who are considered normal and well, and specifically do not suffer from neurological disorders associated with dementia. In one embodiment, the non-dementia control subject or a group of subjects has not developed a neurological disorder associated with dementia within 10 years of providing the sample. In one embodiment, the non-dementia control subject or a group of subjects has not developed a neurological disorder within 5 years of providing the sample. In one embodiment, the non-dementia control subject is the same species as the test subject. In one embodiment, the non-dementia control subject is age-matched, wherein the non-dementia control subject is within 10 years of the age of the test subject. In one embodiment, the non-dementia control subject is within 5 years of the age of the test subject. In one embodiment, the p75ECD and / or p75FL concentration (or the ratio of the two as described herein) of the test subject is compared with a reference value, which is known to be a representative value of p75ECD and / or P75FL (or its ratio) in a non-dementia control subject or a group of non-dementia control subjects. These reference values ​​may be adjusted for factors that may affect the concentrations of p75ECD and / or p75FL (or their ratio), such as age.

[0067] As used herein, the term "concentration" is intended to refer to the amount or level of p75ECD and / or p75FL molecules in a biological sample, and is intended to encompass both absolute amounts (e.g., the amount of p75ECD and / or p75FL molecules in a sample measured in mg / ml, ng / ml, pg / ml, ng / g, etc.) and relative amounts (e.g., as measured by densitometry analysis of staining of specific bands on a Western blot, wherein the results for the test subject can optionally be normalized to the results for a non-dementia control subject). According to the first aspect of the present disclosure, wherein the measured concentration is an absolute amount, the absolute concentration of p75ECD and / or p75FL molecules in the test subject is compared to the absolute concentration of p75ECD and / or p75FL in the non-dementia control subject or a group of non-dementia control subjects to determine whether there is a change in concentration (i.e., an increase or decrease). Alternatively, when the concentration measured is a relative amount (e.g., as measured by densitometry analysis of a Western blot analysis), the staining intensity (or other relative measurement) of the concentration of the p75ECD or p75FL molecule in the test subject is compared to the staining intensity (or such other relative measurement) of the p75ECD and / or p75FL concentration in the non-dementia control subject or a group of non-dementia control subjects to determine if there is a change in concentration (i.e., an increase or decrease).

[0068] It will be apparent to one skilled in the art that the p75ECD:p75FL ratio can be readily calculated by dividing the absolute or relative concentration of p75ECD by the absolute or relative (respectively) concentration of p75FL.

[0069] The biological sample can be any suitable body sample type that can be sampled for determining p75FL and / or p75ECD concentrations. For example, the biological sample can be selected from brain tissue, cerebrospinal fluid (CSF), serum, plasma, whole blood and peripheral blood mononuclear cells (PBMC) and lymphocytes. However, those skilled in the art will recognize that body fluids of other tissue types may be suitable for sampling p75FL and p75ECD levels. In one embodiment, the biological sample is cerebrospinal fluid. In one embodiment, the biological sample is brain tissue. In one embodiment, the biological sample is selected from lymphocytes, plasma, serum and / or whole blood.

[0070] In embodiments where the biological sample is brain tissue, the brain tissue sample can be obtained after death of the subject, or can be a tissue biopsy sample obtained using standard biopsy techniques well known to those skilled in the art. The brain tissue can optionally be processed by methods well known to those skilled in the art such as homogenization and / or ultrasound. Those skilled in the art will recognize that other tissue types can be processed similarly if necessary. In embodiments where the biological sample is CSF, the CSF can be obtained, for example, by lumbar puncture using standard CSF collection techniques well known to those skilled in the art.

[0071] As referred to herein, the term "comparable biological sample" measured in a non-dementia control subject refers to a biological sample of the same or similar type as the biological sample measured in the test subject, or a biological sample in which the p75FL, p75ECD concentration and / or p75ECD: p75FL ratio in one body sample can accurately predict the p75FL, p75ECD concentration and / or p75ECD: p75FL ratio (respectively) in another body sample. For example, one skilled in the art will appreciate that the concentration of an analyte in a serum sample is substantially equivalent to the concentration of the same analyte in a plasma sample, because the difference between plasma and serum is only that serum does not contain fibrinogen and other coagulation factors. Therefore, in one embodiment, plasma and serum are comparable biological samples. In addition, one skilled in the art will appreciate that the concentration of an analyte in a serum or plasma sample corresponds to approximately twice the concentration of the analyte in a whole blood sample, because whole blood contains approximately half of the serum or plasma. Therefore, the concentration of an analyte in whole blood can be compared with plasma or serum because comparable concentrations can be converted by simple calculations.

[0072] As disclosed herein, it has been surprisingly recognized that in AD patients and AD mice, the concentration of p75ECD is reduced compared to their non-demented counterparts, and further, in AD patients and AD mice, the concentration of p75FL is increased compared to their non-demented counterparts. In addition, as disclosed herein, it has been noted that in AD patients and AD mice, the ratio of p75ECD:p75FL is reduced compared to their non-demented counterparts.

[0073] Thus, in one embodiment, a method for diagnosing and / or prognosing Alzheimer's disease (AD) in a test subject comprises:

[0074] (a) obtaining a biological sample from the test subject,

[0075] (b) measuring the concentration of p75 extracellular domain (p75ECD) in the biological sample of the test subject, and

[0076] (c) comparing the concentration of p75ECD in said biological sample from said test subject to the concentration of p75ECD in a comparable biological sample obtained from a non-dementia control subject or a group of non-dementia control subjects,

[0077] Wherein Alzheimer's disease is diagnosed and / or prognosed when the concentration of p75ECD in the test subject is reduced compared to the non-dementia control subject or a group of non-dementia control subjects.

[0078] The concentration of p75ECD in the test subject that can be considered "reduced compared to the non-demented control subject or a group of non-demented control subjects" in order to determine whether Alzheimer's disease is diagnosed or prognosed can vary with the species of the subject, the specific body sample type used, and the age of the subject. In one embodiment, in the test subject, the p75ECD concentration is reduced by about 25% to 99% compared to the non-demented control. In one embodiment, in the test subject, the p75ECD concentration is reduced by about 25% to 90% compared to the non-demented control. In one embodiment, in the test subject, the p75ECD concentration is reduced by about 30% to 80% compared to the non-demented control. In one embodiment, in human AD patients, the p75ECD concentration is reduced by about 50% compared to non-demented human controls. In one embodiment, the reduction is about 50% in cerebrospinal fluid (CSF). In one embodiment, the reduction is about 75% in brain tissue. In one embodiment, in human AD patients, the p75ECD level in human brain tissue is reduced by at least 75% compared to non-demented human controls. In one embodiment, the concentration of p75ECD in the CSF of a test subject for diagnosis or prognosis of AD is less than 500 pg / ml. Similarly, in one embodiment, in AD mice, the p75ECD concentration in the CSF of mice is reduced by about 50% compared to wild-type (wt) littermates, or in an alternative or additional embodiment, the reduction is about 50% in mouse brain tissue.

[0079] As disclosed herein, it is recognized that the normal human concentration of p75ECD in CSF in non-demented controls ranges from about 500 pg / ml to about 1300 pg / ml. In one embodiment, the reduced concentration of p75ECD in the CSF of a human test subject such that AD is diagnosed or prognosticated is less than 500 pg / ml, preferably less than 400 pg / ml. In one embodiment, the reduced concentration of p75ECD in the CSF of a human test subject such that AD is diagnosed or prognosticated is less than 300 pg / ml.

[0080] In another embodiment, the present disclosure provides a method for diagnosing and / or prognosing Alzheimer's disease (AD) in a test subject, the method comprising:

[0081] (a) obtaining a biological sample from the test subject,

[0082] (b) measuring the concentration of p75ECD and full-length p75 (p75FL) in the biological sample of the test subject, and

[0083] (c) comparing the concentration of p75ECD and p75FL in said biological sample from said test subject with the concentration of p75ECD and p75FL in a comparable biological sample obtained from a non-dementia control subject or a group of non-dementia control subjects,

[0084] Wherein Alzheimer's disease is diagnosed and / or prognosed when the ratio of p75ECD:p75FL is reduced in said test subject compared to said non-demented control subject or a group of non-demented control subjects.

[0085] In one embodiment, the ratio of p75ECD:p75FL in the test subject is reduced by at least 30% compared to the non-demented control subject or a group of non-demented control subjects. In one embodiment, the ratio is reduced by at least 50%. In one embodiment, the ratio is reduced by at least 75%. In one embodiment, the ratio is reduced by at least 90%. In one embodiment, the ratio is reduced by at least 99%.

[0086] In yet another embodiment, the present disclosure provides a method for diagnosing and / or prognosing Alzheimer's disease (AD) in a test subject, the method comprising:

[0087] (a) obtaining a biological sample from the test subject,

[0088] (b) measuring the concentration of p75FL in the biological sample of the test subject, and

[0089] (c) comparing the concentration of p75FL in said biological sample from said test subject to the concentration of p75FL in a comparable biological sample obtained from a non-dementia control subject or a group of non-dementia control subjects,

[0090] Wherein Alzheimer's disease is diagnosed and / or prognosed when the concentration of p75FL in the test subject is increased compared to the non-demented control subject or a group of non-demented control subjects.

[0091] In one embodiment, the concentration of p75FL in the test subject is increased by 10% to 500% (i.e., 1.1 to 5-fold) compared to the non-demented control subject or a group of non-demented control subjects. In one embodiment, the concentration of p75FL is increased by 20% to 300% (i.e., 1.2 to 3-fold). In one embodiment, the concentration of p75FL is increased by 30% to 200% (i.e., 1.3 to 2-fold). In a specific embodiment, the concentration of p75FL in human brain tissue is increased by at least 200% (2-fold) or preferably 300% (3-fold).

[0092] However, for the purposes of the methods of the first aspect of the present disclosure, the amount of p75FL and / or p75ECD in the test subject that can be considered to be an elevated concentration, a decreased concentration, or to provide a decreased ratio compared to the non-demented control subject or a group of non-demented control subjects may vary depending on the factors being compared (e.g., p75FL, p75ECD, or the ratio of p75ECD:p75FL), the specific body sample type used, and the age of the subject.

[0093] In one embodiment, the p75FL and / or p75ECD measured in the first aspect of the disclosure is a peptide.

[0094] Quantification or measurement of the concentration of p75FL and / or p75ECD peptides can be performed using suitable techniques known to those skilled in the art, such as by comparing staining to known protein standards to measure the absolute concentration of the molecule, or by comparing the relative staining intensity between the test subject and a normal non-dementia control using densitometry analysis to measure the relative concentration, including using the methods described herein. The concentration of p75FL and / or p75ECD peptides can be determined using techniques well known to those skilled in the art, such as quantification of results obtained from Western blot analysis, immunoassays such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and immunohistochemistry (e.g., using biopsied samples of tissue biopsies, including immunofluorescence staining). However, other methods well known to those skilled in the art can also be used to detect and quantify the level of p75FL and / or p75ECD, such as methods including detection of binding of p75FL and / or p75ECD to any other ligand (e.g., Aβ) that can bind to p75FL and / or p75ECD. Particularly suitable methods for determining the amount of p75FL and / or p75ECD present in a test body sample are immunoassays utilizing labeled molecules in various sandwich, competitive or other assay formats. These immunoassays will generate signals indicating the presence or absence of p75FL and / or p75ECD. In addition, the intensity of the signal generated by these immunoassays may be directly or indirectly (e.g., inversely proportional) related to the amount of p75FL and / or p75ECD present in the sample. Other suitable methods for determining the amount of p75FL and / or p75ECD present in a test body sample are methods including measurements of physical or chemical properties specific to p75FL and / or p75ECD, such as accurate molecular mass or nuclear magnetic resonance (NMR) spectra. Therefore, these methods can be performed using biosensors, optical devices coupled to immunoassays, biochips, analytical devices such as mass spectrometers, NMR analyzers, and chromatographic devices. Other suitable methods may include precipitation (e.g., immunoprecipitation), electrochemiluminescence (i.e., electrically generated chemiluminescence), electrochemiluminescence sandwich immunoassay (ECLIA), dissociation enhanced lanthanide fluorescence immunoassay (DELFIA), scintillation proximity assay (SPA), turbidimetry, nephelometry, latex enhanced turbidimetry, and nephelometry. In addition, methods known to those skilled in the art such as gel electrophoresis, Western blotting, and mass spectrometry may also be used alone or in combination with the above-mentioned other suitable methods. Those skilled in the art will recognize that other methods may be suitable for detecting the concentration of p75 or p75ECD peptides according to the methods described herein.

[0095] As will be appreciated by those skilled in the art, full-length p75FL peptide and p75ECD peptide can be distinguished using specific antibodies that recognize different regions of the p75FL peptide. For example, a first antibody can specifically recognize and bind to the extracellular domain (p75ECD) of p75 but not to the intracellular or transmembrane region, such as the anti-p75ECD antibody (rabbit polyclonal antibody, 9650) used herein; and a second antibody can specifically recognize and bind to the intracellular or transmembrane region (p75ICD) of p75 but not to the extracellular domain, such as the anti-p75NTR antibody (rabbit polyclonal antibody, AB1554, Millipore, US) or anti-NGF receptor p75 antibody (Catalog No. AB1554, Millipore, US) used herein. Those skilled in the art will recognize that in the methods disclosed herein, other suitable antibodies that specifically recognize and bind to the extracellular domain of p75 but not to the intracellular or transmembrane region, or specifically recognize and bind to the intracellular or transmembrane region of p75 but not to the extracellular domain can be easily used.

[0096] However, in one embodiment, there may be at least some p75ECD in the case of full-length p75, and the full-length p75 may be bound to cellular components. That is, in one embodiment, full-length p75 is bound to the cell membrane components of the cell preparation as a transmembrane protein, while soluble ("shed") p75ECD will be bound to the soluble components of the cell, tissue and / or body fluid preparation. In one embodiment, as will be appreciated by those skilled in the art, soluble ("shed") p75ECD can be distinguished from the p75ECD present in the case of full-length p75 on the basis of size (i.e., molecular weight) by Western blotting. Alternatively, in one embodiment, as will be appreciated by those skilled in the art, detergents or similar substances can be omitted from the cell preparation method, so that the cell membrane (and therefore the membrane-bound full-length p75) can be separated from the soluble components of the preparation (and therefore the soluble p75ECD) by centrifugation, and the soluble or "shed" p75ECD can be distinguished from the full-length p75ECD. It is also possible to determine the amount of p75ECD or the ratio of p75ECD:p75FL by quantifying the intensity of binding of the anti-p75ECD antibody to the sample and the intensity of binding of the anti-p75ICD antibody using appropriate controls and using simple calculations to calculate the absolute amount or ratio.

[0097] The method of the first aspect enables changes in the amount of p75FL and / or p75ECD or their ratio to be used as a diagnostic marker for the presence of Alzheimer's disease. However, the sensitivity and specificity of such a method may not only depend on the "quality" of the analysis of the method, but may also depend on the definition of what constitutes an abnormal result. That is, in general, for any particular marker, the distribution of marker levels for subjects with and without the disease overlaps, so that a diagnostic / prognostic test based on the marker cannot absolutely distinguish normal (i.e., non-demented) subjects from diseased subjects with complete accuracy. Therefore, in certain embodiments, the method may further include calculating a receiver operating characteristic (ROC) curve, for example by plotting the change in the amount of p75FL and / or p75ECD against the relative frequency of that value in "normal" and "disease" subjects. The area under the ROC curve calculated in this manner is then used as a measure of the probability that the change in the amount of p75FL and / or p75ECD determined in the test body sample allows a correct diagnosis or prognosis. Furthermore, ROC curves can be used even when the variation in the amount of p75FL and / or p75ECD determined may be considered inaccurate, since ROC curves can still be calculated as long as it is possible to rank the results. For example, the amounts of p75FL and / or p75ECD determined from a test body sample from a subject can be ranked by degree (e.g., 1 = low, 2 = normal, 3 = high). This ranking can then be correlated with the results from normal subjects and a ROC curve generated according to methods well known to those skilled in the art (e.g., Hanley and McNeil, 1982).

[0098] Thus, determination of the amount of p75FL and / or p75ECD in a test body sample may comprise the steps of: (i) contacting p75FL and / or p75ECD with a specific ligand, (ii) optionally removing unbound ligand, and (iii) measuring the amount of bound ligand. Bound ligand (which may be bound by covalent and / or non-covalent binding) will generate an intensity signal. As indicated above, the ligand may be selected from anti-p75FL and / or p75ECD antibodies or fragments thereof, but may also be selected from any other ligand that can bind to p75FL and / or p75ECD, such as any compound that binds to p75FL and / or p75ECD (including peptides, polypeptides, nucleic acids, aptamers (e.g., nucleic acid or peptide aptamers), glycoproteins such as fetuin and small molecules). However, preferably, the ligand is selected from anti-p75FL and / or p75ECD antibodies or fragments thereof (including polyclonal and monoclonal antibodies and fragments thereof capable of binding to p75FL and / or p75ECD such as Fv, Fab and F(ab)2 fragments, and recombinant antibodies such as single-chain antibodies (e.g., scFV antibodies)). Methods for preparing these ligands are well known to those skilled in the art.

[0099] In one embodiment, the ligand specifically binds to p75FL and / or p75ECD. When used in this article, the term "specific binding" means that the ligand should not significantly bind to another peptide, polypeptide or substance present in the test body sample (that is, significantly "cross-react" with it). Preferably, the binding affinity of the specifically bound p75FL and / or p75ECD is at least 3 times higher than any other related peptide, polypeptide or substance, more preferably at least 10 times higher, and most preferably at least 50 times higher. Non-specific binding may be tolerable if it can still be clearly distinguished and measured, for example, based on its size on a Western blot or by the relatively high abundance of p75FL and / or p75ECD in the sample, or if it can be controlled using a negative control sample or a normal subject control sample. The ligand can be covalently or non-covalently coupled to a label to allow the ligand to be detected and measured. Suitable labeling can be carried out by any direct or indirect method known to those skilled in the art.

[0100] As disclosed herein, it is recognized that p75FL and p75ECD concentrations can be easily determined from tissues and body fluids, including CSF and brain tissue. The levels of p75 and p75ECD peptides in serum and lymphocytes can also be easily detected using standard methods known to those skilled in the art, such as ELISA and Western blotting (see, e.g., Zhou et al., 2013). Therefore, similar techniques can also be used to optionally detect p75FL and / or p75ECD in other tissue and body fluid types. In one embodiment, p75FL is a transmembrane molecule and can be detected in cells. For example, p75 can be detected in a variety of different cell types, such as lymphocytes, macrophages, skin cells, Schwann cells, and many neuronal cells. Using techniques known to those skilled in the art, such as ELISA and Western blotting, p75 levels in lymphocytes derived from peripheral blood can be easily detected (see, e.g., Zhou et al., 2013).

[0101] However, those skilled in the art will recognize that p75FL levels can be quantified indirectly by measuring the amount of messenger RNA (mRNA) molecules encoding p75FL within cells. For example, p75 mRNA can be detected in a variety of different cell types, such as lymphocytes, macrophages, skin cells, Schwann cells, and many neuronal cells. In fact, p75 mRNA levels in lymphocytes derived from peripheral blood can be easily detected using techniques known to those skilled in the art, such as real-time reverse transcriptase PCR (real-time RT-PCR) (see, e.g., Zhou et al., 2013) and RT-PCR.

[0102] In one embodiment, the method of the first aspect can be used to perform prognosis of AD, wherein the concentration of p75ECD and / or P75FL and / or the ratio of p75ECD:p75FL in the test subject is altered compared to the concentration of p75ECD and / or P75FL and / or the ratio of p75ECD:p75FL in the non-dementia control subject or a group of non-dementia control subjects, even if there are no signs of AD symptoms or only weak signs of AD symptoms are present in the test subject.

[0103] As disclosed herein, it has also been recognized that p75ECD is a neurotrophic molecule that can provide protection against amyloid peptide β toxicity and reduce tau protein phosphorylation associated with tau disease. As disclosed herein, by administering p75 extracellular domain (p75ECD) in a mouse model overexpressing Aβ, signs and / or symptoms associated with neurological disorders such as Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA) or tau disease can be reduced. This administration can reduce the signs and / or symptoms of the neurological disorder by improving learning and memory, protecting neurons from neurodegeneration caused by amyloid peptide β, and reducing tau protein phosphorylation.

[0104] Therefore, in another aspect, the present disclosure provides a method of treating or preventing cerebral amyloid angiopathy (CAA) or tauopathy, the method comprising administering to a subject in need thereof a therapeutically effective amount of a p75 extracellular domain (p75ECD).

[0105] CAA is a disorder distinct from AD characterized by progressive deposition of Aβ in the walls of small to medium-sized blood vessels in the cerebral cortex and pia mater, causing vascular dementia. As disclosed herein, it has been found that in an AD mouse model, administration of p75ECD reduced CAA lesions following administration of p75ECD. Thus, in one embodiment, the present disclosure provides a method for treating or preventing cerebral amyloid angiopathy (CAA), the method comprising administering a therapeutically effective amount of a p75 extracellular domain (p75ECD) to a subject in need thereof.

[0106] Although unwilling to be bound by theory, it is believed that tauopathy is a class of neurodegenerative diseases, which is accompanied by pathological aggregation of defective hyperphosphorylated tau protein in the brain, which is called neurofibrillary tangles (NFTs). Tauopathy can include any neurodegenerative disease with tau protein hyperphosphorylation, including AD, Parkinson's disease, motor neuron disease, frontotemporal lobar degeneration, Huntington's disease and Pick's Disease. As disclosed herein, it has been found that the administration of p75ECD reduces the phosphorylation of tau protein at multiple sites such as the 396th, 262nd, 199th serine and the 231st threonine. Therefore, in one embodiment, the present disclosure provides a method for treating or preventing tauopathy, the method comprising administering a therapeutically effective amount of p75 extracellular domain (p75ECD) to a subject in need.

[0107] As used herein, the term "treating" is intended to mean that administration of p75ECD reduces or ameliorates at least some signs or symptoms of AD, CAA, or tauopathy.

[0108] As used herein, the term "prevention" is intended to mean that the administration of p75ECD occurs before the diagnosis of AD, CAA and / or tauopathy, and alleviates, improves or delays the onset of at least some signs or symptoms of ADD, CAA and / or tauopathy; or that the administration of p75ECD prevents the worsening of signs or symptoms of ADD, CAA and / or tauopathy. Therefore, the term "prevention" is intended to include preventive treatment.

[0109] The p75ECD administered can be from any target species (e.g., humans, mice, rats, rabbits, cats, dogs, goats, cows, horses, non-human primates, etc.). However, in one embodiment, the p75ECD is human p75ECD. In one embodiment, the p75ECD can have a nucleotide sequence provided in SEQ ID NO: 3 or an amino acid sequence provided in SEQ ID NO: 4.

[0110] In alternative embodiments, the p75ECD may be a functional fragment, variant, analog or derivative of the sequence. For example, a variant, analog or derivative of p75ECD (or a functional fragment thereof) is composed of a molecule that is different from p75ECD (or a functional fragment thereof), but retains similarity to p75ECD in terms of biological activity, particularly the ability to bind to neurotrophic factors and Aβ peptides. A variant, analog or derivative of p75ECD (or a functional fragment thereof) may have significant overall structural similarity to p75ECD (or a functional fragment thereof), or may only have structural similarity to one or more regions of p75ECD (or a functional fragment thereof) that are responsible for the biological activity. Typically, a variant, analog or derivative is provided by modification of one or more amino acids of the relevant amino acid sequence, or is the result of the modification. For example, a variant, analog or derivative comprising a functional fragment of the sequence provided in SEQ ID NO: 4 is provided by adding one or more amino acids to the amino acid sequence, deleting one or more amino acids from the amino acid sequence and / or replacing one or more amino acids from the amino acid sequence, or is the result of the above-mentioned modification. The inversion of amino acids and other mutational changes that cause changes in the amino acid sequence are also covered. Such analogs or derivatives can be prepared by introducing nucleotide changes in the polynucleotide molecule so that the desired amino acid changes are obtained after the mutagenized polynucleotide molecule is expressed, or by synthesizing an amino acid sequence incorporating the desired amino acid changes in other ways. The replacement of amino acids can include conservative or non-conservative amino acid replacements. Conservative amino acid replacement means that the amino acid residue is replaced with another amino acid having similar properties and does not significantly change the biological activity of the peptide or polypeptide. Exemplary conservative amino acid replacements are provided in Table 1 below. The specific conservative amino acids envisioned are: G, A, V, I, L, M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; and P, N α-alkyl amino acids, which may have a small amount of changes in sequence, but do not cause any significant reduction or change in biological activity. These changes can include conservative amino acid substitutions such as Gly, Ala, Val, Ile, Leu, Met; Asp, Glu, Asn, Gln; Ser, Thr; Lys, Arg, His; Phe, Tyr, Trp, His; and Pro, Na-alkyl amino acids; as well as non-conservative amino acid substitutions.

[0111] Table 1 Exemplary conservative amino acid substitutions

[0112]

[0113]

[0114] *Indicates preferred conservative substitutions

[0115] When analogs or derivatives are prepared by synthesis, the analogs or derivatives may include one or more amino acids not encoded by the genetic code, such as carboxyglutamic acid and hydroxyproline, or may include D-amino acids in place of L-amino acids. Thus, the analogs or derivatives may be mimetics, such as peptide mimetics, of p75ECD (or its functional fragments). However, analogs or derivatives of p75ECD (or its functional fragments) do not necessarily have amino acid sequence identity and / or similarity, and in fact, analogs or derivatives may not be proteins at all.

[0116] It should be understood that peptides encoded by amino acid sequences having minor changes from the amino acid sequences provided in SEQ ID NO: 4 and SEQ ID NO: 6 may still fall within the scope of the present invention, as long as the peptides are able to bind to p75 and prevent signal transduction through p75. Specifically, natural variants (or isoforms) of p75ECD or p75ECD peptides derived from non-human species are considered to fall within the scope of the p75ECD or p75ECD-Fc fusion peptides of the disclosed methods.

[0117] In one embodiment, p75ECD comprises p75ECD fusion, wherein p75ECD is fused to a fusion partner. The fusion partner can be any suitable fusion partner known to those skilled in the art, such as the Fc region of an immunoglobulin, human serum albumin (HSA) and bovine serum albumin (BSA), etc., as long as the p75ECD retains at least some of its biological activity. The fusion product can be expressed by expressing the product from a suitable construct encoding the fusion product, or produced by otherwise chemically connecting p75ECD to a fusion partner. The fusion partner should improve the stability and serum half-life of the p75ECD in vivo. In one embodiment, p75ECD is fused to the Fc domain of an immunoglobulin to form a p75ECD-Fc fusion. The Fc domain can be any suitable Fc domain from any suitable immunoglobulin. However, in a preferred embodiment, the Fc domain comes from human IgG.

[0118] In one embodiment, the Fc region can dimerize to produce a dimeric form of p75ECD. However, other fusion partners may be able to form other types of oligomers, such as trimers, tetramers, pentamers, etc. Therefore, p75ECD may be an oligomer such as a dimer, trimer, tetramer, pentamer, etc. In one embodiment, the p75ECD may be a dimer. In one embodiment, the p75ECD-Fc fusion may be a p75ECD-Fc dimer.

[0119] In one embodiment, the p75ECD-Fc fusion comprises a human p75ECD molecule linked to the Fc portion of human IgG1. One skilled in the art will recognize that the fusion may have a wide variety of nucleotide and amino acid sequences, depending on, for example, the linker used. However, in one embodiment, the nucleotide sequence of the human p75ECD-human IgG1 Fc domain fusion (human p75ECD-Fc) is provided in SEQ ID NO: 5.

[0120] Similarly, in one embodiment, the amino acid sequence of the human p75ECD-Fc fusion is provided in SEQ ID NO: 6. In one embodiment, SEQ ID NO: 6 comprises the p75ECD peptide at amino acids 1-169 of SEQ ID NO: 6, followed by a linker "DKTHTCPPCP" at residues 170 to 179, followed by the Fc domain at residues 180 to 396. However, it should be recognized that the p75ECD-Fc fusion can be a functional fragment, variant, analog or derivative of the sequence provided by SEQ ID NO: 6. Functional fragments, variants, analogs or derivatives are described elsewhere herein.

[0121] In one embodiment, p75ECD or p75ECD fusion is administered to the subject as a nucleotide molecule. Such a nucleotide molecule can be composed, for example, of an expression vector or an expression cassette. Preferred expression vectors include viral vectors capable of achieving transduction of the desired host cell type, such as retroviral vectors, adenoviral vectors, and vectors derived from adeno-associated viruses (AAV), such as those known to those skilled in the art. Viral vectors comprising nucleotide sequences encoding p75ECD or p75ECD fusions of the present disclosure can be used to provide an in vivo source of p75ECD or p75ECD fusions at the desired site for the treatment or prevention of neurological disorders.

[0122] In a preferred embodiment, p75ECD or p75ECD fusion is administered by a viral vector, which is known to provide a tool for delivering nucleotides to cells in a manner suitable for gene therapy. As will be appreciated by those skilled in the art, adeno-associated virus (AAV) vectors, lentiviral vectors or herpes simplex virus vectors can be used as viral vectors because they can all deliver nucleotides to cells for the purpose of gene therapy. Delivery of p75ECD or p75ECD fusions by viral vectors can mediate long-term expression of agents in the CNS (e.g., brain), and due to the chronic nature of many neurological disorders, the continuous secretion of p75ECD or p75ECD fusions is desirable. In one embodiment, the use of AAV vectors is advantageous because the vectors can be administered as a single injection (e.g., intracranial or intraventricular injection), can provide long-term secretion of agents, and are also considered to be very safe, and pathological events have not been reported in animals or humans. AAV vectors are known to be tools for gene therapy delivery. Therefore, in one embodiment, the viral vector can be an AAV vector. The AAV vector can be any suitable adeno-associated virus vector known to those skilled in the art, such as AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, etc. In one embodiment, the AAV vector is AAV8.

[0123] A "therapeutically effective amount" is any amount that elicits a beneficial or therapeutic effect in a subject. In one embodiment, the dose of an AAV vector for any suitable subject is in the range of 1 x 10 6 Up to 6x10 13 In one embodiment, the dose of AAV vector for any suitable subject is between 1×10 11 Up to 6x10 12 viral genomes / kg subject body weight.

[0124] As is well known to those skilled in the art, the dose of adeno-associated viral vector administered can vary. For example, for human subjects, in clinical trials conducted by the US National Institutes of Health, a "safe" dose of 6 x 10 12 Viral genomes (vg) / kg body weight. However, for the methods described herein, the dose of adenoviral vector administered to a human subject may be 1x10 6 Up to 6x10 13 In one embodiment, the dose of adenoviral vector administered to a human subject is in the range of 1 x 1010 Up to 6x10 12 In one embodiment, the dose of adenoviral vector administered to a human subject is between 1×10 11 Up to 3x10 12 In one embodiment, the dose of adenovirus can be higher, for example, 1x10 14 , 1x10 15 , 1x10 16 , 1x10 17 , 1x10 18 , 1x10 18 , 1x10 19 , 1x10 20 etc., as long as those doses are considered safe.

[0125] The dose of AAV vectors used in human subjects can be converted to doses suitable for use in other animals based on body surface area using the US Food and Drug Administration (FDA) criteria for converting pharmaceutical equivalent doses between species [human equivalent dose in mg / kg = animal dose in mg / kg × (animal body weight in kg / human body weight in kg) 0.33] (FDA, 2003). Thus, a "safe" dose in mice may be 8 × 10 10 However, those skilled in the art will recognize that the dose of adeno-associated viral vector administered to a mouse subject may be 1×10 5 Up to 1x10 13 In one embodiment, the dose of adeno-associated virus vector administered to a mouse subject can be 1x10 8 Up to 1x10 12 In one embodiment, the dose of adeno-associated virus vector administered to a mouse subject can be 1x10 9 Up to 1x10 11 within the viral genome.

[0126] In one embodiment of this aspect of the invention, the p75ECD or p75ECD-Fc fusion is a recombinant peptide. The recombinant p75ECD or p75ECD-Fc fusion peptide can be produced using any suitable technique known to those skilled in the art, such as by expression in a cultured bacterial system (e.g., Escherichia coli), a yeast expression system, an insect expression system, a viral expression system, or in a eukaryotic cell expression system, or the peptide can be artificially synthesized, etc., all of which are known to those skilled in the art.

[0127] In one embodiment, the dosage of the recombinant p75ECD or p75ECD-Fc fusion peptide administered can be any suitable amount that induces a beneficial or therapeutic effect in a subject, and may vary with the route of administration. In one embodiment, the dosage is about 0.01 to about 500 mg / kg of subject body weight per day, which can be administered in a single dose or multiple doses. Preferably, the dosage is about 0.1 to about 250 mg / kg per day, more preferably about 0.5 to about 20 mg / kg per day.

[0128] In one embodiment, the p75ECD, p75ECD fusion, p75ECD-Fc or AAV-p75ECD-Fc of this aspect of the disclosure is provided in combination with a pharmaceutically acceptable carrier, excipient and / or diluent. Suitable pharmaceutically acceptable carriers, excipients and / or diluents are well known to those skilled in the art.

[0129] The p75ECD, p75ECD fusion, p75ECD-Fc or AAV-p75ECD-Fc of this aspect of the disclosure can be administered to the subject by any suitable route, such as oral, intravenous, intramuscular, intraperitoneal, intranasal, intracranial, intrathecal or intracerebroventricular routes. However, preferably, the p75ECD or p75ECD-Fc is administered intraperitoneally, intramuscularly, intrathecally or intracerebroventricularly.

[0130] In another aspect, a method for protecting neurons from apoptosis and / or neurodegeneration by blocking signal transduction through p75 is provided, the method comprising administering a therapeutically effective amount of p75ECD or an analog thereof to a subject in need thereof. In one embodiment, the p75ECD or an analog thereof prevents the binding of a neurotoxic molecule selected from Aβ, proNGF, proBDNF and proNT3 to p75. The method of this aspect uses the embodiments described above.

[0131] In another aspect, a method for treating and / or preventing a neurodegenerative disorder or tauopathy is provided, the method comprising administering to a subject in need thereof a therapeutically effective amount of a p75 extracellular domain (p75ECD) or an analog thereof, wherein the neurodegenerative disorder or tauopathy is selected from the group consisting of Alzheimer's disease, CAA, amyotrophic lateral sclerosis (ALS), Down syndrome, Huntington's disease, Parkinson's disease, motor neuron disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Pick's disease, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, tangle-only dementia, white matter tauopathy containing glomerular ganglia, frontotemporal dementia, and Parkinson's disease linked to chromosome 17. The method of this aspect uses the embodiments described above.

[0132] Example

[0133] Example 1 Diagnosis and treatment of neurological diseases

[0134] Materials and methods

[0135] Reagents and antibodies

[0136] The vectors of AAV8-CBA-p75NTR-ECD-Fc (AAV-ECD-Fc) and AAV8-CBA-GFP (AAV-GFP) were produced and purified by Virovek (Hayward, CA, USA). pcDNA3-human full-length p75NTR came from Dr. Rainer Niedenthal (Institute for Physiological Chemistry / Biochemistry, Hannover Medical School, Hannover, Germany). Synthetic Aβ42 (Catalog No. 62080) and mixed Aβ42 (Catalog No. 62046) were purchased from American Peptide (Sunnyvale, CA, USA).

[0137] Human p75ECD-Fc and Fc recombinant proteins were prepared as follows: Human p75ECD and Fc nucleotide constructs were produced using primers (Table 2) and PCR, respectively, under standard conditions using human cDNA as the DNA source, and then for the p75ECD-Fc fusion protein, the fragments were fused together by overlapping PCR using standard conditions.

[0138] Table 2 PCR primer pair sequences for human p75ECD-Fc and Fc cloning

[0139]

[0140] After the p75ECD-Fc and Fc constructs were generated, they were cloned into pET-28a(+) vector (Merck Millipore) using Nco-I and EcoR-I restriction enzymes and then transformed into E. coli DH10B competent cells and subsequently transformed into E. coli BL21 competent cells. To express the p75ECD-F and Fc recombinant peptides, E. coli BL21 stocks of p75ECD-Fc and Fc were inoculated in a large volume of Luria Broth (LB) medium and incubated at 37°C until the OD value reached 0.4-0.6, and then 1 mM IPTG was used to induce the expression of p75ECD and Fc in E. coli for 3 hours at 37°C. E. coli was centrifuged at 5,000RPM for 15 min at 4°C, and the precipitate was resuspended in a hypotonic lysis buffer (10 mM tris, 2 mM EDTA, 25 mM NaCl, 0.1% Triton X-100, 1-2 mg / ml lysozyme, pH = 8.0), and the supernatant was extracted by high-speed centrifugation (15,000RPM) at 4°C for 30 min after sonication. To harvest p75ECD-Fc and Fc, the supernatant was passed through a protein-G sepharose column, and then p75ECD-Fc and Fc were eluted from the protein-G sepharose by applying 100 mM glycine pH 2.5. Subsequently, the protein in the glycine solution was dialyzed in PBS at 4°C, and the purity of the peptide was verified by Coomassie blue staining. P75EC-Fc can also be produced in yeast, mammalian cells, and insect cells using any conventional production method for recombinant proteins.

[0141] The antibodies used in this study were: anti-p75NTR antibody (rabbit polyclonal, AB1554, Millipore, US), anti-p75ECD antibody (rabbit polyclonal, 9650, a generous gift from Professor Moses Chao (Department of Cell Biology, Skirball Institute, New York, USA)), anti-TACE (ADAM-17) antibody (rabbit polyclonal, ab2051, Abcom, US, for detection of tumor necrosis factor α converting enzyme), anti-human IgG Fc antibody (mouse monoclonal, MAB1302, Millipore, US, for detection of p75ECD-Fc), anti-β-amyloid peptide antibody (mouse monoclonal, 6E10, Covance, US), anti-sAPPβ antibody (rabbit polyclonal, SIG-39138, Covance, US), anti-amyloid precursor protein C-terminal antibody (751-770aa) (rabbit polyclonal, 171610, Calbiochem, US, for detection of APP full length and CTF cleavage products), anti-APP C-terminal (678-695aa) antibody (rabbit polyclonal, 6687, kindly provided by Prof. C. Haass (Ludwig-MaximiliansUniversity Munich, Munich, Germany)), anti-BACE C-terminal antibody (mouse monoclonal, clone 61-3E7, Millipore, US, for detection of β-site APP cleavage enzyme), anti-BACE1 Extracellular domain antibody (mouse monoclonal, MAB931, R&D, US, for detecting cellular BACE1 levels in in vitro studies), anti-neprilysin antibody (rabbit polyclonal, AB5458, Millipore, US), anti-RAGE antibody (rabbit polyclonal, clone DD / A11, Millipore, US, for detecting receptor for advanced glycation end products), anti-LRP (mouse monoclonal, 5A6, Calbiochem, US, for detecting low-density lipoprotein receptor-related protein), anti-IDE N-terminal (97-273) antibody (rabbit polyclonal, PC730, Millipore, US, for detecting insulin-degrading enzyme), anti-NeuN antibody (mouse monoclonal, MAB377, Millipore, US, for detecting neuronal nuclei), anti-ChAT antibody (rabbit polyclonal, ab137349, Abcam, US, for detecting choline acetyltransferase), anti-MAP2 antibody (mouse monoclonal, 05-346, Millipore, US, for detection of microtubule-associated protein 2), anti-glial fibrillary acidic protein (GFAP) antibody (rabbit monoclonal, 04-1062, Millipore, US,for detecting active astrocytes), anti-active caspase-3 antibody (rabbit polyclonal, ab13847, Abcam, US, for detecting apoptosis), anti-CD45 antibody (rat monoclonal, ab25386, Abcam, US, for detecting active microglia), anti-SNAP25 antibody (rabbit polyclonal, AB1762, Millipore, US, for detecting synaptotagmin-25), anti-PSD95 antibody (mouse monoclonal, MAB1596, Millipore, US, for detecting postsynaptic density protein 95), anti-SYP antibody (mouse monoclonal, MAB332, Millipore, US, for detecting synaptic vesicle protein), anti-synapsin I antibody (rabbit polyclonal, 574778, Millipore, US), anti-VAMP1 antibody (rabbit monoclonal, ab151712, Abcam, US, for detection of vesicle-associated membrane protein 1), anti-pS396 antibody (rabbit polyclonal, 11102, Signalway Antibody, US, for detection of tau protein phosphorylated at serine 396), anti-pT231 antibody (rabbit polyclonal, 11110, Signalway Antibody, US, for detection of tau protein phosphorylated at threonine 231), anti-pS262 antibody (rabbit monoclonal, ab92627, Abcam, US, for detection of tau protein phosphorylated at serine 262), anti-pS199 antibody (rabbit monoclonal, ab81268, Abcam, US, for detection of tau protein phosphorylated at serine 199), anti-total tau antibody (rabbit polyclonal, ab39524, Abcam, US), anti-GSK3 (α+β) (phospho Y279+Y216) antibody (rabbit polyclonal, ab52188, Abcam, US), anti-GSK3β antibody (rabbit monoclonal, ab18893, Abcam, US, for detecting glycogen synthase kinase-3β), anti-GSK3β (phospho S9) antibody (rabbit monoclonal, ab75814, Abcam, US, for detecting glycogen synthase kinase-3β phosphorylated at serine 9), anti-3-phosphoglyceraldehyde dehydrogenase (GAPDH) antibody (sheep polyclonal, OSG00034W, Osenses, Australia) and anti-β-actin antibody (rabbit polyclonal, ab8227, Abcam, US).

[0142] Human CSF and brain tissue sample preparation

[0143] Studies on human cerebrospinal fluid (CSF) samples were approved by the Ethics Committee of Daping Hospital of the Third Military Medical University. For CSF sampling, a total of 56 study participants (n=29 for AD and n=27 for ND) were recruited. Written informed consent for lumbar puncture and CSF sampling was obtained from all participants or their agents. The diagnosis of AD was made according to the criteria of the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS-ADRDA) (Raffi, 2013; McKhann et al., 1984), including the Mini-Mental State Examination (MMSE). CSF samples were collected in sterile glass tubes at 8:00 AM after an overnight fast and immediately stored at -80°C for future biochemical analysis. Studies on human brain samples were approved by the Ethics Committee of University of South Florida (Pro00011605).Post-mortem human brain samples from histologically confirmed Alzheimer's disease cases and age-matched non-demented individuals were obtained from Banner Sun Health Research Institute (Sun City, AZ, USA).

[0144] animal

[0145] APPswe / PS1DE9 transgenic mice (which are used as an AD model and are referred to herein as "AD mice") overexpress the Swedish mutation of APP and also have a PS1 deletion in exon 9, which causes an increased parenchymal Aβ load accompanied by the appearance of Aβ plaques from 4 months of age, glial cell activation, and cognitive impairment demonstrated by the radial arm water maze at 6 months of age and observed using the Morris water maze test at 12-13 months of age.

[0146] For some experiments, AD / p75- / - and AD / p75+ / + mice were used to investigate the effect of p75 on amyloid precursor protein (APP) processing. Transgenic (Tg) mouse strains expressing APPswe / PS1DE9 (AD) on a C57BL / 6 background (Castellani and Perry, 2012) and p75- / - (p75NTR / exon III- / -) mice on a 129sv background were obtained from Jackson Laboratories (no. 005864) and maintained at the Third Military Medical University Animal House and the University of South Australia. To obtain AD / p75- / - and AD / p75+ / + mice to investigate the effect of p75 on amyloid precursor protein (APP) processing, p75- / - mice were crossed with AD mice and the animals were studied after PCR genotyping according to the supplier's instructions.

[0147] All animals were maintained under standard conditions at 22°C and a 12 h light / dark cycle with ad libitum access to food and water. All mouse husbandry procedures performed were approved by the Third Military Medical University Animal Welfare Committee and the Animal Ethics Committee of South Australian Pathology, Department of Pathology, University of South Australia (16b / 12) in accordance with NHMRC guidelines. AD mice were randomly divided into groups in the in vivo experiments.

[0148] Measuring p75ECD levels in human cerebrospinal fluid (CSF) and in human and mouse brain

[0149] The levels of p75ECD in human CSF (n=29 for AD and n=27 for ND) and human brain tissue samples (n=12 for AD and n=12 for ND) were quantified by ELISA and Western blot. ELISA quantification of human p75ECD in CSF was performed using the NGFR / TNFRSF16 kit (Catalog No. DY367, R&D Systems, US) according to the manufacturer's instructions. p75ECD in human brain homogenate samples was detected by Western blot using anti-NGF receptor p75 antibody (Catalog No. AB1554, Millipore, US). The same process was used to detect p75ECD in Tris-buffered saline (TBS) fractions of brain homogenates of 12-month-old AD mice (Tg) and wild-type littermates (Wt) (n=13 for Tg and n=14 for Wt).

[0150] Measuring TACE activity in human and mouse brain

[0151] Tumor necrosis factor alpha converting enzyme (TACE) is the major enzyme that cleaves p75 and releases p75ECD. TACE activity in human CSF (n=29 for AD and n=27 for ND) was measured using 520TACE activity assay kit (Catalog No. 72085, ANASPEC, US) was used to measure. Enzyme activity was monitored at excitation / emission = 490 nm / 520 nm according to the manufacturer's instructions. TACE antibody (Catalog No. ab2051, Abcom, US) was also used to detect the expression level of TACE in the brain of AD mice by Western blotting.

[0152] Preparation of oligomeric forms of Aβ42

[0153] In this study, the oligomeric form of Aβ42 was used. Synthetic Aβ42 and mixed Aβ42 were prepared according to the previously described process. Briefly, the Aβ42 peptide was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, catalog number 105228, Sigma, US) at a concentration of 1 mg / ml and divided into aliquots (100 μg / tube) in eppendorf tubes. HFIP was allowed to evaporate in a fume hood, and the resulting transparent peptide film was dried under vacuum overnight, and then the deposited particles were stored at -20 ° C until use. For the oligomerization of Aβ42, the dried deposited particles were resuspended in Dulbecco's modified Eagle medium (DMEM) to a final concentration of 100 μM, and then incubated at 4 ° C for 24 hours. The oligomeric form of Aβ42 was checked by Western blotting before use (Saadipour, K. et al., 2013).

[0154] SH-SY5Y cell culture and assays

[0155] Human neuroblastoma SH-SY5Y was purchased from the Cell Resource Center (CRC) of Peking Union Medical College and cultured in a growth medium consisting of a 1:1 mixture of DMEM / Ham's F-12 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% antibiotic-antimycotic mixture at 37° C. in a humidified incubator with 5% CO 2. All in vitro assays were performed at least three times.

[0156] To evaluate the effect of p75ECD against Aβ toxicity in vitro, cell viability assay was performed using 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT). After 24 h of growth in 96-well culture plates (50,000 cells / well), SH-SY5Y cells were treated with 0.3-10uM Aβ42 or 10ug / ml recombinant p75ECD-Fc combined with 0.3-10uM Aβ42 for 24 h, and then incubated with 0.5mg / ml MTT (Catalog No. M5655, Sigma, US) at 37°C for 4 h. Insoluble purple formazan crystals produced by mitochondrial dehydrogenases in living cells were then dissolved in 100 μl of 10% (w / v) SDS in HCl (0.01 M) solution at 37°C for 15 min, and the optical density at 563 nm was measured by a microplate reader (Synergy H4, Bio Tek).

[0157] In order to evaluate the protective effect of p75ECD against neurite collapse, neurite growth assay was performed. After incubation in DMEM / F-12 for 24 hours, SH-SY5Y cells were cultured in a medium containing 1% FBS and 10 μM all-trans retinoic acid (Catalog No. R2625, Sigma, US) diluted in DMSO for 7 days, then treated with 5 μM Aβ for 24 hours in the presence or absence of 10 μg / ml recombinant ECD-Fc, and then fixed with 4% polyformaldehyde for 10 min. Images of 50 neurons in total from different groups were obtained by microscopy, and the length of neurites was measured using ImageJ and the data were expressed as mean ± sem, and significance was obtained when p < 0.05. The variability between groups was determined by one-way ANOVA and Tukey's post hoc test.

[0158] To examine the effect of p75ECD on tau phosphorylation in vitro, SH-SY5Y cells were differentiated in 10 μM all-trans retinoic acid for 3 days and then incubated with Aβ42 oligomers at different concentrations from 0.1 to 10 uM in the presence or absence of 10 μg / ml recombinant p75ECD-Fc or rabbit anti-p75ECD antibody (9650, Professor Moses Chao of New York University) for 24 hours. Cell lysates were subjected to Western blotting using pS262 antibody (Catalog No. ab92627, Abcam, US), total tau antibody (Catalog No. ab39524, Abcam, US), GSK3 (α+β) antibody (Catalog No. ab52188, Abcam, US), pSer9 GSK3β antibody (ab75814, Abcam, US) and GAPDH antibody (Catalog No. OSG00034W, Osenses, Australia) as a loading control.

[0159] Primary cortical neuron cultures and assays

[0160] Cortical neurons were isolated from 1-day-old 129sv mouse brains and cultured on coverslips pre-coated with poly-D-lysine (Catalog No. P7280, Sigma, US). 24 hours after inoculation, neurons were treated with 5 μM Aβ42 oligomers for 24 hours in the presence or absence of recombinant p75ECD-Fc (10 μg / ml), and then neurons were fixed with 4% polyformaldehyde for 10 min, followed by staining with DAPI (Catalog No. D9542, Sigma, US) and MAP-2 (Catalog No. 05-346, Millipore, US). Neuron images were collected and analyzed in the manner described above.

[0161] To investigate the effect of Aβ on TACE expression, cortical neurons were isolated from 1-day-old 129sv mice and cultured in 6-well plates. At 72 hours after inoculation, neurons were incubated with Aβ42 oligomers at concentrations of 0.3, 1, 3, and 10 μM for 24 hours, and then cell lysates were harvested in RIPA buffer containing a protease inhibitor cocktail. Cell lysates were sonicated and then centrifuged at 14,000 rpm for 10 min at 4 ° C. The total protein concentration of the supernatant was determined using a BCA protein assay kit (Thermo Scientific, Rockford, USA), and 30 μg of total protein was Western blotted using a TACE antibody (Catalog No. ab2051, Abcom, US) to quantify the expression level of TACE protein.

[0162] To elucidate whether Aβ mediates the amyloidogenic processing of APP through p75, cortical neurons were isolated from 1-day-old AD / p75- / - and AD / p75+ / + mice carrying overexpressed APPswe / PS1DE9 with / without the p75 gene and cultured in 6-well plates. 72 hours after plating, neurons were treated with 0.1 and 0.3 μM Aβ42 oligomers for 24 hours, and then cell lysates were harvested in RIPA buffer and Western blotting was performed using APP antibody (Catalog No. 171610, Calbiochem, US), sAPPβ antibody (Catalog No. SIG-39138, Covance, US) and BACE1 antibody (Catalog No. MAB931, R&D, US) to quantify endogenous APP.

[0163] AAV-ECD-Fc vector production and purification

[0164] Recombinant baculovirus was generated using the Bac-to-bac system following the manufacturer's protocol (Invitrogen). First, DH10Bac competent bacteria were transformed individually with the pFastBac shuttle plasmid containing the target gene, two recombinant bacmid clones (white colonies) from each construct were selected, and DNA "minipreps" were prepared using standard techniques. The DNA minipreps were then used to transfect Sf9 cells to generate recombinant baculovirus. The recombinant baculovirus was amplified once and used for AAV production. Specifically, Sf9 cells were cultured to approximately 1x10 7 cells / ml and diluted to approximately 5x10 6Cells / ml. A 200 ml volume of diluted Sf9 culture was double infected with an auxiliary recombinant baculovirus containing AAV Rep and Cap genes and a second recombinant baculovirus containing the target gene for AAV vector production. Three days after infection, Sf9 cells were harvested and lysed in SF9 lysis buffer (50 mM Tris-HCl, pH 7.8, 50 mM NaCl, 2 mM MgCl2, 1% Sarkosyl, 1% Triton X-100 and 140 units / ml of Benzonase). Genomic DNA was digested by incubation at 37 ° C for 1 hour. Cell debris was removed by centrifugation at 8,000 rpm for 30 min. The clarified lysate was loaded onto a CsCl stepwise gradient and ultracentrifuged at 28,000 rpm for 20 hours. The virus band was extracted by a syringe with an 18-gauge needle, loaded onto a second CsCl and subjected to linear ultracentrifugation at 65,000 rpm for 20 hours. The virus band was then drawn off and passed through 2 PD-10 desalting columns (GE HealthCare) to remove CsCl and detergent, and simultaneously exchanged with PBS buffer containing 0.001% pluronic F-68. Quantitative real-time PCR (qPCR) was performed using QPCR primers corresponding to the target gene to determine the AAV vector genome copy number.

[0165] Plasmids containing full-length p75 (NM_002507) and plasmids containing p75-ECD-Fc purchased from the American Type Culture Collection (ATCC) were provided to Virovek, and p75ECD-Fc was inserted into an AAV8 vector (provided by Virovek) containing an AAV8 capsid sequence with an AAV2 phospholipase domain to improve the potency of AAV8. The sequence of the AAV8 capsid sequence with an AAV2 phospholipase domain is provided in SEQ ID NO: 13, wherein the AAV2 phospholipase domain sequence comprises nucleotides 26 to 431. The AAV-ECD-Fc vector was generated, produced, and purified by Virovek. Briefly, the target gene was cloned into Virovek's AAV production shuttle plasmid. Recombinant baculovirus was generated using Virovek's proprietary BAC-TO-AAV technology and used to infect Sf9 cells to produce AAV vectors. AAV vectors were purified by double CsCl ultracentrifugation and buffer exchanged using PD-10 desalting columns. AAV titers were determined using quantitative real-time PCR and purity was verified by SDS-PAGE followed by the SimplyBlue Safestain assay ( Figure 1). The p75-ECD fragment was PCR amplified using the p75 plasmid as a template and the primers provided in SEQ ID NO: 7 and SEQ ID NO: 8. The Fc fragment was PCR amplified using p75-ECD-Fc as a template and the primers provided in SEQ ID NO: 11 and 12. The PCR was performed under standard conditions.

[0166] Intraventricular injection

[0167] APP / PS1 transgenic mice were injected with AAV-ECD-Fc or AAV-GFP into the left lateral ventricle once at 3 months of age for "prevention" or once at 9 months of age for "treatment" of AD. The injection coordinates were obtained from the bregma according to the stereotaxic coordinates: anterior-posterior, -0.6 mm; lateral, 1.2 mm; ventral, 2.2 mm. The AAV injection dose was 8×10 10 The viral genomes were converted from safe doses used in patients at the US National Institutes of Health (ClinicalTrials.gov) on the basis of body surface area according to US Food and Drug Administration (FDA) standards for converting drug equivalent doses between species [human equivalent dose in mg / kg = animal dose in mg / kg × (animal body weight in kg / human body weight in kg) 0.33] (Food and Drug Administration, 2003).

[0168] Behavioral phenotyping

[0169] Operators and investigators were completely blinded to grouping and intervention information. All animals underwent behavioral tasks at 12 months of age, including Morris water maze, Y-maze and open field test. The Morris water maze test was performed according to the previously reported protocol with minor modifications (Wang YJ et al., 2010; Yau JL et al., 2007; Markowska, AL et al., 1993). In brief, it consists of 4 platform tests per day for 4 consecutive days and subsequent exploration tests. Performance was video recorded and analyzed by image analysis software (ANY-maze, Stoelting). In the platform test, path distance and latency were measured; and in the exploration test, quadrant time and ring crossing were measured (Markowska, AL et al., 1993). Spontaneous alternation tests and new arm exploration were performed in the Y-maze. For the spontaneous alternation test, mice were allowed to move freely through the maze within 5 minutes. Alternation is defined as entering 3 arms in sequence in an overlapping three-group setting (Sarter, M. et al., 1988). The alternation percentage is calculated as the ratio of the actual (total number of alternations) to the possible number of alternations (defined as the number of arm entries minus 2) multiplied by 100. For the new arm exploration, the mouse is placed at the end of the selected starting arm (starting arm) and allowed to explore the maze for 5 min (Jung, WR et al., 2008) when one arm (new arm) is blocked. After an interval of 2 hours, the mouse is allowed to freely explore all 3 arms. The number of new arm entries and the time spent in the new arm are calculated as the percentage of the total time in all 3 arms during the 3min retrieval test. For the open field test, each mouse is placed in the center of the open field device (Dellu, F. et al., 1992; Conrad, CD et al., 1999). The path is tracked for 3 min using a computer tracking system (Limelight, ActiMetrics). In addition, standing, combing, defecation and urination are recorded.

[0170] Tissue sampling

[0171] Animals were humanely killed by overdose of 6% chloral hydrate (6ml / kg). Blood samples were obtained from the right atrium, and then intracardiac perfusion was performed with 100ml of 0.1% NaNO2 in normal saline. The brain was separated and weighed on a digital electronic balance (BX-420H, Shimadzu Scientific Instruments) with a readability of 1mg. The right cerebral hemisphere for histological analysis was fixed in 4% polyoxymaldehyde (pH 7.4) for 24h, and incubated in 30% sucrose for 24h for subsequent cryoprotection. Coronal sections of the brain were cut out using a freezing ultrathin slicer with a thickness of 35μm, and stored in PBS containing 0.1% sodium azide at 4°C until use. The left cerebral hemisphere was snap-frozen in liquid nitrogen, and stored at -80°C for future biochemical analysis.

[0172] Analysis of p75ECD-Fc expression

[0173] The expression of p75ECD-Fc in the brain of AD mice after intraventricular injection of AAV-ECD-Fc was examined by ELISA, Western blotting and immunohistochemistry. For ELISA, fresh brains were weighed and homogenized in TBS. The homogenate was centrifuged at 10,000 × g for 10 minutes at 4 ° C, and the resulting supernatant was collected. p75ECD-Fc in TBS was detected as described above. For Western blotting, 10 μl TBS fractions of brain extracts were mixed with an equal volume of 2 × loading buffer, loaded on a 10% SDS-PAGE gel and electrophoresed. The gel was transferred to a nitrocellulose membrane, which was then incubated with anti-human IgG Fc antibody (Millipore) or anti-NGF receptor p75 antibody (Millipore), followed by incubation with IRDye 800CW secondary antibody (LI-COR), and scanned using an Odyssey fluorescence scanner. For immunohistochemistry, anti-human IgG Fc antibody (catalog number MAB1302, Millipore, US) was used, and tissue sections were stained according to the free-floating immunohistochemistry protocol described previously (Wang YJ et al., 2009b). Immunofluorescence was performed to check the colocalization between ECD-Fc and neurons or astrocytes or microglia, and human IgG Fc antibody (catalog number MAB1302, Millipore, US) was used for p75 ECD-Fc, NeuN antibody (catalog number MAB377, Millipore, US) was used for neurons, GFAP antibody (catalog number 04-1062, Millipore, US) was used for astrocytes, and CD45 antibody (ab25386, Abcam, US) was used for microglia. Confocal fluorescence microscope (Radiance 2000 MP, Bio-Rad) was used to observe the sections.

[0174] Pathology and quantitative image analysis

[0175] For dense Aβ plaque staining, a series of sections were fixed and stained with Congo red (Catalog No. C6277, Sigma, US). Briefly, a series of 5 equally spaced tissue sections (~1.3 mm apart) across the entire brain were treated with sodium chloride working solution (containing sodium chloride saturated in 80% ethanol and 0.01% sodium hydroxide) at room temperature for 20 minutes, then placed directly in Congo red working solution (containing Congo red saturated in sodium chloride working solution) for 45 minutes, and rapidly dehydrated in anhydrous ethanol. Images were collected at 4× magnification using constant bulb temperature and exposure, with all images acquired during the same observation period. Staining for total Aβ, microgliosis, astrogliosis, and microhemorrhage was processed as described previously (Wang, YJ et al., 2009b). Briefly, sections were randomly selected and stained for total Aβ (Catalog No. 6E10, Covance, US), microglia (Catalog No. ab25386, Abcam, US), astrocytes (Catalog No. 04-1062, Millipore, US) and tau phosphorylation (pS396 antibody, Catalog No. 11102, Signalway Antibody, US) using free-floating immunohistochemistry. The sections were incubated overnight at 4 ° C with the first antibody and further developed with biotin-labeled secondary antibodies and ABC kits (Catalog No. PK6200, Vector Laboratories, US), using diaminobenzidine and glucose oxidase as substrates. The neocortex and hippocampal regions were selected for automatic quantification of Aβ, microglia, astrocyte immunostaining, and Congo red-positive Aβ plaques by ImageJ to obtain the area fraction of total positive staining for the analyzed tissue area. The group mean and sem were calculated using the average of the individual measurements.

[0176] For cerebral amyloid angiopathy (CAA) staining, a series of 5 consecutive sections were stained using the Congo red method. Microhemorrhage staining and quantification were performed as previously described (Wang, YJ et al., 2009a). Briefly, a series of 5 sections were fixed on slides and stained for hemosiderin using 2% potassium ferrocyanide (Catalog No. 455989, Aldrich, US) in 2% hydrochloric acid for 15 min, then counterstained for 10 min at room temperature in 1% neutral red (Catalog No. N4638, Sigma, US) solution. Microhemorrhages on all sections of each brain were counted under a microscope, and the average number of hemosiderin deposits per section was calculated. All image analyses were processed in a blinded manner.

[0177] Colocalization of Aβ plaques and ECD-Fc

[0178] A series of 3 equally spaced tissue sections covering the hippocampus of each animal in 8 mice were subjected to immunohistochemistry or immunofluorescence staining. For immunohistochemistry, sections were stained with Congo red and subsequently incubated with anti-human IgG Fc antibodies. For immunofluorescence, sections were stained with thioflavin S (Cat. No. T3516, Sigma, US) and anti-human IgG Fc antibodies and observed using a confocal fluorescence microscope (Radiance 2000MP, Bio-Rad).

[0179] ELISA quantification of Aβ

[0180] ELISA analysis of brain Aβ was processed as described previously (Wang YJ et al., 2009b). Briefly, frozen brains were homogenized and sonicated in TBS containing protease inhibitors. The homogenate was centrifuged at 100,000g for 1 h at 4°C and the resulting supernatant was collected, which represented the TBS soluble fraction. The resulting precipitate was suspended in water containing 2% SDS and protease inhibitors and sonicated. The SDS-solubilized homogenate was centrifuged at 100,000 × g for 1 h at 4°C and the resulting supernatant was collected, which represented the SDS soluble fraction. The resulting precipitate was then extracted and centrifuged in 70% formic acid (FA), and the resulting supernatant was collected, which represented the FA insoluble fraction. Prior to the ELISA assay, the formic acid extract was neutralized by diluting 1:20 in 1M Tris phosphate buffer (pH 11) and then diluted in sample buffer. The concentrations of Aβ40 and Aβ42 in brain extracts and serum were quantitatively measured by ELISA according to the manufacturer's instructions (SIG-38954 for Aβ40 and SIG-38956 for Aβ42, Covance, US). The final values ​​of brain Aβ were expressed as pmoles per gram of brain wet weight using the wet weight of brain tissue in the original homogenate.

[0181] β-Secretase activity assay

[0182] Beta-secretase activity was measured according to the manufacturer's protocol (Catalog No. K360-100, Bio Vision, US). In brief, fresh brain tissue was pulverized in liquid nitrogen and divided into aliquots for secretase determination. Secretase was extracted in the extraction buffer provided in the product kit, and their activity was measured by adding secretase-specific peptides coupled to reporter molecules EDANS and DABCYL. The peptide was physically separated from EDANS and DABCYL by the cutting of secretase, allowing the release of fluorescent signals, which were proportional to the enzyme activity level of secretase. Fluorescent signals were read at an excitation wavelength of 355nm and an emission wavelength of 510nm, with a cutoff of 495nm.

[0183] Analysis of neuronal apoptosis

[0184] In order to measure apoptotic cells, TUNEL staining and immunofluorescence using anti-activated caspase-3 antibodies were used. Five sections of each animal were used for TUNEL or activated caspase-3 staining. For TUNEL staining, apoptotic cells in 35 μm formalin-fixed coronal brain sections were labeled using an in situ death detection kit POD (catalog number 11684817910, Roche) according to the manufacturer's instructions. The sections were fixed on slides and dried in a fume hood, then incubated with a blocking solution for 10 minutes at room temperature, and washed twice with PBS. After incubation for 2 min in a permeabilization solution on ice, the sections were rinsed twice with PBS, and the area around the sections was dried. TUNEL reaction mixture was added to the sections, and in a humidified incubator, incubated 60 min at 37 ° C under humidified conditions and in the dark. The slides were rinsed three times with PBS, and Converter-POD was added to the sections. After incubation at 37°C for 30 min, DAB (3,3'-diaminobenzidine) substrate or alternative POD substrate was added to the sections and incubated at room temperature for 10 min. For immunofluorescence staining, briefly, the sections were rinsed 3×2 min in PBS-Tween 20, incubated with 3% BSA in PBS for 30 min at room temperature, incubated with primary antibody against active caspase-3 (Catalog No. ab13847, Abcam, US) at 4°C overnight, rinsed twice with PBS, and incubated with Alexa Fluor in PBS. The second antibody was incubated at room temperature for 60 min. The sections on the slides were protected from light using a black box, rinsed in PBS for 3×5 min, and mounted with anti-fluorescence quenching mounting solution. The sections were observed using a confocal fluorescence microscope (Radiance 2000 MP, Bio-Rad).

[0185] Quantification of dendritic spines by Golgi staining

[0186] Animals were anesthetized with 6% chloral hydrate (6 ml / kg) and perfused intracardially with 0.9% sodium chloride containing 0.5% sodium nitrite and then 4% formaldehyde. The mouse brain was further perfused with a Golgi dye solution containing 5% potassium dichromate, 5% chloral hydrate and 4% formaldehyde. After perfusion, the brain was cut into 0.1 cm × 0.1 cm sections and transferred to a 50 ml opaque tube containing a Golgi dye solution for 3 days at room temperature, then immersed in a 1% silver nitrate solution for another 3 days in an opaque tube. The brain was sliced ​​into 35 μm thick slices using a vibrating ultramicrotome (Leica, VT1000 S, Germany). The number of dendritic spines was estimated at high magnification (1000×) by a researcher who was blind to the injection conditions.

[0187] Western blotting

[0188] Western blotting was used to analyze the levels of full-length p75 (p75FL), p75ECD, molecules involved in Aβ production and degradation, tau protein phosphorylation, and synaptic proteins associated with synaptic plasticity (SNAP25, synapsin 1, VAMP1, and PSD95). Proteins in animal brain homogenates were extracted using RIPA buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM sodium pyrophosphate, 1 mM EDTA, 25 mM β-glycerophosphate, 1 mM Na3VO4, 0.5 μg / ml leupeptin, 1-2 mM PMSF, based on the ratio of 9 μl RIPA buffer added per mg of brain tissue. The samples were subjected to SDS-PAGE (8–15% acrylamide) gel electrophoresis. The separated proteins were transferred to nitrocellulose membranes. The blots were probed with the following antibodies: anti-APP C-terminal antibody recognizing APPfl, CTFβ and CTFα (171610, Calbiochem, US), anti-BACE1 antibody (clone 61-3E7, Millipore, US), anti-neprilysin antibody (Cat. No. AB5458, Millipore, US), anti-RAGE antibody (clone DD / A11, Millipore, US), anti-LRP antibody (5A6, Calbiochem, US), anti-IDE antibody (PC730, Millipore, US), anti-phospho-tau antibodies including pS396 (Cat. No. 11102, Signalway Antibody, US), pT231 (Cat. No. 11110, Signalway Antibody, US), pS199 (Cat. No. 11111, Signalway Antibody, US), pS231 (Cat. No. 11111, Signalway Antibody, US), pS396 (Cat. No. 11110, Signalway Antibody, US), pS231 (Cat. No. 1 ... (Catalog number ab81268, Abcam, US), pS262 (Catalog number ab92627, Abcam, US), anti-SNAP25 antibody (Catalog number AB1762, Millipore, US), anti-synaptobrevin antibody (Catalog number MAB332, Millipore, US), anti-synapsin I antibody (Catalog number 574778, Millipore, US), anti-VAMP1 antibody (ab151712, Abcam, US), anti-PSD95 antibody (MAB1596, Millipore, US), anti-β-actin antibody (ab8227, Abcam, US). The band density was normalized to that of β-actin. The membrane was incubated with IRDye 800CW secondary antibody (LI-COR) and scanned using an Odyssey fluorescence scanner.

[0189] ELISA quantification of IL-6, IL-1β, INF-γ and TNF-α concentrations

[0190] The levels of IL-6, IL-1β, INF-γ, TNF-α in TBS fractions and serum of brain extracts were quantitatively measured using BMS603, BMS6002, BMS606, and BMS607 ELISAs, respectively, according to the manufacturer's instructions (eBioscience). The final values ​​of brain IL-6, IL-1β, INF-γ, and TNF-α were expressed as pmoles per gram of brain wet weight using the wet weight of brain tissue in the original homogenate.

[0191] Statistical analysis

[0192] No data were excluded from the statistical analysis.For data collection, all samples were numbered and the analyzing investigators were blinded to the identity of the samples.

[0193] Unless otherwise stated, data in the text and figures are expressed as mean ± sem. Statistical comparisons between groups were analyzed using Tukey's test, Students t-test, one-way ANOVA or two-way repeated measures ANOVA to test the significance of values. Spearman's rank correlation coefficient was used to assess the correlation of p75ECD levels with Aβ load, inflammation and behavioral performance. P values ​​<0.05 were considered significant. All of these analyses were performed using SPSS for Windows, version 13.0.

[0194] result

[0195] Shedding of p75ECD is downregulated in the brains of AD subjects and APPswe / PS1dE9 mice

[0196] In Wt mice, the levels of p75ECD in the whole brain or in different regions increased with age ( Figure 2 a, b). Soluble p75ECD is present in many different regions of the brain of wild-type (Wt) mice ( Figure 2 b, c), in peripheral ganglia and organs (data not shown). The data indicate that in normal non-demented control individuals, shedding of p75 ECD is a physiological process and is associated with aging.

[0197] p75ECD levels in cerebrospinal fluid (CSF) from AD patients and ND controls were measured by ELISA. The sensitivity of the p75ECD ELISA reached 150 pg / ml and it was specific (r=0.8198, p<0.0001) as confirmed by Western blot on the same samples (data not shown). The p75ECD levels in the cerebrospinal fluid (CSF) of AD patients (n=29) were significantly lower than those in the CSF of ND controls (n=27, P<0.01) ( Figure 3 a). For non-demented controls, the mean ± sem of p75ECD concentration in CSF was 864 ± 30 pg / ml and ranged from 549 to 1233 pg / ml. For AD patients, the mean ± sem of p75ECD concentration in CSF was 343 ± 33 pg / ml and ranged from 94 to 848 pg / ml. Thus, in AD patients, the mean of p75ECD concentration in CSF was 2.22-fold lower compared to ND controls.

[0198] In the brain of AD subjects ( Figure 3 b, c, d) and APPswe / PS1dE9(AD) mice brain ( Figure 3 e, f, g), the presence of p75ECD was confirmed by Western blotting. In human AD patients, p75ECD levels in the brain (parietal cortex) were significantly reduced compared with age- and sex-matched non-demented controls (ND, n = 12) (P < 0.05, Figure 3 c). The mean ± sem of the relative intensity of p75ECD concentration in AD patients normalized to non-demented controls was 22 ± 3%, meaning that the intensity of p75ECD in AD was reduced more than 4-fold compared to non-demented controls.

[0199] As a comparison, full-length (FL) p75 (p75FL) was significantly increased in human AD patients compared with ND controls (P < 0.01, n = 12; Figure 3 c). The mean ± sem of the relative intensity of p75FL concentration in AD patients normalized to non-demented controls was 315 ± 20%, meaning that the intensity of p75FL in AD was increased more than 3-fold compared to non-demented controls.

[0200] In particular, the ratio of p75ECD to p75FL (p75ECD: p75FL) was significantly decreased in AD patients compared with ND controls ( Figure 3d). For AD patients, the mean ± sem of the ratio (normalized to the mean ± sem of non-demented controls) was 0.11 ± 0.02%. Thus, the ratio was 9-fold lower in AD patients compared to non-demented controls. These results suggest that p75 processing to remove ECD is significantly reduced in AD patients compared to normal non-demented controls.

[0201] Table 3 Raw data values ​​of p75ECD in CSF obtained from Alzheimer's disease (AD) patients and non-demented (ND) controls, in pg / ml

[0202]

[0203]

[0204] In the brains of 12-month-old AD mice, the abnormal expression of p75ECD and p75FL was significantly similar when compared with their Wt littermates ( Figure 3 e, f, g, h). Specifically, the mean ± SEM level of p75ECD in the mouse brain determined by ELISA was 75 ± 5 ng / ml; while the p75ECD concentration in wt littermates was 201 ± 10 ng / ml ( Figure 3 e). Thus, p75ECD was 2.7-fold lower in AD mice compared to wt mice.

[0205] p75FL is expressed at significantly higher levels in the brains of AD mice compared to wt littermates ( Figure 3 f, g); and p75ECD was expressed at significantly lower levels in AD mice compared to wt littermates ( Figure 3 f, g). Specifically, the mean ± sem of the relative intensity of p75ECD in AD mice was 65 ± 4% compared to wt littermates ( Figure 3 g). Thus, p75ECD concentrations were approximately 0.35-fold lower in AD mice compared to wt controls.

[0206] The ratio of p75ECD to p75FL was significantly reduced in AD mice compared to wt counterparts ( Figure 3 h). Specifically, the mean ± sem of the p75ECD:p75FL ratio in AD mice normalized to wt mice was 48 ± 4%, meaning that the ratio was 2.1-fold lower in AD mice compared to wt littermates ( Figure 3 h).

[0207] In AD animals, brain p75ECD levels were negatively correlated with cognitive impairment, brain Aβ burden, and inflammation (Tables 3, 4, and 5).

[0208] Table 4 Correlation between brain p75ECD levels and brain inflammation

[0209]

[0210] Table 5 Correlation between brain p75ECD levels and Aβ load in the brain

[0211]

[0212] Table 6 Correlation between brain p75ECD levels and behavioral performance

[0213] variable coefficient Standard error t-value P-value 95.0% Confidence Interval Latency in MWM -0.127 0.039 -3.229 0.005 -0.210―-0.044 Alternation in the Y-maze 0.159 0.040 4.002 0.001 -0.076―0.243 Standing in the open field 0.064 0.016 4.078 0.001 -0.031―-0.096

[0214] MWM, Morris water maze

[0215] While not wanting to be bound by theory, these results suggest that ECD-depleted p75 processing is significantly reduced in AD compared to normal wild-type littermates.

[0216] It is reported that tumor necrosis factor alpha converting enzyme (TACE) is the main enzyme that cuts the ECD of p75 (Weskamp, ​​G. et al., 2004). As disclosed herein, it has been found that TACE activity is significantly reduced in the brain of AD mice (data not shown) and in the CSF of AD patients (data not shown) compared with their ND or wt counterparts. Compared with age-matched Wt controls, the expression of TACE in AD mice is also reduced (data not shown). In contrast to the nerve growth factor (NGF) (Urra, S. et al., 2007; Kommaddi, RP et al., 2011) that increases TACE dependence, Aβ treatment of mouse cortical neurons significantly reduces TACE expression in a dose-dependent manner (data not shown). Although unwilling to be bound by theory, these results indicate that the reduction of soluble p75ECD in AD is a downstream toxic effect of Aβ.

[0217] Restoration of brain p75ECD levels in AD mice by delivery of recombinant human ECD-Fc gene

[0218] Considering that recombinant ECD-Fc can destroy Aβ-p75 interaction and inhibit Aβ aggregation (Wang, YJ et al., 2011), and as mentioned above, p75ECD levels are reduced in AD brains, it is believed that the recovery of p75ECD levels in AD brains may protect neurons against the pathological effects of Aβ. Adeno-associated virus 8 (AAV8) was used to deliver the fusion gene (AAV-ECD-Fc) of human p75ECD and human IgG Fc fragments to the lateral ventricle of AD mice at 3 months of age (before amyloid plaques are produced in the brain) to check whether ECD delivery can be used to prevent Aβ-related diseases, and also at 9 months of age (when amyloid plaques have been clearly produced) to deliver the fusion gene to check whether ECD delivery can be used as a treatment to alleviate the symptoms of Aβ-related diseases. The dose of AAV used is equivalent to that used in human clinical trials of AAV. AAV expressing the green fluorescent protein (GFP) gene was used as a control. Expression of the ECD-Fc transgene was detected one week after delivery and remained stable in neurons of the neocortex, hippocampus, and thalamus until animals were sacrificed at 12 months of age, as detected using an anti-human Fc antibody that detects only ECD-Fc expressed from the transgene but not endogenous p75ECD (data not shown).

[0219] When AAV-ECD-Fc was injected into the left lateral ventricle at 3 months of age, immunofluorescence of p75ECD-Fc was detected in brain sections of animals injected with AAV using an anti-human IgG Fc antibody (α-IgG-Fc), where ECD-Fc was expressed in the cortex, hippocampus, and thalamus (data not shown). p75ECD-Fc was expressed in neurons, but not in microglia or astrocytes (data not shown). ELISA analysis of p75ECD in TBS fractions of AD mouse brain homogenates showed stable expression (1 week (1w), 2 weeks (2w), 3 weeks (3w), and 9 months (9m) after AAV-ECD-Fc injection. Figure 4 a). At 12 months of age, the levels of ECD-Fc in the brains of transfected AD mice were similar to those of age-matched Wt mice ( Figure 4 b).

[0220] Restoration of brain p75ECD levels in AD mice prevents cognitive decline

[0221] AD mice treated with AAV-ECD-Fc at 3 or 9 months of age (as either prophylaxis (Pre) or treatment (Tre), respectively) performed better in the Morris water maze compared with AAV-GFP treated control mice (con). This was demonstrated by a significant decrease in escape latency and travel distance taken to escape to the platform with progressive platform learning trials ( Figure 5 a, b, c), as well as increased number of platform area crossings and longer time spent in the platform quadrant in exploration trials without the platform ( Figure 5 d, e, f). These data indicate that p75ECD-Fc significantly improved spatial learning and memory in AD mice, whether administered as a preventive at 3 months of age or as a treatment at 9 months of age. Mice in both the preventive and treatment groups also performed better in the Y-maze and open field tests compared to GFP-administered controls, as reflected by a greater number of entries into the novel arm and higher spontaneous alternations in the Y-maze test ( Figure 5 g, h, i, j). This indicates that the working memory of mice administered p75ECD-Fc was better than that of control mice administered GFP. In the open field test, mice administered ECD-Fc also had a higher number of upright movements, a longer travel distance, and a reduced time spent in the central zone compared to control mice administered GFP ( Figure 5 k, l), which is comparable to Wt mice. The data show that mice administered p75ECD-Fc have better overall locomotor activity than control AD ​​mice administered GFP. Therefore, in AD mice, ECD administration provides a protective effect on memory and behavior.

[0222] Restoration of brain p75ECD levels reduces Aβ levels in the brain and blood of AD mice

[0223] In AD mice administered p75ECD-Fc at 3 months of age (prevention) or 9 months of age (treatment), the area fraction and density of Congo red-stained plaques in the hippocampus (HC) and neocortex (NC) were reduced by approximately 50% compared with controls administered GFP ( Figure 6 a, b, c, d), meaning that amyloid plaques were reduced by 50% after p75ECD treatment. In AD mice administered with p75ECD-Fc in both the prevention and treatment groups, the load of amyloid plaques in the hippocampus and cortex identified by immunohistochemistry using antibody 6E10 was also reduced compared to controls ( Figure 6 e).

[0224] Congo red staining was also used to examine cerebral amyloid angiopathy (CAA) in mice. As shown in FIG6( f ), p75ECD-Fc reduced the severity of CAA, as confirmed by Congo red staining in blood vessels. Figure 6 (g) It is clearly seen that p75ECD-Fc reduced the number of CAA lesions by about 50%, with the mean ± SEM of the number of CAA lesions identified for the control group being 3.95 ± 0.12, while the prevention group was 2.05 ± 0.13, and the treatment group was 1.75 ± 0.20. The mean ± SEM of the number of microhemorrhages was also reduced by about 50% in both the prevention and treatment groups ( Figure 6 h), the mean ± SEM of the number of microbleeds in the control group was 0.63 ± 0.03, while the mean ± SEM of the number of microbleeds in the prevention group was 0.375 ± 0.13, and the mean ± SEM of the number of microbleeds in the treatment group was 0.22 ± 0.02. This suggests that administration of p75ECD-Fc as a treatment or prevention in subjects with increased Aβ load reduces CAA pathology.

[0225] Interestingly, p75ECD-Fc was localized around or within Congo red or thioflavin S-positive plaques as detected by immunohistochemistry ( Figure 6 i), indicating that the expressed human p75ECD-Fc binds and interacts with Aβ plaques. Consistent with the histological results, ELISA assays also showed that the levels of total Aβ and Aβ40 or Aβ42 in TBS, SDS and formic acid fractions of brain homogenates and in serum were significantly reduced in both the prevention and treatment groups ( Figure 6 i). Therefore, this result indicates that p75ECD-Fc administration reduces Aβ levels in the brain and blood.

[0226] Restoration of brain p75ECD levels in AD mice reduces APP amyloidogenic processing by inhibiting BACE1 expression

[0227] In sporadic AD, Aβ upregulates the expression of β-site APP cleavage enzyme (BACE1) and drives the vicious cycle that causes AD onset. However, it is not known which receptor mediates the Aβ-JNK-BACE1 vicious cycle. Although unwilling to be bound by theory, it has been proposed that p75 may upregulate Aβ production (Wang YJ et al., 2011), forming a positive feedback. It was investigated whether the disruption of the interaction between Aβ-p75 and p75ECD-Fc can affect APP processing and Aβ production. In both the prevention and treatment groups, p75ECD-Fc treatment significantly reduced the expression and activity of BACE1, the β-cleavage product (CTFβ) of APP, and Aβ production (data not shown). However, Aβ degrading enzymes neprilysin (NEP) and insulin degrading enzyme (IDE) and Aβ blood-brain barrier (BBB) ​​transport molecules low-density lipoprotein receptor-related protein (LRP) and receptor for advanced glycation end products (RAGE) did not change (data not shown).

[0228] To determine the mechanism by which p75ECD inhibits amyloid peptide generation in vivo, the expression of BACE1, APP and APP fragments in primary neurons was examined. They realized that in response to Aβ42β or Aβ25-35, BACE1 expression and sAPPβ levels in cultured primary neurons increased in a dose-dependent manner (data not shown). This increase was abolished by the presence of recombinant ECD-Fc or in p75 KO neurons (data not shown). Aβ also increased APP expression in a dose-dependent manner in AD / p75+ / + neurons (p<0.05) but not in AD / p75- / - neurons (data not shown). In AD / p75- / - neurons, in the presence or absence of Aβ, the level of full-length APP increased (p<0.001) but the level of sAPPβ decreased (p<0.001) compared to ADp75+ / + neurons (data not shown), indicating that p75 is required for APP processing of amyloid peptide production promoted by Aβ. Thus, the results indicate that p75 is a key receptor mediating Aβ-induced BACE1 upregulation and plays a key role in the Aβ-BACE1 vicious cycle that drives AD pathogenesis. As disclosed herein, it is recognized that p75ECD-Fc treatment can break this vicious cycle, as the data indicate that p75ECD-Fc reduces Aβ production by inhibiting BACE1 expression.

[0229] Restoration of brain p75ECD levels in AD mice attenuates neurite degeneration, neuronal death, and Tau phosphorylation

[0230] Compared with control AD ​​mice administered with GFP, the area fraction (neurons) stained for NeuN as well as MAP-2 dendrites and ChAT-positive axons in the hippocampus increased in both the prevention and treatment groups ( Figure 7 a), indicating that p75ECD-Fc treatment can protect the neuronal structure in the hippocampus of AD mice. In both the prevention and treatment groups, the number of dendritic spines detected by Golgi staining in the hippocampus increased significantly ( Figure 7 b), indicating that p75ECD-Fc can protect cholinergic neurons and hippocampal neurons against Aβ-induced toxicity. The levels of synaptic proteins, including the major synaptic vesicle protein p38 synaptobrevin, vesicle-associated membrane protein (VAMP) 1, synaptosome binding protein 25 (SNAP25), postsynaptic density protein 95 (PSD95), and synapsin I (Syn I), were significantly increased in the brains of both the prevention and treatment groups ( Figure 7 b), indicating that p75ECD-Fc can protect neuronal synapses and the connectivity of the central nervous system against the toxicity of Aβ. The area fractions of activated caspase-3 and TUNEL staining identifying apoptotic cells in the hippocampus were significantly lower in the prevention and treatment groups than in the control ( Figure 7c). Thus, consistent with the in vivo data, recombinant p75ECD-Fc protected primary cortical neurons from neurite collapse triggered by Aβ ( Figure 7 e).

[0231] The area fraction of Tau-phospho-Ser396 positive neurons in the hippocampus of both the prevention and treatment groups was lower than that of the control group ( Figure 7 d, upper and middle panels). In the brains of both the prevention and treatment groups, the levels of Tau phosphorylation at multiple sites including Ser 396, 262, 199 and Thr 231 were consistently and significantly reduced ( Figure 7 d, middle and lower panels), indicating that administration of p75ECD-Fc reduced tau phosphorylation associated with tauopathy. Although unwilling to be bound by theory, it is believed that the effect of p75ECD on Tau phosphorylation is through blocking Aβ that activates upstream GSK3β (Chakravarthy, B. et al., 2012). In fact, it has been found that in the human SH-SY5Y cell line, both recombinant p75ECD-Fc peptide and p75 receptor neutralizing antibody can block the increase in Ser262 phosphorylation of Tau protein in response to Aβ ( Figure 8 a). In addition, recombinant p75ECD-Fc dose-dependently inhibited the phosphorylation of S262 and the increase of p-Ser9-GSK3β triggered by Aβ42 ( Figure 8 b, c). These data indicate that p75ECD-Fc reduces Aβ-induced Tau hyperphosphorylation in SH-SY5Y cells by inhibiting GSK3β activity. Therefore, elevated p75ECD levels may provide protection against Aβ-induced axonal and neurite degeneration, neuronal death, and Tau hyperphosphorylation associated with Tauopathy.

[0232] Restoration of brain p75ECD levels attenuates microgliosis, astrogliosis, and inflammation in the brains of AD mice

[0233] Brain inflammation (including microgliosis and astrogliosis) is a secondary sign of AD. Compared with the control group, in the two groups of prevention and treatment, the area fraction of CD45 (microgliosis) and GFAP (astrogliosis) staining in the cortex and hippocampus was significantly reduced (data not shown). In the two groups of prevention and treatment, the levels of inflammatory cytokines TNF-α, IFN-γ, IL-1β and IL-6 in TBS fractions and serum were also lower than those in the control group (data not shown). Better performance and reduced neuroinflammation in learning and memory tasks are possible secondary results of reduced amyloid peptide load, Tau protein phosphorylation and neurodegeneration after ECD-Fc treatment.

[0234] Delivery of the ECD-Fc gene via AAV8 is well tolerated

[0235] During the animal study, no animal deaths occurred, and no obvious abnormal behavior was observed. Gene delivery did not significantly affect animal body weight or liver enzymes (data not shown). No discernible pathological morphology was observed in major organs (including brain, muscle, heart, liver, lung, kidney and intestine) (data not shown). These data show that the long-term expression of ECD-Fc fusion gene in the brain is well tolerated by AD mice, indicating the safety of AAV-ECD-Fc treatment.

[0236] discuss

[0237] As disclosed herein, it has been recognized that soluble p75ECD (i.e., after falling off from p75) is a potentially useful therapeutic agent and marker for AD. Soluble p75ECD is physiologically distributed in both the central and peripheral nervous systems and is subject to developmental regulation of aging. It has been recognized that p75ECD levels are significantly reduced in the brains of AD subjects and mice, which may be caused by the reduction in the expression and activity of TACE induced by Aβ. In addition, it has been found that p75ECD protects neurons against Aβ-induced neurotoxicity and inhibits BACE1 expression and Tau protein phosphorylation. In addition, p75ECD levels are restored in the brains of AD mice by administering p75ECD-Fc to protect the brain against neurodegeneration.

[0238] The function of soluble p75ECD after shedding was not previously understood. As disclosed herein, it has been recognized that p75ECD is a neurotrophic molecule that protects neurons against neurodegeneration in AD brains. Surprisingly, p75ECD levels are significantly reduced in AD, indicating that the cleavage process in AD is impaired by Aβ. Since TACE is a physiological shedding enzyme that regulates the production of p75ECD (Westkamp et al., 2004), the reduction in p75ECD levels in AD brains may be the result of impaired p75 cleavage caused by reduced expression and activity of TACE triggered by Aβ.

[0239] As disclosed herein, p75 has been found to mediate Aβ-induced BACE1 upregulation, as both p75ECD-Fc and p75 knockout mice can block Aβ-induced BACE1 upregulation and inhibit APP processing that produces amyloid peptides. The data described herein indicate that p75FL is a central player in driving the Aβ-BACE cycle. Reduction of p75ECD levels in AD brains may contribute to the overproduction of Aβ, exacerbating the vicious cycle; and restoration of p75ECD levels in vivo can inhibit BACE1 expression and related amyloid peptide pathways. p75ECD can interrupt the Aβ-BACE1 vicious cycle that contributes to the overproduction of Aβ in AD.

[0240] p75ECD protects neurons at multiple levels of the Aβ cascade that causes AD pathogenesis, including inhibition of Aβ aggregation and deposition (Wang, YJ et al., 2011), overproduction and neurotoxicity of Aβ, and Aβ-induced Tau phosphorylation. Treatment with p75ECD-Fc and p75 antibodies in vitro and in vivo was able to block Tau phosphorylation, indicating that Aβ-induced Tau phosphorylation may be mediated by p75. Taken together, p75ECD is a new neurotrophic molecule that is reduced in AD and protects the brain against Aβ toxicity. Restoring p75ECD levels in the AD brain may be an ideal treatment for AD.

[0241] Example 2 Specificity of the effect of p75ECD

[0242] In order to investigate the specificity of p75ECD-Fc to amyloid peptide load, additional experiments were performed by injecting recombinant p75ECD-Fc or one of two control proteins (human IgG or recombinant human fibroblast growth factor receptor 4ECD (FGFR4-Fc) fused to human IgG Fc) in the hippocampus of 8-month-old AD mice. A single injection of recombinant p75ECD-Fc, but not human IgG or FGFR4-Fc, also significantly reduced the area fraction of amyloid plaques on the injection site one week after injection (data not shown). In order to investigate the potential mechanism of how p75ECD-Fc affects the amyloid peptide load in the brain, the effects of p75ECD-Fc on Aβ fibril formation and disaggregation were visually examined using ThT assay and transmission electron microscopy (TEM). Data show that p75ECD-Fc, but not control protein human IgG or FGFR4-Fc, inhibited the fluorescence of Aβ fluorescence and preformed Aβ fibers in fibril formation (data not shown). TEM visualization determined that p75ECD-Fc, but not human IgG or FGFR4-Fc, prevented Aβ fibril formation and disaggregated preformed Aβ fibrils (data not shown). The data suggest that the effect of p75ECD-Fc on amyloid peptide load in vivo is a specific event and is not caused by nonspecific protein-Aβ interactions.

[0243] In addition, consistent with the in vivo data, in vitro, recombinant p75ECD-Fc, but not human IgG and recombinant FGFR4-Fc, inhibited cell death of SH-SY5Y cells and protected cortical neurons from neurite collapse triggered by Aβ (data not shown).

[0244] These experiments suggest that the effect of p75ECD-Fc on amyloid peptide loading is due to the specificity of the action of p75ECD.

[0245] Example 3 Intramuscular delivery of p75ECD-Fc in AD mice reduces AD pathology and improves cognitive function

[0246] AD mice (APP / PS1 transgenic mice) or wild-type mice were injected intramuscularly with AAV-ECD-Fc as follows: at 3 months of age, female APP / PS1 transgenic mice were injected intramuscularly with 2×10 9Each AAV-ECD-Fc or AAV-GFP vector genome was injected intramuscularly. At 12 months of age, all mice were sacrificed and the brains were sampled. The control group received AAV-GFP, but the other treatments were the same. After treatment, the mice were tested using the Morris water maze test. In some experiments, mice were injected once intraperitoneally with recombinant p75ECD-Fc peptide at 50μg / 50μl per mouse. Other experiments were performed as described above.

[0247] Intramuscular delivery of p75ECD improves cognitive decline in AD mice

[0248] Compared with AAV-GFP treated mice, mice treated with AAV-ECD-Fc performed better in the Morris water maze test, as evidenced by a significant decrease in escape latency and distance traveled to escape onto the platform, as well as a greater number of platform zone crossings with progressive platform learning trials ( Fig. 9 a, b, c). The data show that AAV-ECD-Fc treatment significantly improves spatial learning and memory in AD mice. In addition, AAV-ECD-Fc treated mice also performed better in the Y-maze and open field tests compared to AAV-GFP treated controls. This was reflected by a higher number of entries into the new arm in the Y-maze test and higher spontaneous alternations ( Fig. 9 d and e). This indicates that the working memory of AAV-ECD-Fc treated mice was better than that of control mice. In the open field test, longer travel distance was observed in AAV-ECD-Fc treated mice compared to AAV-GFP treated controls ( Fig. 9 f), indicating that ECD-Fc treated mice had better exploratory activity compared to control AD ​​mice. However, there was no significant difference in swimming speed between the groups, indicating that the mice had comparable motor activity. Therefore, this data shows that intramuscular p75ECD delivery improves cognitive decline in AD mice.

[0249] Intramuscular delivery of p75ECD reduces Aβ burden in the brain and blood of AD mice

[0250] In AAV-ECD-Fc-treated AD mice, the burden of amyloid plaques in the hippocampus and neocortex, as identified by Congo red staining or immunohistochemistry, was reduced compared with AAV-GFP-treated controls ( Fig.10 a to d). Consistent with the histological results, ELISA assays also showed that Aβ40 and Aβ42 were significantly reduced in Tris-buffered saline, SDS, and formic acid fractions of brain homogenates and in serum in AAV-ECD-Fc-treated AD mice ( Fig.10e). However, in an acute experiment in AD transgenic mice injected intraperitoneally with p75ECD-Fc, serum Aβ40 and Aβ42 levels were significantly increased 24 hours after a single intraperitoneal injection of p75ECD peptide at 50ug / 50ul per mouse ( Fig.10 f), indicating that p75ECD can promote the efflux of Aβ from the brain to the periphery.

[0251] Intramuscular delivery of p75ECD-Fc attenuates microgliosis, astrogliosis, and inflammation in the brains of AD mice

[0252] Brain inflammation (including microgliosis and astrogliosis) is a secondary hallmark of AD pathology after Aβ. Compared with AAV-GFP treated AD mice, the area fraction of CD45 (microgliosis) and GFAP (astrogliosis) staining in the neocortex and hippocampus was significantly reduced in AAV-ECD-Fc treated AD mice ( Fig.11 ), as reflected by a reduction in the fraction of GFAP- and CD45-positive staining areas. Thus, in addition to a reduction in amyloid burden, improved performance in learning and memory tasks and reduced neuroinflammation are possible outcomes of ECD-Fc treatment.

[0253] Example 4 Intramuscular delivery of p75ECD-Fc attenuates Tau protein phosphorylation in AD mice

[0254] To evaluate Tau pathology, phosphorylation of Tau at pS396, pT231, pS199, and pS262 in AAV-ECD-Fc treated AD mice was examined as described above and compared with AAV-GFP treated controls. Phosphorylation at these sites was investigated using the anti-pS396, pT231, pS262, and pS199 antibodies described above. These phosphorylation sites were selected in this study because they are common sites of Tau hyperphosphorylation in AD mice. The area fraction of Tau-phospho-Ser396 positive neurons in the cortex and hippocampus was significantly lower in the p75ECD treated group compared to the GFP group ( Fig.12 a, b, c and d). In the brains of the treated groups, the levels of Tau phosphorylation at multiple sites including Ser 396, 262, 199 and Thr 231 were consistently and significantly reduced (Figure 12e and f). These findings suggest that p75ECD provides protection against Tau hyperphosphorylation.

[0255] Example 5 Treatment of Tauopathy Mice with p75ECD-Fc

[0256] In the brain of patients with Alzheimer's disease or frontotemporal dementia (FTD), aggregates of hyperphosphorylated tau protein are significant. After identifying tau mutations in familial FTD cases, they have been reproduced in animal models. This includes the transgenic model pR5 we previously generated, which expresses FTD (P301L) mutant microtubule-associated protein tau (MAPT) (Deters et al., 2008) in neurons. The mouse is characterized by tau protein aggregation, including tangle (NFT) formation, memory impairment and mitochondrial dysfunction. In this experiment, 3 month old pR5 mice were randomly divided into two different groups. In the first week of treatment, 10mg / kg of p75ECD-Fc or 5mg / kg of Fc recombinant protein were administered to mice by intraperitoneal injection twice a week to provide sufficient amounts of protein in mouse plasma. The mice then received 5 mg / kg of recombinant p75ECD-Fc or 2.5 mg / kg of recombinant Fc protein in a volume of 150-200 μl of PBS twice weekly by intraperitoneal injection for the remainder of the total treatment time of 3 months.

[0257] Effects of p75ECD-Fc on cognitive function in mice with tauopathy

[0258] To evaluate the effect of p75ECD-Fc on learning and memory function in PR5 mice, 12 3-month-old mice were divided into two groups and received p75ECD-Fc or Fc recombinant protein for 3 months. Wild-type mice were tested in parallel. A group of age- and sex-matched wild-type mice were used as controls. The Morris water maze (MWM) test of PR5 mice showed that p75ECD-Fc significantly reduced escape latency during days 2, 4, and 5 of the learning test compared with the Fc group ( Fig.13 A), and significantly reduced travel distance during days 2, 3, and 5 of the learning trial ( Fig.13 (B and C). The learning process was improved in the wild-type and p75ECD-Fc groups. In the wild-type group, a significant reduction in escape latency and travel distance was observed on days 3, 4, and 5 during the learning process compared to day 1. In the p75ECD-Fc group, during the learning process, the escape latency and travel distance decreased on days 4 and 5 compared to day 1, while no significant improvement in learning was observed in the Fc group during the learning process. However, p75ECD-Fc did not change the average time in the target quadrant during the exploration trial ( Fig.13 D) The number of times entering the platform quadrant ( Fig.13 E). The number of platform crossings during the exploration trial was constant across groups ( Fig.13 F). The results indicate that p75ECD-Fc can improve the learning process in PR5 mice.

[0259] p75ECD-Fc reduces BACE1 expression and sAPPβ production in the brain of PR5 mice

[0260] To evaluate the effect of p75ECD-Fc on AD hallmarks in PR5 mice, brain lysates from both p75ECD-Fc and Fc treated groups were subjected to Western blotting and incubated with different primary antibodies for measuring AD biomarker proteins BACE1, sAPPβ, and full-length APP (APP.FL). In the Western blotting data, the expression levels of BACE1 and sAPPβ in the brain of PR5 mice confirmed that p75ECD-Fc significantly reduced BACE1 expression (p<0.05) ( Fig.14 A and B). In addition, it was observed that in PR5 mice, the level of sAPPβ was decreased in the p75ECD-Fc treated group compared with the Fc group ( Fig.14 A and C).

[0261] p75ECD-Fc inhibits Tau phosphorylation (p.Ser202 and Thr205) in PR5 mouse brain

[0262] To test whether p75ECD-Fc inhibits Tau phosphorylation in PR5 mice, brain lysates from PR5 mice treated with p75ECD-Fc or Fc were subjected to Western blot analysis to detect Tau phosphorylated proteins using mouse phosphor-PHF-TaupSer202+Thr205 monoclonal (AT8) antibody. Our data showed that p75ECD-Fc significantly reduced Tau (Ser202 and Thr205) phosphorylation in PR5 mice (p<0.01) (Figure 15A and B).

[0263] Throughout the specification and claims, unless the context requires otherwise, the phrases "comprises" and "comprising" and variations thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0264] The reference to any prior art in this specification is not, and should not be taken as, any form of admission that such prior art forms part of the common general knowledge.

[0265] Those skilled in the art will recognize that the use of the present invention is not limited to the specific applications described. In preferred embodiments, the present invention is also not limited to the specific elements and / or features described or depicted herein. It should be appreciated that the present invention is not limited to the disclosed one or more embodiments, but is capable of a large number of rearrangements, modifications and substitutions without departing from the scope of the present invention as set forth and defined by the claims.

[0266] References

[0267] Castellani,RJ&Perry,G.(2012)Arch Med Res 43:694-698

[0268] Chakravarthy, B. et al., (2012) J Alzheimer's disease: JAD 30: 675-684

[0269] Charidimou A et al., (2012) J Neurol Neurosurg Psychiatry 83:124–37

[0270] Conrad, CD et al., (1999) Neurobiol of learning and memory 72: 39-46 (1999)

[0271] Dellu, F. et al., (1992) Brain Res 588:132-139

[0272] Detters et al., (2008) Eur J Neurosci.28(1):137-47

[0273] Food and Drug Administration(2003)Fed Regist 68:2340–2341

[0274] Gotz et al. (2001) Science 293(5534):1491-5

[0275] Jellinger KA.(2002)J Neural Transm 109:813

[0276] Jung, WR et al., (2008) Neurosci Lett 439, 220-225

[0277] Keage HA et al. (2009) BMC Neurol 9:3.

[0278] Kommaddi, RP et al. (2011) FASEB J 25:2061-2070

[0279] Markowska AL et al. (1993) Behavioral Neurosci 107:627-632.

[0280] McKhann G. et al., (1984) Neurology 34:939-944.

[0281] Rafii,MS(2013)Reviews on Recent ClinicalTtrials 8:110-118

[0282] Saadipour, K. et al., (2013) J Neurochem 127:152-162

[0283] Sarter M. et al., (1988) Psychopharmacology 94:491-495

[0284] Schor,NF(2005).Prog Neurobiol 77(3):201-214

[0285] Urra, S. et al., (2007) J Biol Chem 282:7606-7615

[0286] Wang, YJ et al., (2009a) Neurotoxicity Res 15:3-14

[0287] Wang YJ et al., (2009b) Neurobiology of Aging 30:364-376

[0288] Wang YJ et al., (2010) Brain, Behavior, and Immunity 24:1281-1293

[0289] Wang, YJ et al. (2011) J Neurosci 31:2292-2304

[0290] Weskamp, ​​G. et al., (2004) J Biol Chem 279:4241-4249.

[0291] Yau J.L. et al., (2007) J Neurosci.27:10487-10496

[0292] Zhou L et al. (2013) J Affect Disorder 150:776-784. Sequence Listing <110> Suzhou Aozong Biotechnology Co., Ltd. <120> Methods of diagnosing or treating neurological disorders using P75ECD and / or P75 <130> SPI204720-01 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 1284 <212> DNA <213> Homo sapiens <400> 1 atgggggcag gtgccaccgg ccgcgccatg gacgggccgc gcctgctgct gttgctgctt 60 ctgggggtgt cccttggagg tgccaaggag gcatgcccca caggcctgta cacacacagc 120 ggtgagtgct gcaaagcctg caacctgggc gagggtgtgg cccagccttg tggagccaac 180 cagaccgtgt gtgagccctg cctggacagc gtgacgttct ccgacgtggt gagcgcgacc 240 gagccgtgca agccgtgcac cgagtgcgtg gggctccaga gcatgtcggc gccgtgcgtg 300 gaggccgacg acgccgtgtg ccgctgcgcc tacggctact accaggatga gacgactggg 360 cgctgcgagg cgtgccgcgt gtgcgaggcg ggctcgggcc tcgtgttctc ctgccaggac 420 aagcagaaca ccgtgtgcga ggagtgcccc gacggcacgt attccgacga ggccaaccac 480 gtggacccgt gcctgccctg caccgtgtgc gaggacaccg agcgccagct ccgcgagtgc 540 acacgctggg ccgacgccga gtgcgaggag atccctggcc gttggattac acggtccaca 600 cccccagagg gctcggacag cacagccccc agcacccagg agcctgaggc acctccagaa 660 caagacctca tagccagcac ggtggcaggt gtggtgacca cagtgatggg cagctcccag 720 cccgtggtga cccgaggcac caccgacaac ctcatccctg tctattgctc catcctggct 780 gctgtggttg tgggccttgt ggcctacata gccttcaaga ggtggaacag ctgcaagcag 840 aacaagcaag gagccaacag ccggccagtg aaccagacgc ccccaccaga gggagaaaaa 900 ctccacagcg acagtggcat ctccgtggac agccagagcc tgcatgacca gcagccccac 960 acgcagacag cctcgggcca ggccctcaag ggtgacggag gcctctacag cagcctgccc 1020 ccagccaagc gggaggaggt ggagaagctt ctcaacggct ctgcggggga cacctggcgg 1080 cacctggcgg gcgagctggg ctaccagccc gagcacatag actcctttac ccatgaggcc 1140 tgccccgttc gcgccctgct tgcaagctgg gccacccagg acagcgccac actggacgcc 1200 ctcctggccg ccctgcgccg catccagcga gccgacctcg tggagagtct gtgcagtgag 1260 tccactgcca catccccggt gtga 1284 <210> 2 <211> 420 <212> PRT <213> Homo sapiens <400> 2 Met Gly Ala Gly Ala Thr Gly Arg Ala Met Asp Gly Pro Arg Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Gly Val Ser Leu Gly Gly Ala Lys Glu Ala Cys 20 25 30 Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys Lys Ala Cys Asn 35 40 45 Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn Gln Thr Val Cys 50 55 60 Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val Val Ser Ala Thr 65 70 75 80 Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu Gln Ser Met Ser 85 90 95 Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg Cys Ala Tyr Gly 100 105 110 Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala Cys Arg Val Cys 115 120 125 Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp Lys Gln Asn Thr 130 135 140 Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp Glu Ala Asn His 145 150 155 160 Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp Thr Glu Arg Gln 165 170 175 Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys Glu Glu Ile Pro 180 185 190 Gly Arg Trp Ile Thr Arg Ser Thr Pro Pro Glu Gly Ser Asp Ser Thr 195 200 205 Ala Pro Ser Thr Gln Glu Pro Glu Ala Pro Pro Glu Gln Asp Leu Ile 210 215 220 Ala Ser Thr Val Ala Gly Val Val Thr Thr Val Met Gly Ser Ser Gln 225 230 235 240 Pro Val Val Thr Arg Gly Thr Thr Asp Asn Leu Ile Pro Val Tyr Cys 245 250 255 Ser Ile Leu Ala Ala Val Val Val Gly Leu Val Ala Tyr Ile Ala Phe 260 265 270 Lys Arg Trp Asn Ser Cys Lys Gln Asn Lys Gln Gly Ala Asn Ser Arg 275 280 285 Pro Val Asn Gln Thr Pro Pro Pro Glu Gly Glu Lys Leu His Ser Asp 290 295 300 Ser Gly Ile Ser Val Asp Ser Gln Ser Leu His Asp Gln Gln Pro His 305 310 315 320 Thr Gln Thr Ala Ser Gly Gln Ala Leu Lys Gly Asp Gly Gly Leu Tyr 325 330 335 Ser Ser Leu Pro Pro Ala Lys Arg Glu Glu Val Glu Lys Leu Leu Asn 340 345 350 Gly Ser Ala Gly Asp Thr Trp Arg His Leu Ala Gly Glu Leu Gly Tyr 355 360 365 Gln Pro Glu His Ile Asp Ser Phe Thr His Glu Ala Cys Pro Val Arg 370 375 380 Ala Leu Leu Ala Ser Trp Ala Thr Gln Asp Ser Ala Thr Leu Asp Ala 385 390 395 400 Leu Leu Ala Ala Leu Arg Arg Ile Gln Arg Ala Asp Leu Val Glu Ser 405 410 415 Leu Cys Ser Glu 420 <210> 3 <211> 1188 <212> DNA <213> Homo sapiens <400> 3 aaggaggcat gccccacagg cctgtacaca cacagcggtg agtgctgcaa agcctgcaac 60 ctgggcgagg gtgtggccca gccttgtgga gccaaccaga ccgtgtgtga gccctgcctg 120 gacagcgtga cgttctccga cgtggtgagc gcgaccgagc cgtgcaagcc gtgcaccgag 180 tgcgtggggc tccagagcat gtcggcgccg tgcgtggagg ccgacgacgc cgtgtgccgc 240 tgcgcctacg gctactacca ggatgagacg actgggcgct gcgaggcgtg ccgcgtgtgc 300 gaggcgggct cgggcctcgt gttctcctgc caggacaagc agaacaccgt gtgcgaggag 360 tgccccgacg gcacgtattc cgacgaggcc aaccacgtgg acccgtgcct gccctgcacc 420 gtgtgcgagg acaccgagcg ccagctccgc gagtgcacac gctgggccga cgccgagtgc 480 gaggagatcc ctggccgttg gattacagac aaaactcaca catgcccacc gtgcccagca 540 cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 600 atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 660 gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 720 Glutamic acid Alanine Cysteine Proline Threonine Glycine Leucine Tyrosine Threonine Histidine Serine Glycine Glutamic acid Cysteine Cysteine Lysine cgggaggagc agtacaacag cacgtaccgg gtggtcagcg tcctcaccgt cctgcaccag 780 Arginine Glutamic acid Glutamic acid Glutamine Tyrosine Asparagine Serine Histidine Tyrosine Arginine Glycine Valine Serine Serine Serine Threonine Valine Leucine Histidine Glutamine gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc 840 Aspartic acid Tryptophan Leucine Methionine Alanine Lysine Glutamic acid Tyrosine Lysine Cysteine Lysine Valine Serine Asparagine Lysine Proline Serine Glutamine Proline Proline atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg 900 Isoleucine Glutamic acid Lysine Threonine Isoleucine Serine Lysine Alanine Lysine Glycine Glutamine Proline Glutamic acid Asparagine Histidine Arginine Valine Threonine Leucine cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 960 Proline Proline Isoleucine Proline Glycine Aspartic acid Glutamic acid Leucine Threonine Lysine Asparagine Glutamine Valine Serine Leucine Threonine Cysteine Leucine Valine Lysine Glycine ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac 1020 Phenylalanine Tyrosine Proline Serine Aspartic acid Isoleucine Alanine Valine Glutamic acid Tryptophan Glutamic acid Serine Asparagine Glycine Glutamine Proline Glutamic acid Asparagine Asparagine Tyrosine aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc 1080 Lysine Aspartic acid Histidine Arginine Leucine Proline Valine Leucine Aspartic acid Serine Aspartic acid Glycine Leucine Phenylalanine Phenylalanine Leucine Threonine Serine Lysine Leucine Threonine gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct 1140 Valine Aspartic acid Lysine Serine Glutamine Glycine Glycine Glutamine Glycine Asparagine Valine Phenylalanine Serine Cysteine Serine Valine Methionine Histidine Glutamic acid Alanine ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaa 1188 Leucine Cysteine Threonine Histidine Tyrosine Threonine Histidine Glutamic acid Glutamic acid Serine Serine Proline Valine Serine Glycine Lysine <210> 4 <211> 221 <212> PRT <213> Homo sapiens <400> 4 Glu Ala Cys Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys Lys Glutamic acid Alanine Cysteine Proline Threonine Glycine Leucine Tyrosine Threonine Histidine Serine Glycine Glutamic acid Cysteine Cysteine Lysine 1 5 10 15 Ala Cys Asn Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn Gln 20 25 30 Thr Val Cys Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val Val 35 40 45 Ser Ala Thr Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu Gln 50 55 60 Ser Met Ser Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg Cys 65 70 75 80 Ala Tyr Gly Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala Cys 85 90 95 Arg Val Cys Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp Lys 100 105 110 Gln Asn Thr Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp Glu 115 120 125 Ala Asn His Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp Thr 130 135 140 Glu Arg Gln Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys Glu 145 150 155 160 Glu Ile Pro Gly Arg Trp Ile Thr Arg Ser Thr Pro Pro Glu Gly Ser 165 170 175 Asp Ser Thr Ala Pro Ser Thr Gln Glu Pro Glu Ala Pro Pro Glu Gln 180 185 190 Asp Leu Ile Ala Ser Thr Val Ala Gly Free Mp3 Download 195 200 205 Ser Ser Gln Pro Val Val Thr Arg Gly Thr Thr Asp Asn 210 215 220 <210> 5 <211> 1188 <212> DNA <213> The snowstorm <220> <223> The p75ECD-transforming complex-an Fc fragment <400> 5 aaggaggcat gccccacagg cctgtacaca cacagcggtg agtgctgcaa agcctgcaac ctgggcgagg gtgtggccca gccttgtgga gccaaccaga ccgtgtgtga gccctgcctg 180. gacagcgtga cgttctccga cgtggtgagc gcgaccgagc cgtgcaagcc gtgcaccgag tgcgtggggc tccagagcat gtcggcgccg tgcgtggagg ccgacgacgc cgtgtgccgc 240 tgcgcctacg gctactacca ggatgagacg actgggcgct gcgaggcgtg ccgcgtgtgc 300 gaggcgggct cgggcctcgt gttctcctgc caggacaagc agaacaccgt gtgcgaggag 360 tgccccgacg gcacgtattc cgacgaggcc aaccacgtgg acccgtgcct gccctgcacc 420 gtgtgcgagg acaccgagcg ccagctccgc gagtgcacac gctgggccga cgccgagtgc 480 gaggagatcc ctggccgttg gattacagac aaaactcaca catgcccacc gtgcccagca 540 cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 600 atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 660 gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 720 cgggaggagc agtacaacag cacgtaccgg gtggtcagcg tcctcaccgt cctgcaccag 780 gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc 840 atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg 900 cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 960 ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac 1020 aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc 1080 gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct 1140 ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaa 1188 <210> 6 <211> 396 <212> PRT <213> Artificial Sequence <220> <223> Human p75ECD - linker - Human Fc fusion <400> 6 Lys Glu Ala Cys Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys 1 5 10 15 Lys Ala Cys Asn Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn 20 25 30 Gln Thr Val Cys Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val 35 40 45 Val Ser Ala Thr Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu 50 55 60 Gln Ser Met Ser Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg 65 70 75 80 Cys Ala Tyr Gly Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala 85 90 95 Cys Arg Val Cys Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp 100 105 110 Lys Gln Asn Thr Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp 115 120 125 Glu Ala Asn His Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp 130 135 140 Thr Glu Arg Gln Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys 145 150 155 160 Glu Glu Ile Pro Gly Arg Trp Ile Thr Asp Lys Thr His Thr Cys Pro 165 170 175 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 180 185 190 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 195 200 205 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 210 215 220 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 225 230 235 240 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 245 250 255 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 260 265 270 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 275 280 285 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 290 295 300 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 305 310 315 320 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 325 330 335 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 340 345 350 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 355 360 365 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 370 375 380 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 385 390 395 <210> 7 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Forward PCR primer for human p75ECD <400> 7 gcgctctaga ccaccatggg ggcaggtgcc accgg 35 <210> 8 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Reverse PCR primers for human p75ECD <400> 8 ctctgtcgac ttatcattta cccggagaca ggg 33 <210> 9 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Forward PCR primer for human Fc <400> 9 aaccatgggc gacaaaactc acacatg 27 <210> 10 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Reverse PCR primers for human Fc <400> 10 aagaattctc atttacccgg agacag 26 <210> 11 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Forward PCR primer for human Fc <400> 11 ttggattaca gacaaaactc acacatgccc 30 <210> 12 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Reverse PCR primers for human Fc <400> 12 ctctgtcgac ttatcattta cccggagaca ggg 33 <210> 13 <211> 2217 <212> DNA <213> Artificial Sequence <220> <223> Adeno-associated virus (AAV) 8 capsid sequence with AAV2 phospholipase domain <400> 13 atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga aggaataaga 60 cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120 gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180 aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctacgac 240 cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300 caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360 gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg 420 ggaaagaaga gaccggtaga gccatcaccc cagcgttctc cagactcctc tacgggcatc 480 ggcaagaaag gccaacagcc cgccagaaaa agactcaatt ttggtcagac tggcgactca 540 gagtcagttc cagaccctca acctctcgga gaacctccag cagcgccctc tggtgtggga cctaacaa tggctgcagg cggtggcgca ccaatggcag acaataacga aggcgccgac ggagtgggta gttcctcggg aaattggcat tgcgattcca catggctggg cgacagagtc 720 atcaccacca gcacccgac ctgggccctg cccacctaca acaaccacct ctacaagcaa atctccaacg ggacatcggg aggagccacc aacgacaaca cctacttcgg ctacagcacc ccctgggggt attttgactt taacagattc cactgccact tttcaccacg tgactggcag cgactcatca acaacaactg gggattccgg cccaagagac tcagcttcaa gctcttcaac atccaggtca aggaggtcac gcagaatgaa ggcaccaaga ccatcgccaa taacctcacc agcaccatcc aggtgtttac ggactcggag taccagctgc cgtacgttct cggctctgcc 1080. caccagggct gcctgcctcc gttcccggcg gacgtgttca tgattcccca gtacggctac 1140 ctaacactca acaacggtag tcaggccgtg ggacgctcct ccttctactg cctggaatac tttccttcgc agatgctgag aaccggcaac aacttccagt ttacttacac cttcgaggac gtgcctttcc acagcagcta cgcccacagc cagagcttgg accggctgat gaatcctctg 1320 attgaccagt acctgtacta cttgtctcgg actcaaacaa caggaggcac ggcaaatacg 1380 cagactctgg gcttcagcca aggtgggcct aatacaatgg ccaatcaggc aaagaactgg 1440 ctgccaggac cctgttaccg ccaacaacgc gtctcaacga caaccgggca aaacaacaat 1500 agcaactttg cctggactgc tgggaccaaa taccatctga atggaagaaa ttcattggct 1560 aatcctggca tcgctatggc aacacacaaa gacgacgagg agcgtttttt tcccagtaac 1620 gggatcctga tttttggcaa acaaaatgct gccagagaca atgcggatta cagcgatgtc 1680 atgctcacca gcgaggaaga aatcaaaacc actaaccctg tggctacaga ggaatacggt 1740 atcgtggcag ataacttgca gcagcaaaac acggctcctc aaattggaac tgtcaacagc 1800 cagggggcct tacccggtat ggtctggcag aaccgggacg tgtacctgca gggtcccatc 1860 tgggccaaga ttcctcacac ggacggcaac ttccacccgt ctccgctgat gggcggcttt 1920 ggcctgaaac atcctccgcc tcagatcctg atcaagaaca cgcctgtacc tgcggatcct 1980 ccgaccacct tcaaccagtc aaagctgaac tctttcatca cgcaatacag caccggacag 2040 gtcagcgtgg aaattgaatg ggagctgcag aagaaaaca gcaagcgctg gaacccgag 2100 atccagtaca cctccaacta ctacaaatct acaagtgtgg actttgctgt taatacagaa 2160 ggcgtgtact ctgaaccccg ccccattggc acccgttacc tcacccgtaa tctgtaa 2217

Claims

1. Use of a p75ECD-Fc fusion as the sole active ingredient in the preparation of a medicament for treating and / or preventing frontotemporal dementia, wherein the amino acid sequence of the p75ECD-Fc fusion is shown in SEQ ID NO:

6.

2. The use of claim 1, wherein the p75ECD-Fc fusion is administered in the form of a p75ECD-Fc fusion adeno-associated virus (AAV) vector.

3. The use of claim 1, wherein the p75ECD-Fc fusion is a recombinant peptide.

4. The use of claim 1, wherein the p75ECD-Fc fusion is administered by intraperitoneal, intracerebroventricular, intrathecal or intramuscular injection.

5. The use according to claim 1, wherein the subject for which the medicament is used to treat and / or prevent frontotemporal dementia is a human being.

Images