Neuroprotective effects of Cerebrolysin in triple repeat Tau transgenic model of Pick's disease and fronto-temporal tauopathies.

BackgroundTauopathies are a group of neurodegenerative disorders with accumulation of three-repeat (3R) or four-repeat (4R) Tau. While 3R tau is found in Pick's disease and Alzheimer's disease (AD), 4R tau is more abundant in corticobasal degeneration, progressive supranuclear palsy, and AD. We have previously shown that Cerebrolysin™ (CBL), a neuropeptide mixture with neurotrophic effects, ameliorates the pathology in amyloid precursor protein transgenic (tg) mouse model of AD and 4R tau, however it is unclear if CBL ameliorates the deficits and neuropathology in the mouse model of Pick's disease over expressing 3R tau.ResultsMice expressing 3R tau (L266V and G272V mutations) under the mThy-1 promoter were treated with CBL in two separate groups, the first was 3 months old (treated for 3 months, IP) and the second was 6 months old (treated for 3 months, IP) at the start of the treatment. We found that although the levels of total 3R tau were unchanged, CBL reduced the levels of hyper-phosphorylated tau in both groups of mice. This was accompanied by reduced neurodegenerative pathology in the neocortex and hippocampus in both groups and by improvements in the behavioral deficits in the nest-building test and water maze in the 3-6 month group.ConclusionTaken together these results support the notion that CBL may be beneficial in other taupathy models by reducing the levels of aberrantly phosphorylated tau

Neuroprotective effects of the immunomodulatory drug FK506 in a model of HIV1-gp120 neurotoxicity.

BackgroundHIV-associated neurocognitive disorders (HAND) continue to be a common morbidity associated with chronic HIV infection. It has been shown that HIV proteins (e.g., gp120) released from infected microglial/macrophage cells can cause neuronal damage by triggering inflammation and oxidative stress, activating aberrant kinase pathways, and by disrupting mitochondrial function and biogenesis. Previous studies have shown that FK506, an immunophilin ligand that modulates inflammation and mitochondrial function and inhibits calcineurin, is capable of rescuing the neurodegenerative pathology in models of Parkinson's disease, Alzheimer's disease, and Huntington's disease. In this context, the main objective of this study was to evaluate if FK506 could rescue the neuronal degeneration and mitochondrial alterations in a transgenic (tg) animal model of HIV1-gp120 neurotoxicity.MethodsGFAP-gp120 tg mice were treated with FK506 and analyzed for neuropathology, behavior, mitochondrial markers, and calcium flux by two-photon microscopy.ResultsWe found that FK506 reduced the neuronal cell loss and neuro-inflammation in the gp120 tg mice. Moreover, while vehicle-treated gp120 tg mice displayed damaged mitochondria and increased neuro-inflammatory markers, FK506 rescued the morphological mitochondrial alterations and neuro-inflammation while increasing levels of optic atrophy 1 and mitofusin 1. By two-photon microscopy, calcium levels were not affected in the gp120 tg mice and no effects of FK506 were detected. However, at a functional level, FK506 ameliorated the gp120 tg mice hyperactivity in the open field.ConclusionsTogether, these results suggest that FK506 might be potentially neuroprotective in patients with HAND by mitigating inflammation and mitochondrial alterations

Cerebrolysin™ efficacy in a transgenic model of tauopathy: role in regulation of mitochondrial structure.

BackgroundAlzheimer's Disease (AD) and Fronto temporal lobar dementia (FTLD) are common causes of dementia in the aging population for which limited therapeutical options are available. These disorders are associated with Tau accumulation. We have previously shown that Cerebrolysin™ (CBL), a neuropeptide mixture with neurotrophic effects, ameliorates the behavioral deficits and neuropathological alterations in amyloid precursor protein (APP) transgenic (tg) mouse model of AD by reducing hyper-phosphorylated Tau. CBL has been tested in clinical trials for AD, however it's potential beneficial effects in FTLD are unknown. For this purpose we sought to investigate the effects of CBL in a tg model of tauopathy. Accordingly, double tg mice expressing mutant Tau under the mThy-1 promoter and GSK3β (to enhance Tau phosphorylation) were treated with CBL and evaluated neuropathologically.ResultsCompared to single Tau tg mice the Tau/GSK3β double tg model displayed elevated levels of Tau phosphorylation and neurodegeneration in the hippocampus. CBL treatment reduced the levels of Tau phosphorylation in the dentate gyrus and the degeneration of pyramidal neurons in the temporal cortex and hippocampus of the Tau/GSK3β double tg mice. Interestingly, the Tau/GSK3β double tg mice also displayed elevated levels of Dynamin-related protein-1 (Drp-1), a protein that hydrolyzes GTP and is required for mitochondrial division. Ultrastructural analysis of the mitochondria in the Tau/GSK3β double tg mice demonstrated increased numbers and fragmentation of mitochondria in comparison to non-tg mice. CBL treatment normalized levels of Drp-1 and restored mitochondrial structure.ConclusionsThese results suggest that the ability of CBL to ameliorate neurodegenerative pathology in the tauopathy model may involve reducing accumulation of hyper-phosphorylated Tau and reducing alterations in mitochondrial biogenesis associated with Tau

Age-dependent molecular alterations in the autophagy pathway in HIVE patients and in a gp120 tg mouse model: reversal with beclin-1 gene transfer.

Aged (>50 years old) human immunodeficiency virus (HIV) patients are the fastest-growing segment of the HIV-infected population in the USA and despite antiretroviral therapy, HIV-associated neurocognitive disorder (HAND) prevalence has increased or remained the same among this group. Autophagy is an intracellular clearance pathway for aggregated proteins and aged organelles; dysregulation of autophagy is implicated in the pathogenesis of Parkinson's disease, Alzheimer's disease, and HAND. Here, we hypothesized that dysregulated autophagy may contribute to aging-related neuropathology in HIV-infected individuals. To explore this possibility, we surveyed autophagy marker levels in postmortem brain samples from a cohort of well-characterized <50 years old (young) and >50 years old (aged) HIV+ and HIV encephalitis (HIVE) patients. Detailed clinical and neuropathological data showed the young and aged HIVE patients had higher viral load, increased neuroinflammation and elevated neurodegeneration; however, aged HIVE postmortem brain tissues showed the most severe neurodegenerative pathology. Interestingly, young HIVE patients displayed an increase in beclin-1, cathepsin-D and light chain (LC)3, but these autophagy markers were reduced in aged HIVE cases compared to age-matched HIV+ donors. Similar alterations in autophagy markers were observed in aged gp120 transgenic (tg) mice; beclin-1 and LC3 were decreased in aged gp120 tg mice while mTor levels were increased. Lentivirus-mediated beclin-1 gene transfer, that is known to activate autophagy pathways, increased beclin-1, LC3, and microtubule-associated protein 2 expression while reducing glial fibrillary acidic protein and Iba1 expression in aged gp120 tg mice. These data indicate differential alterations in the autophagy pathway in young versus aged HIVE patients and that autophagy reactivation may ameliorate the neurodegenerative phenotype in these patients

Early neuronal accumulation of DNA double strand breaks in Alzheimer's disease.

The maintenance of genomic integrity is essential for normal cellular functions. However, it is difficult to maintain over a lifetime in postmitotic cells such as neurons, in which DNA damage increases with age and is exacerbated by multiple neurological disorders, including Alzheimer's disease (AD). Here we used immunohistochemical staining to detect DNA double strand breaks (DSBs), the most severe form of DNA damage, in postmortem brain tissues from patients with mild cognitive impairment (MCI) or AD and from cognitively unimpaired controls. Immunostaining for γH2AX-a post-translational histone modification that is widely used as a marker of DSBs-revealed increased proportions of γH2AX-labeled neurons and astrocytes in the hippocampus and frontal cortex of MCI and AD patients, as compared to age-matched controls. In contrast to the focal pattern associated with DSBs, some neurons and glia in humans and mice showed diffuse pan-nuclear patterns of γH2AX immunoreactivity. In mouse brains and primary neuronal cultures, such pan-nuclear γH2AX labeling could be elicited by increasing neuronal activity. To assess whether pan-nuclear γH2AX represents DSBs, we used a recently developed technology, DNA damage in situ ligation followed by proximity ligation assay, to detect close associations between γH2AX sites and free DSB ends. This assay revealed no evidence of DSBs in neurons or astrocytes with prominent pan-nuclear γH2AX labeling. These findings suggest that focal, but not pan-nuclear, increases in γH2AX immunoreactivity are associated with DSBs in brain tissue and that these distinct patterns of γH2AX formation may have different causes and consequences. We conclude that AD is associated with an accumulation of DSBs in vulnerable neuronal and glial cell populations from early stages onward. Because of the severe adverse effects this type of DNA damage can have on gene expression, chromatin stability and cellular functions, DSBs could be an important causal driver of neurodegeneration and cognitive decline in this disease

Oral N-Acetyl-Cysteine Attenuates Loss of Dopaminergic Terminals in α-Synuclein Overexpressing Mice

Levels of glutathione are lower in the substantia nigra (SN) early in Parkinson's disease (PD) and this may contribute to mitochondrial dysfunction and oxidative stress. Oxidative stress may increase the accumulation of toxic forms of α-synuclein (SNCA). We hypothesized that supplementation with n-acetylcysteine (NAC), a source of cysteine – the limiting amino acid in glutathione synthesis, would protect against α-synuclein toxicity. Transgenic mice overexpressing wild-type human α-synuclein drank water supplemented with NAC or control water supplemented with alanine from ages 6 weeks to 1 year. NAC increased SN levels of glutathione within 5–7 weeks of treatment; however, this increase was not sustained at 1 year. Despite the transient nature of the impact of NAC on brain glutathione, the loss of dopaminergic terminals at 1 year associated with SNCA overexpression was significantly attenuated by NAC supplementation, as measured by immunoreactivity for tyrosine hydroxylase in the striatum (p = 0.007; unpaired, two-tailed t-test), with a similar but nonsignificant trend for dopamine transporter (DAT) immunoreactivity. NAC significantly decreased the levels of human SNCA in the brains of PDGFb-SNCA transgenic mice compared to alanine treated transgenics. This was associated with a decrease in nuclear NFκB localization and an increase in cytoplasmic localization of NFκB in the NAC-treated transgenics. Overall, these results indicate that oral NAC supplementation decreases SNCA levels in brain and partially protects against loss of dopaminergic terminals associated with overexpression of α-synuclein in this model

Active immunization against alpha-synuclein ameliorates the degenerative pathology and prevents demyelination in a model of multiple system atrophy.

BackgroundMultiple system atrophy (MSA) is a neurodegenerative disease characterized by parkinsonism, ataxia and dysautonomia. Histopathologically, the hallmark of MSA is the abnormal accumulation of alpha-synuclein (α-syn) within oligodendroglial cells, leading to neuroinflammation, demyelination and neuronal death. Currently, there is no disease-modifying treatment for MSA. In this sense, we have previously shown that next-generation active vaccination technology with short peptides, AFFITOPEs®, was effective in two transgenic models of synucleinopathies at reducing behavioral deficits, α-syn accumulation and inflammation.ResultsIn this manuscript, we used the most effective AFFITOPE® (AFF 1) for immunizing MBP-α-syn transgenic mice, a model of MSA that expresses α-syn in oligodendrocytes. Vaccination with AFF 1 resulted in the production of specific anti-α-syn antibodies that crossed into the central nervous system and recognized α-syn aggregates within glial cells. Active vaccination with AFF 1 resulted in decreased accumulation of α-syn, reduced demyelination in neocortex, striatum and corpus callosum, and reduced neurodegeneration. Clearance of α-syn involved activation of microglia and reduced spreading of α-syn to astroglial cells.ConclusionsThis study further validates the efficacy of vaccination with AFFITOPEs® for ameliorating the neurodegenerative pathology in synucleinopathies

Long-term neprilysin gene transfer is associated with reduced levels of intracellular Abeta and behavioral improvement in APP transgenic mice

<p>Abstract</p> <p>Background</p> <p>Proteolytic degradation has emerged as a key pathway involved in controlling levels of the Alzheimer's disease (AD)-associated amyloid-β (Aβ) peptide in the brain. The endopeptidase, neprilysin, has been implicated as a major Aβ degrading enzyme in mice and humans. Previous short and intermediate term studies have shown the potential therapeutic application of neprilysin by delivering this enzyme into the brain of APP transgenic mice using gene transfer with viral vectors. However the effects of long-term neprilysin gene transfer on other aspects of Aβ associated pathology have not been explored yet in APP transgenic mice.</p> <p>Results</p> <p>We show that the sustained expression of neprilysin for up to 6 months lowered not only the amyloid plaque load but also reduced the levels of intracellular Aβ immunoreactivity. This was associated with improved behavioral performance in the water maze and ameliorated the dendritic and synaptic pathology in the APP transgenic mice.</p> <p>Conclusion</p> <p>These data support the possibility that long-term neprilysin gene therapy improves behavioral and neurodegenerative pathology by reducing intracellular Aβ.</p

Combined Active Humoral and Cellular Immunization Approaches for the Treatment of Synucleonopathies

Objectives: Parkinson’s Disease (PD), Dementia with Lewy bodies (DLB), and Multiple System Atrophy (MSA) are neurodegenerative disorders of the aging population characterized by the progressive accumulation of alpha-synuclein. Jointly these disorders have been denominated synucleinopathies and currently no disease modifying treatments are available. Previous in vivo studies in transgenic (tg) mice have shown that active and passive immunization targeting alpha-synuclein ameliorates to some extent deficits and synuclein accumulation, however it’s unknown if combining humoral and cellular immunization might synergize and also reduce inflammation and improve microglial cell mediated synuclein clearance. Methods: PDGF- alpha-synuclein tg mice and control non-tg mice were immunized with: 1) Glucan Particle (GP) adjuvant alone, 2) GP human (hu)- alpha-synuclein (active immunization), 3) GP plus rapamycin and 4) GP plus rapamycin and hu-alpha-synuclein (combined active and humoral) and analyzed by neuropathological and biochemical markers. Results: Compared to tg mice treated with adjuvant alone, mice immunized with GP hu-alpha-synuclein displayed a 30% reduction in alpha-synuclein accumulation. Combined immunotherapy with GP plus rapamycin and hu-alpha-synuclein resulted in 50% reduction in alpha-synuclein accumulation which was accompanied by reduced neuro-inflammation (Iba-1, GFAP, IL6, TNFalpha), phospho and insoluble alpha-synuclein, microglia and astroglia cell numbers, and retention of CD25, FoxP3 and CD4 positive cells. Levels of TGFb1 were also increased. Serological studies showed that active immunization resulted in higher levels of total IgG, IgG1 and IgG2 titers, levels were slightly higher in the combined group. Conclusions: In vivo studies targeting alpha-synuclein support the hypothesis that cellular immunization might enhance the effects of active immunotherapy for the treatment of synucleionopathies