Fig. 2.Transformants releasing E␤C suffered less damage than control lines when EPNs were present. (A) Root damage measured on plants that had received neither WCR eggs nor nematodes was minimal, and there was no difference between transformed and nontransformed plants (n ϭ 5, P ϭ 0.87). (B) Root damage on plants that received only WCR eggs, but no nematodes, was substantial. Again, no significant difference was found between the transformed and nontransformed plants (n ϭ 5, P ϭ 0.18). (C) In plots that received WCR eggs and H. megidis, roots from transformed plants (pooled) had significantly less damage than roots from control lines (n ϭ 30, P ϭ 0.007). Approximately one-quarter of the transformed plants were found not to emit E␤C. Removing these plants from the statistical analysis did not significantly affect the results. The letters above the bars indicate significant differences within a graph. Error bars indicate standard errors.

The aggregation of proteins into oligomers and amyloid fibrils is characteristic of several neurodegenerative diseases, including Parkinson disease (PD). In PD, the process of aggregation of α-synuclein (α-syn) from monomers, via oligomeric intermediates, into amyloid fibrils is considered the disease-causative toxic mechanism. We developed α-syn mutants that promote oligomer or fibril formation and tested the toxicity of these mutants by using a rat lentivirus system to investigate loss of dopaminergic neurons in the substantia nigra. The most severe dopaminergic loss in the substantia nigra is observed in animals with the α-syn variants that form oligomers (i.e., E57K and E35K), whereas the α-syn variants that form fibrils very quickly are less toxic. We show that α-syn oligomers are toxic in vivo and that α-syn oligomers might interact with and potentially disrupt membranes. P arkinson disease (PD) is the most common movement disorder, currently affecting approximately 2% of the population older than age 60 y. Prominent neuropathological hallmarks of PD are the loss of dopaminergic neurons in the substantia nigra (SN) region of the midbrain (1) and the presence of α-syn-containing intracellular inclusions: Lewy bodies (LBs) and Lewy neurites (2). α-Syn, a 140-aa protein physiologically found in presynaptic terminals of neurons, is the major fibrillar protein in LBs and Lewy neurites in sporadic and inherited PD. Moreover, point mutations (A53T, A30P, E46K) and gene multiplications of human WT (hWT) α-syn are related to rare familial autosomal-dominant forms of early-onset PD (3-6), suggesting that increased gene dosage and aberrant protein structure may accelerate disease onset and progression.Recent reports indicate that the accumulation of α-syn can result in the formation of intermediate-state oligomers, and oligomers of different shapes and sizes have been described (7-10). These oligomers interact with lipids, disrupt membranes (7,8), and cause cell death in vitro (10, 11) and in nonmammalian models, such as Caenorhabditis elegans and Drosophila melanogaster (12). However, we are aware of no previous direct in vivo demonstration of the toxicity of α-syn oligomers in mammals.We aim to establish a model that allows specific testing of the effects of α-syn oligomerization in vitro and in vivo. To elucidate the causal structure-toxicity relationship of these oligomeric protein assemblies in a mammalian system, we designed "conformation-trapped" mutants based on structural modeling of α-syn fibrils (13, 14). Structurally, amyloid fibrils of α-syn are composed of cross-β-sheets (15). Residues from approximately 30 to 110 of α-syn form the core of the fibrils, whereas the approximately 30 N-terminal residues are heterogeneous and the approximately 30 C-terminal residues are flexible (13,14,16,17). Based on our structural model, recently developed from NMR data, the core of α-syn fibrils comprises five β-strands reminiscent of a five-layered "β-sandwich" (14). Several loops adjacent to and between the strands ar...

Abnormal deposition and intercellular propagation of α-synuclein plays a central role in the pathogenesis of disorders such as Parkinson's Disease (PD) and dementia with Lewy bodies (DLB). Previous studies demonstrated that immunization against α-synuclein resulted in reduced α-synuclein accumulation and synaptic loss in a transgenic (tg) mouse model, highlighting the potential for immunotherapy. However, the mechanism by which immunization prevents synucleinopathy-associated deficits remains unknown. Here, we show that antibodies against α-synuclein specifically target and aid in clearance of extracellular α-synuclein proteins by microglia, thereby preventing their actions on neighboring cells. Antibody-assisted clearance occurs mainly in microglia through the Fcγ receptor, and not in neuronal cells or astrocytes. Stereotaxic administration of antibody into the brains of α-synuclein tg mice prevented neuron-to-astroglia transmission of α-synuclein and led to increased localization of α-synuclein and the antibody in microglia. Furthermore, passive immunization with α-synuclein antibody reduced neuronal and glial accumulation of α-synuclein and ameliorated neurodegeneration and behavioral deficits associated with α-synuclein overexpression. These findings provide an underlying mechanistic basis for immunotherapy for PD/DLB and suggest extracellular forms of α-synuclein as potential therapeutic targets.

BackgroundLewy body disease is a heterogeneous group of neurodegenerative disorders characterized by α-synuclein accumulation that includes dementia with Lewy bodies (DLB) and Parkinson's Disease (PD). Recent evidence suggests that impairment of lysosomal pathways (i.e. autophagy) involved in α-synuclein clearance might play an important role. For this reason, we sought to examine the expression levels of members of the autophagy pathway in brains of patients with DLB and Alzheimer's Disease (AD) and in α-synuclein transgenic mice.Methodology/Principal FindingsBy immunoblot analysis, compared to controls and AD, in DLB cases levels of mTor were elevated and Atg7 were reduced. Levels of other components of the autophagy pathway such as Atg5, Atg10, Atg12 and Beclin-1 were not different in DLB compared to controls. In DLB brains, mTor was more abundant in neurons displaying α-synuclein accumulation. These neurons also showed abnormal expression of lysosomal markers such as LC3, and ultrastructural analysis revealed the presence of abundant and abnormal autophagosomes. Similar alterations were observed in the brains of α-synuclein transgenic mice. Intra-cerebral infusion of rapamycin, an inhibitor of mTor, or injection of a lentiviral vector expressing Atg7 resulted in reduced accumulation of α-synuclein in transgenic mice and amelioration of associated neurodegenerative alterations.Conclusions/SignificanceThis study supports the notion that defects in the autophagy pathway and more specifically in mTor and Atg7 are associated with neurodegeneration in DLB cases and α-synuclein transgenic models and supports the possibility that modulators of the autophagy pathway might have potential therapeutic effects.

BackgroundMisfolding and pathological aggregation of neuronal proteins has been proposed to play a critical role in the pathogenesis of neurodegenerative disorders. Alzheimer's disease (AD) and Parkinson's disease (PD) are frequent neurodegenerative diseases of the aging population. While progressive accumulation of amyloid β protein (Aβ) oligomers has been identified as one of the central toxic events in AD, accumulation of α-synuclein (α-syn) resulting in the formation of oligomers and protofibrils has been linked to PD and Lewy body Disease (LBD). We have recently shown that Aβ promotes α-syn aggregation and toxic conversion in vivo, suggesting that abnormal interactions between misfolded proteins might contribute to disease pathogenesis. However the molecular characteristics and consequences of these interactions are not completely clear.Methodology/Principal FindingsIn order to understand the molecular mechanisms involved in potential Aβ/α-syn interactions, immunoblot, molecular modeling, and in vitro studies with α-syn and Aβ were performed. We showed in vivo in the brains of patients with AD/PD and in transgenic mice, Aβ and α-synuclein co-immunoprecipitate and form complexes. Molecular modeling and simulations showed that Aβ binds α-syn monomers, homodimers, and trimers, forming hybrid ring-like pentamers. Interactions occurred between the N-terminus of Aβ and the N-terminus and C-terminus of α-syn. Interacting α-syn and Aβ dimers that dock on the membrane incorporated additional α-syn molecules, leading to the formation of more stable pentamers and hexamers that adopt a ring-like structure. Consistent with the simulations, under in vitro cell-free conditions, Aβ interacted with α-syn, forming hybrid pore-like oligomers. Moreover, cells expressing α-syn and treated with Aβ displayed increased current amplitudes and calcium influx consistent with the formation of cation channels.Conclusion/SignificanceThese results support the contention that Aβ directly interacts with α-syn and stabilized the formation of hybrid nanopores that alter neuronal activity and might contribute to the mechanisms of neurodegeneration in AD and PD. The broader implications of such hybrid interactions might be important to the pathogenesis of other disorders of protein misfolding.

Transcriptional dysregulation has emerged as a core pathologic feature of Huntington's disease (HD), one of several triplet-repeat disorders characterized by movement deficits and cognitive dysfunction. Although the mechanisms contributing to the gene expression deficits remain unknown, therapeutic strategies have aimed to improve transcriptional output via modulation of chromatin structure. Recent studies have demonstrated therapeutic effects of commercially available histone deacetylase (HDAC) inhibitors in several HD models; however, the therapeutic value of these compounds is limited by their toxic effects. Here, beneficial effects of a novel pimelic diphenylamide HDAC inhibitor, HDACi 4b, in an HD mouse model are reported. Chronic oral administration of HDACi 4b, beginning after the onset of motor deficits, significantly improved motor performance, overall appearance, and body weight of symptomatic R6/2 300Q transgenic mice. These effects were associated with significant attenuation of gross brain-size decline and striatal atrophy. Microarray studies revealed that HDACi 4b treatment ameliorated, in part, alterations in gene expression caused by the presence of mutant huntingtin protein in the striatum, cortex, and cerebellum of R6/2 300Q transgenic mice. For selected genes, HDACi 4b treatment reversed histone H3 hypoacetylation observed in the presence of mutant huntingtin, in association with correction of mRNA expression levels. These findings suggest that HDACi 4b, and possibly related HDAC inhibitors, may offer clinical benefit for HD patients and provide a novel set of potential biomarkers for clinical assessment.rologic disorder caused by a CAG repeat expansion within the coding region of the HD gene (Htt), resulting in a mutant protein (htt) with a lengthened polyglutamine tract (1). Mutant htt protein has been shown to disrupt transcription by multiple mechanisms, but it is unclear which are most important to pathology (2-4). By interacting with specific transcription factors, htt can alter the expression of clusters of genes controlled by those factors. For example, several genes driven by Sp1, which has been shown to interact with htt (5, 6), show decreased mRNA expression in human HD and in mouse models of HD (7). Alternatively, htt may have more global effects on transcription by disrupting core transcriptional machinery (8, 9) or by altering posttranslational modifications of histones, resulting in condensed chromatin structure (10-13). Understanding the basis for transcriptional dysregulation is important for choosing appropriate drug-discovery strategies.Manifestations of transcriptional dysregulation are evident from several gene-profiling studies, which have revealed alterations in the expression of large numbers of genes in the brains of different HD mouse models and in human subjects with HD (7, 14-16). Many of the expression changes in mouse models are observed in early stages of illness before the onset of symptoms, suggesting that gene expression alterations may be pathogenic.Because o...

Neurodegenerative disorders such as Alzheimer's Disease, Parkinson's Disease, fronto-temporal dementia, Huntington's Disease and Creutzfeldt-Jakob Disease (CJD) are characterized by progressive accumulation of protein aggregates in selected brain regions. Protein misfolding and templated assembly into aggregates might result from an imbalance between protein synthesis, aggregation and clearance. While protein misfolding and aggregation occur in most neurodegenerative disorders, the concept of spreading and infectivity of aggregates in the CNS has been reserved to prion diseases such as CJD and bovine spongiform encephalopathy. Emerging evidence suggests that prion-like spreading may occur in other neurodegenerative disorders, taking place with secreted proteins, such as amyloid-β,) and cytosolic proteins, such as tau, huntingtin and α-synuclein. Underlying molecular mechanisms and therapeutic implications are discussed.

Objective: We aimed to investigate whether HIV latency in the CNS might have adverse molecular, pathologic, and clinical consequences.Methods: This was a case-control comparison of HIV-1 seropositive (HIV1) patients with clinical and neuropathologic examination. Based on the levels of HIV-1 DNA, RNA, and p24 in the brain, cases were classified as controls, latent HIV CNS infection, and HIV encephalitis (HIVE). Analysis of epigenetic markers including BCL11B, neurodegeneration, and neuroinflammation was performed utilizing immunoblot, confocal microscopy, immunochemistry/image analysis, and qPCR. Detailed antemortem neurocognitive data were available for 23 out of the 32 cases.Results: HIV1 controls (n 5 12) had no detectable HIV-1 DNA, RNA, or p24 in the CNS; latent HIV1 cases (n 5 10) showed high levels of HIV-1 DNA but no HIV RNA or p24; and HIVE cases (n 5 10) had high levels of HIV-1 DNA, RNA, and p24. Compared to HIV1 controls, the HIV1 latent cases displayed moderate cognitive impairment with neurodegenerative and neuroinflammatory alterations, although to a lesser extent than HIVE cases. Remarkably, HIV1 latent cases showed higher levels of BCL11B and other chromatin modifiers involved in silencing. Increased BCL11B was associated with deregulation of proinflammatory genes like interleukin-6, tumor necrosis factor-a, and CD74. Conclusion:Persistence of latent HIV-1 infection in the CNS was associated with increased levels of chromatin modifiers, including BCL11B. Alteration of these epigenetic factors might result in abnormal transcriptomes, leading to inflammation, neurodegeneration, and neurocognitive impairment. BCL11B and other epigenetic factors involved in silencing might represent potential targets for HIV-1 involvement of the CNS.