Search

Parkinson disease (PD) is a multifactorial neurodegenerative disorder with high incidence in the elderly, where environmental and genetic factors are involved in etiology. In addition, epigenetic mechanisms, including deregulation of DNA methylation have been recently associated to PD. As accurate diagnosis cannot be achieved pre-mortem, identification of early pathological changes is crucial to enable therapeutic interventions before major neuropathological damage occurs. Here we investigated genome-wide DNA methylation in brain and blood samples from PD patients and observed a distinctive pattern of methylation involving many genes previously associated to PD, therefore supporting the role of epigenetic alterations as a molecular mechanism in neurodegeneration. Importantly, we identified concordant methylation alterations in brain and blood, suggesting that blood might hold promise as a surrogate for brain tissue to detect DNA methylation in PD and as a source for biomarker discovery.

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.

Parkinson disease is characterized by the loss of dopaminergic neurons mainly in the substantia nigra. Accumulation of α-synuclein and cell loss has been also reported in many other brain regions including the hippocampus, where it might impair adult neurogenesis, contributing to nonmotor symptoms. However, the molecular mechanisms of these alterations are still unknown. In this report we show that α-synuclein-accumulating adult rat hippocampus neural progenitors present aberrant neuronal differentiation, with reduction of Notch1 expression and downstream signaling targets. We characterized a Notch1 proximal promoter that contains p53 canonical response elements. In vivo binding of p53 represses the transcription of Notch1 in neurons. Moreover, we demonstrated that α-synuclein directly binds to the DNA at Notch1 promoter vicinity and also interacts with p53 protein, facilitating or increasing Notch1 signaling repression, which interferes with maturation and survival of neural progenitors cells. This study provides a molecular basis for α-synuclein-mediated disruption of adult neurogenesis in Parkinson disease.

Abnormal accumulation of α-synuclein is centrally involved in the pathogenesis of many disorders with Parkinsonism and dementia. Previous in vitro studies suggest that α-synuclein dysregulates intracellular calcium. However, it is unclear whether these alterations occur in vivo. For this reason, we investigated calcium dynamics in transgenic mice expressing human WT α-synuclein using two-photon microscopy. We imaged spontaneous and stimulus-induced neuronal activity in the barrel cortex. Transgenic mice exhibited augmented, long-lasting calcium transients characterized by considerable deviation from the exponential decay. The most evident pathology was observed in response to a repetitive stimulation in which subsequent stimuli were presented before relaxation of calcium signal to the baseline. These alterations were detected in the absence of significant increase in neuronal spiking response compared with age-matched controls, supporting the possibility that α-synuclein promoted alterations in calcium dynamics via interference with intracellular buffering mechanisms. The characteristic shape of calcium decay and augmented response during repetitive stimulation can serve as in vivo imaging biomarkers in this model of neurodegeneration, to monitor progression of the disease and screen candidate treatment strategies.

It is now recognized that the cell-to-cell transmission of misfolded proteins such as α-synuclein contributes to the neurodegenerative phenotype in neurological disorders such as idiopathic Parkinson's disease, Dementia with Lewy bodies, and Parkinson's disease dementia. Thus, establishing cell-based models for the transmission of α-synuclein is of importance to understand the mechanisms of neurodegeneration in these disorders and to develop new therapies. Here we describe methods to study the neuron-to-neuron propagation of α-synuclein in an in vitro setting that also has in vivo applications.

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.

DNA methylation is a major epigenetic modification that regulates gene expression. Dnmt1, the maintenance DNA methylation enzyme, is abundantly expressed in the adult brain and is mainly located in the nuclear compartment, where it has access to chromatin. Hypomethylation of CpG islands at intron 1 of the SNCA gene has recently been reported to result in overexpression of α-synuclein in Parkinson disease (PD) and related disorders. We therefore investigated the mechanisms underlying altered DNA methylation in PD and dementia with Lewy bodies (DLB). We present evidence of reduction of nuclear Dnmt1 levels in human postmortem brain samples from PD and DLB patients as well as in the brains of α-synuclein transgenic mice models. Furthermore, sequestration of Dnmt1 in the cytoplasm results in global DNA hypomethylation in human and mouse brains, involving CpG islands upstream of SNCA, SEPW1, and PRKAR2A genes. We report that association of Dnmt1 and α-synuclein might mediate aberrant subcellular localization of Dnmt1. Nuclear Dnmt1 levels were partially rescued by overexpression of Dnmt1 in neuronal cell cultures and in α-synuclein transgenic mice brains. Our results underscore a novel mechanism for epigenetic dysregulation in Lewy body diseases, which might underlie the decrease in DNA methylation reported for PD and DLB.

Dementia with Lewy bodies (DLB) and Parkinson's Disease (PD) are neurodegenerative disorders of the aging population characterized by the abnormal accumulation of alpha-synuclein (alpha-syn). Previous studies have suggested that excitotoxicity may contribute to neurodegeneration in these disorders, however the underlying mechanisms and their relationship to alpha-syn remain unclear. For this study we proposed that accumulation of alpha-syn might result in alterations in metabotropic glutamate receptors (mGluR), particularly mGluR5 which has been linked to deficits in murine models of PD. In this context, levels of mGluR5 were analyzed in the brains of PD and DLB human cases and alpha-syn transgenic (tg) mice and compared to age-matched, unimpaired controls, we report a 40% increase in the levels of mGluR5 and beta-arrestin immunoreactivity in the frontal cortex, hippocampus and putamen in DLB cases and in the putamen in PD cases. In the hippocampus, mGluR5 was more abundant in the CA3 region and co-localized with alpha-syn aggregates. Similarly, in the hippocampus and basal ganglia of alpha-syn tg mice, levels of mGluR5 were increased and mGluR5 and alpha-syn were co-localized and co-immunoprecipitated, suggesting that alpha-syn interferes with mGluR5 trafficking. The increased levels of mGluR5 were accompanied by a concomitant increase in the activation of downstream signaling components including ERK, Elk-1 and CREB. Consistent with the increased accumulation of alpha-syn and alterations in mGluR5 in cognitive- and motor-associated brain regions, these mice displayed impaired performance in the water maze and pole test, these behavioral alterations were reversed with the mGluR5 antagonist, MPEP. Taken together the results from study suggest that mGluR5 may directly interact with alpha-syn resulting in its over activation and that this over activation may contribute to excitotoxic cell death in select neuronal regions. These results highlight the therapeutic importance of mGluR5 antagonists in alpha-synucleinopathies.

BACKGROUND Lewy body disease is a heterogeneous group of neurodegenerative disorders characterized by alpha-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 alpha-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 alpha-synuclein transgenic mice. METHODOLOGY/PRINCIPAL FINDINGS By 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 alpha-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 alpha-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 alpha-synuclein in transgenic mice and amelioration of associated neurodegenerative alterations. CONCLUSIONS/SIGNIFICANCE This 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 alpha-synuclein transgenic models and supports the possibility that modulators of the autophagy pathway might have potential therapeutic effects.

Neuronal accumulation of α-synuclein and Lewy body formation are characteristic to many neurodegenerative diseases, including Parkinson's disease (PD). This Lewy pathology appears to spread throughout the brain as the disease progresses. Furthermore, recent studies showed the occurrence of Lewy pathology in neurons grafted into the brains of PD patients, suggesting the spread of pathology from the host tissues to the grafts. The mechanism underlying this propagation is unknown. Here, we show that α-synuclein is transmitted via endocytosis to neighboring neurons and neuronal precursor cells, forming Lewy-like inclusions. Moreover, α-synuclein was transmitted from the affected neurons to engrafted neuronal precursor cells in a transgenic model of PD-like pathology. Failure of the protein quality control systems, especially lysosomes, promoted the accumulation of transmitted α-synuclein and inclusion formation. Cells exposed to neuron-derived α-synuclein showed signs of apoptosis, such as nuclear fragmentation and caspase 3 activation, both in vitro and in vivo. These findings demonstrate the cell-to-cell transmission of α-synuclein aggregates and provide critical insights into the mechanism of pathological progression in PD and other proteinopathies.

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.

Misfolding 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 beta protein (Abeta) oligomers has been identified as one of the central toxic events in AD, accumulation of alpha-synuclein (alpha-syn) resulting in the formation of oligomers and protofibrils has been linked to PD and Lewy body Disease (LBD). We have recently shown that Abeta promotes alpha-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.In order to understand the molecular mechanisms involved in potential Abeta/alpha-syn interactions, immunoblot, molecular modeling, and in vitro studies with alpha-syn and Abeta were performed. We showed in vivo in the brains of patients with AD/PD and in transgenic mice, Abeta and alpha-synuclein co-immunoprecipitate and form complexes. Molecular modeling and simulations showed that Abeta binds alpha-syn monomers, homodimers, and trimers, forming hybrid ring-like pentamers. Interactions occurred between the N-terminus of Abeta and the N-terminus and C-terminus of alpha-syn. Interacting alpha-syn and Abeta dimers that dock on the membrane incorporated additional alpha-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, Abeta interacted with alpha-syn, forming hybrid pore-like oligomers. Moreover, cells expressing alpha-syn and treated with Abeta displayed increased current amplitudes and calcium influx consistent with the formation of cation channels.These results support the contention that Abeta directly interacts with alpha-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.

Altered expression and mutations in alpha-synuclein (alpha-syn) have been linked to Parkinson's disease (PD) and related disorders. The neurological alterations in PD patients have been associated with degeneration of dopaminergic cells and other neuronal populations. Moreover, recent studies in murine models have shown that alterations in neurogenesis might also contribute to the neurodegenerative phenotype. However, the mechanisms involved and the effects of alpha-syn expression on neurogenesis are not yet clear. To this end, murine embryonic stem (mES) cells were infected with lentiviral (LV) vectors expressing wild-type (WT) and mutant alpha-syn. Compared with mES cells infected with LV-green fluorescent protein (GFP), cells expressing WT and mutant alpha-syn showed reduced proliferation as indicated by lower 5-bromo-2'-deoxyuridine uptake, increased apoptosis, and reduced expression of neuronal markers such as neuron specific enolase and beta-III tubulin. The alterations in neurogenesis in alpha-syn-expressing mES cells were accompanied by a reduction in Notch-1 and Hairy and enhancer of split-5 (Hes-5) mRNA and protein levels. Moreover, levels of total Notch-1 and Notch intracellular domain (NICD) were lower in mES cells expressing WT and mutant alpha-syn compared with GFP controls. The reduced survival of alpha-syn-expressing mES cells was reverted by overexpressing constitutively active NICD. Similarly, in alpha-syn transgenic mice, the alterations in neurogenesis in the hippocampal subgranular zone were accompanied by decreased Notch-1, NICD, and Hes-5 expression. Together, these results suggest that accumulation of alpha-syn might impair survival of NPCs by interfering with the Notch signaling pathway. Similar mechanisms could be at play in PD and Lewy body disease.

Transcriptional dysregulation has emerged as a central pathogenic mechanism in Huntington's disease (HD), which is associated with neuropathological changes predominantly in the striatum. Here we demonstrate that expression of Bcl11b (a.k.a. CTIP2), a transcription factor exhibiting highly-enriched localization in adult striatum, is significantly decreased in HD cells, mouse models and human subjects and that overexpression of Bcl11b attenuates toxic effects of mutant huntingtin in cultured striatal neurons. We show that Bcl11b directly activates the proximal promoter regions of striatal-enriched genes and can increase mRNA levels of striatal-expressing genes. We further demonstrate an interaction between Bcl11b and huntingtin protein in cultured cells and brain homogenates from HD R6/1 and YAC72 transgenic mice. We propose that sequestration and/or decreased expression of Bcl11b in HD is responsible, at least in part, for the dysregulation of striatal gene expression observed in HD and may contribute to the specificity of pathology observed in this disease.

Abnormal accumulation of α-synuclein is centrally involved in the pathogenesis of many disorders with Parkinsonism and dementia. Although the mechanism through which α-synuclein triggers neuronal vulnerability is not entirely clear, in vitro studies suggest that neuronal accumulation of α-synuclein might lead to increase in intracellular calcium levels. In this study, we investigated in vivo calcium dynamics in an α-synuclein transgenic mouse model of Parkinson's Disease and dementia with Lewy bodies using two photon microscopy. We imaged spontaneous and stimulus-induced neuronal activity in the barrel cortex. Transgenic mice exhibited abnormal calcium activity characterized by augmented, long-lasting transients with considerable deviation from the exponential decay. The most evident pathology was observed in response to a repetitive stimulation when subsequent stimuli were presented before relaxation of calcium signal to the baseline. Augmented calcium entry can result from an increase in neuronal spiking or a decrease in a cell buffering capacity. To address the former possibility, we performed in vivo electrophysiological recordings of population spiking activity. Our measurements revealed no significant increase in neuronal spiking response compared to age-matched controls. To investigate potential impairment in calcium buffering, we conducted experiments in a neuronal cell line expressing α-synuclein. Inhibitors of the calcium influx into the intracellular calcium stores (mitochondria and endoplasmic reticulum) rescued cytosolic calcium levels, elevated by α-synuclein, while inhibitors of the calcium influx via channels located on the plasma membrane were ineffective. These studies support the notion that α-synuclein dysregulates calcium buffering capacity in vivo. Moreover, the characteristic shape of calcium decay and augmentation of the response amplitude during repetitive stimulation can serve as in vivo imaging biomarkers for screening of potential therapeutic agents in this model of neurodegeneration.

The first identification and characterization of a prokaryotic gene ( spsA ) encoding sucrose-phosphate synthase (SPS) is reported for Synechocystis sp. strain PCC 6803, a unicellular non-nitrogen-fixing cyanobacterium. Comparisons of the deduced amino acid sequence and some relevant biochemical properties of the enzyme with those of plant SPSs revealed important differences in the N-terminal and UDP-glucose binding site regions, substrate specificities, molecular masses, subunit compositions, and regulatory properties.

Background Parkinson's disease (PD) is a multifactorial disease where environmental factors act on genetically predisposed individuals. Although only 5% of PD manifestations are associated with specific mutations, majority of PD cases are of idiopathic origin, where environment plays a prominent role. Concurrent exposure to Paraquat (PQ) and Maneb (MB) in rural workers increases the risk for PD and exposure of adult mice to MB/PQ results in dopamine fiber loss and decreased locomotor activity. While PD is characterized by neuronal loss in the substantia nigra, we previously showed that accumulation of α-synuclein in the limbic system contributes to neurodegeneration by interfering with adult neurogenesis. Results We investigated the effect of pesticides on adult hippocampal neurogenesis in two transgenic models: Line 61, expressing the human wild type SNCA gene and Line LRRK2(G2019S), expressing the human LRRK2 gene with the mutation G2019S. Combined exposure to MB/PQ resulted in significant reduction of neuronal precursors and proliferating cells in non-transgenic animals, and this effect was increased in transgenic mice, in particular for Line 61, suggesting that α-synuclein accumulation and environmental toxins have a synergistic effect. We further investigated the transcription of 84 genes with direct function on neurogenesis. Overexpresion of α-synuclein resulted in the downregulation of 12% of target genes, most of which were functionally related to cell differentiation, while LRRK2 mutation had a minor impact on gene expression. MB/PQ also affected transcription in non-transgenic backgrounds, but when transgenic mice were exposed to the pesticides, profound alterations in gene expression affecting 27% of the studied targets were observed in both transgenic lines. Gene enrichment analysis showed that 1:3 of those genes were under the regulation of FoxF2 and FoxO3A, suggesting a primary role of these proteins in the response to genetic and environmental cues. Conclusions We report that adult neurogenesis is highly susceptible to multiple “risk factors” for PD, including α-synuclein accumulation, LRRK2 G2019 mutation and exposure to environmental toxins. We identified specific groups of genes that are responsive to each stressor, while uncovering a novel function for Fox transcription factors in PD.

Nat. Rev. Neurol. 6, 702–706 (2010); doi: 10.1038/nrneurol.2010.145 In the December 2010 issue of Nature Reviews Neurology, the 'Correspondence to:' details on page 706 were incorrect. The corresponding author should be S.-J. Lee sjlee@konkuk.ac.kr.