Sarah Michael

α-Synuclein in Central Nervous System and from Erythrocytes, Mammalian Cells, and Escherichia coli Exists Predominantly as Disordered Monomer

Background: The oligomeric state of α-syn in vivo remains unknown. Results: α-syn in the CNS and produced by erythrocytes, mammalian cells, and Escherichia coli exists predominantly as a disordered monomer. Conclusion: Native α-syn from various sources behaves as unstructured and monomeric. Significance: Stabilizing monomeric α-syn, lowering its levels, and/or inhibiting its fibrillization remain viable therapeutic strategies for Parkinson disease. Since the discovery and isolation of α-synuclein (α-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that α-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in α-helical structure (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107–110; Wang, W., Perovic, I., Chittuluru, J., Kaganovich, A., Nguyen, L. T. T., Liao, J., Auclair, J. R., Johnson, D., Landeru, A., Simorellis, A. K., Ju, S., Cookson, M. R., Asturias, F. J., Agar, J. N., Webb, B. N., Kang, C., Ringe, D., Petsko, G. A., Pochapsky, T. C., and Hoang, Q. Q. (2011) Proc. Natl. Acad. Sci. 108, 17797–17802). However, it remains unknown whether or not this putative tetramer is the main physiological form of α-syn in the brain. In this study, we investigated the oligomeric state of α-syn in mouse, rat, and human brains. To assess the conformational and oligomeric state of native α-syn in complex mixtures, we generated α-syn standards of known quaternary structure and conformational properties and compared the behavior of endogenously expressed α-syn to these standards using native and denaturing gel electrophoresis techniques, size-exclusion chromatography, and an oligomer-specific ELISA. Our findings demonstrate that both human and rodent α-syn expressed in the central nervous system exist predominantly as an unfolded monomer. Similar results were observed when human α-syn was expressed in mouse and rat brains as well as mammalian cell lines (HEK293, HeLa, and SH-SY5Y). Furthermore, we show that α-syn expressed in E. coli and purified under denaturing or nondenaturing conditions, whether as a free protein or as a fusion construct with GST, is monomeric and adopts a disordered conformation after GST removal. These results do not rule out the possibility that α-syn becomes structured upon interaction with other proteins and/or biological membranes.

Aβ induces astrocytic glutamate release, extrasynaptic NMDA receptor activation, and synaptic loss

Significance Communication between nerve cells occurs at specialized cellular structures known as synapses. Loss of synaptic function is associated with cognitive decline in Alzheimer’s disease (AD). However, the mechanism of synaptic damage remains incompletely understood. Here we describe a pathway for synaptic damage whereby amyloid-β1–42 peptide (Aβ1–42) releases, via stimulation of α7 nicotinic receptors, excessive amounts of glutamate from astrocytes, in turn activating extrasynaptic NMDA-type glutamate receptors (eNMDARs) to mediate synaptic damage. The Food and Drug Administration-approved drug memantine offers some beneficial effect, but the improved eNMDAR antagonist NitroMemantine completely ameliorates Aβ-induced synaptic loss, providing hope for disease-modifying intervention in AD. Synaptic loss is the cardinal feature linking neuropathology to cognitive decline in Alzheimer’s disease (AD). However, the mechanism of synaptic damage remains incompletely understood. Here, using FRET-based glutamate sensor imaging, we show that amyloid-β peptide (Aβ) engages α7 nicotinic acetylcholine receptors to induce release of astrocytic glutamate, which in turn activates extrasynaptic NMDA receptors (eNMDARs) on neurons. In hippocampal autapses, this eNMDAR activity is followed by reduction in evoked and miniature excitatory postsynaptic currents (mEPSCs). Decreased mEPSC frequency may reflect early synaptic injury because of concurrent eNMDAR-mediated NO production, tau phosphorylation, and caspase-3 activation, each of which is implicated in spine loss. In hippocampal slices, oligomeric Aβ induces eNMDAR-mediated synaptic depression. In AD-transgenic mice compared with wild type, whole-cell recordings revealed excessive tonic eNMDAR activity accompanied by eNMDAR-sensitive loss of mEPSCs. Importantly, the improved NMDAR antagonist NitroMemantine, which selectively inhibits extrasynaptic over physiological synaptic NMDAR activity, protects synapses from Aβ-induced damage both in vitro and in vivo.

Neuron-released oligomeric α-synuclein is an endogenous agonist of TLR2 for paracrine activation of microglia

Abnormal aggregation of α-synuclein and sustained microglial activation are important contributors to the pathogenic processes of Parkinsons disease. However, the relationship between disease-associated protein aggregation and microglia-mediated neuroinflammation remains unknown. Here, using a combination of in silico, in vitro and in vivo approaches, we show that extracellular α-synuclein released from neuronal cells is an endogenous agonist for Toll-like receptor 2 (TLR2), which activates inflammatory responses in microglia. The TLR2 ligand activity of α-synuclein is conformation-sensitive; only specific types of oligomer can interact with and activate TLR2. This paracrine interaction between neuron-released oligomeric α-synuclein and TLR2 in microglia suggests that both of these proteins are novel therapeutic targets for modification of neuroinflammation in Parkinsons disease and related neurological diseases.

α-Synuclein Sequesters Dnmt1 from the Nucleus A NOVEL MECHANISM FOR EPIGENETIC ALTERATIONS IN LEWY BODY DISEASES

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.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.

Progressive accumulation of amyloid-beta oligomers in Alzheimer's disease and in amyloid precursor protein transgenic mice is accompanied by selective alterations in synaptic scaffold proteins.

The cognitive impairment in patients with Alzheimer’s disease is closely associated with synaptic loss in the neocortex and limbic system. Although the neurotoxic effects of aggregated amyloid‐β oligomers in Alzheimer’s disease have been studied extensively in experimental models, less is known about the characteristics of these aggregates across the spectrum of Alzheimer’s disease. In this study, postmortem frontal cortex samples from controls and patients with Alzheimer’s disease were fractionated and analyzed for levels of oligomers and synaptic proteins. We found that the levels of oligomers correlated with the severity of cognitive impairment (blessed information‐memory‐concentration score and mini‐mental state examination) and with the loss of synaptic markers. Reduced levels of the synaptic vesicle protein, vesicle‐associated membrane protein‐2, and the postsynaptic protein, postsynaptic density‐95, correlated with the levels of oligomers in the various fractions analyzed. The strongest associations were found with amyloid‐β dimers and pentamers. Co‐immunoprecipitation and double‐labeling experiments supported the possibility that amyloid‐β and postsynaptic density‐95 interact at synaptic sites. Similarly, in transgenic mice expressing high levels of neuronal amyloid precursor protein, amyloid‐β co‐immunoprecipitated with postsynaptic density‐95. This was accompanied by a decrease in the levels of the postsynaptic proteins Shank1 and Shank3 in patients with Alzheimer’s disease and in the brains of amyloid precursor protein transgenic mice. In conclusion, this study suggests that the presence of a subpopulation of amyloid‐β oligomers in the brains of patients with Alzheimer’s disease might be related to alterations in selected synaptic proteins and cognitive impairment.

Progressive accumulation of amyloid-β oligomers in Alzheimer’s disease and APP transgenic mice is accompanied by selective alterations in synaptic scaffold proteins

The cognitive impairment in patients with Alzheimer’s disease is closely associated with synaptic loss in the neocortex and limbic system. Although the neurotoxic effects of aggregated amyloid‐β oligomers in Alzheimer’s disease have been studied extensively in experimental models, less is known about the characteristics of these aggregates across the spectrum of Alzheimer’s disease. In this study, postmortem frontal cortex samples from controls and patients with Alzheimer’s disease were fractionated and analyzed for levels of oligomers and synaptic proteins. We found that the levels of oligomers correlated with the severity of cognitive impairment (blessed information‐memory‐concentration score and mini‐mental state examination) and with the loss of synaptic markers. Reduced levels of the synaptic vesicle protein, vesicle‐associated membrane protein‐2, and the postsynaptic protein, postsynaptic density‐95, correlated with the levels of oligomers in the various fractions analyzed. The strongest associations were found with amyloid‐β dimers and pentamers. Co‐immunoprecipitation and double‐labeling experiments supported the possibility that amyloid‐β and postsynaptic density‐95 interact at synaptic sites. Similarly, in transgenic mice expressing high levels of neuronal amyloid precursor protein, amyloid‐β co‐immunoprecipitated with postsynaptic density‐95. This was accompanied by a decrease in the levels of the postsynaptic proteins Shank1 and Shank3 in patients with Alzheimer’s disease and in the brains of amyloid precursor protein transgenic mice. In conclusion, this study suggests that the presence of a subpopulation of amyloid‐β oligomers in the brains of patients with Alzheimer’s disease might be related to alterations in selected synaptic proteins and cognitive impairment.

Peripheral delivery of a CNS targeted, metalo-protease reduces aβ toxicity in a mouse model of Alzheimer's disease.

Alzheimers disease (AD), an incurable, progressive neurodegenerative disorder, is the most common form of dementia. Therapeutic options have been elusive due to the inability to deliver proteins across the blood-brain barrier (BBB). In order to improve the therapeutic potential for AD, we utilized a promising new approach for delivery of proteins across the BBB. We generated a lentivirus vector expressing the amyloid β-degrading enzyme, neprilysin, fused to the ApoB transport domain and delivered this by intra-peritoneal injection to amyloid protein precursor (APP) transgenic model of AD. Treated mice had reduced levels of Aβ, reduced plaques and increased synaptic density in the CNS. Furthermore, mice treated with the neprilysin targeting the CNS had a reversal of memory deficits. Thus, the addition of the ApoB transport domain to the secreted neprilysin generated a non-invasive therapeutic approach that may be a potential treatment in patients with AD.

Lentivirus Mediated Delivery of Neurosin Promotes Clearance of Wild-type α-Synuclein and Reduces the Pathology in an α-Synuclein Model of LBD

Neurosin is a predominant serine protease in the central nervous system (CNS) and has been shown to play a role in the clearance of α-synuclein (α-syn) which is centrally involved in the pathogenesis of Parkinsons disease (PD) and dementia with Lewy bodies (DLB). Although it has been previously shown that neurosin and α-syn colocalize and that neurosin degrades α-syn aggregates in vitro, it is not clear if neurosin is dysregulated in the brains of patients with PD/DLB and to what extent delivery of neurosin into the CNS might ameliorate the deficits associated with α-syn accumulation in vivo. We analyzed the levels of neurosin in the brains of patients with PD/DLB and in α-syn transgenic (tg) models. With increased accumulation of α-syn, we observed decreased neurosin expression. Lentiviral vector (LV) driven expression of neurosin in neuronal cell cultures reduced the accumulation of wild type but not A53T α-syn and prevented α-syn associated toxicity. Neuropathological analysis following delivery of LV-Neurosin to α-syn tg mice resulted in reduced accumulation of α-syn and reversal of neurodegenerative alterations in wild type but not A53T α-syn tg mice. Therefore, viral vector driven expression of neurosin may warrant further investigation as a potential therapeutic tool for DLB.

ESCRT-mediated Uptake and Degradation of Brain-targeted α-synuclein Single Chain Antibody Attenuates Neuronal Degeneration In Vivo

Parkinsons disease and dementia with Lewy bodies are neurodegenerative disorders characterized by accumulation of α-synuclein (α-syn). Recently, single-chain fragment variables (scFVs) have been developed against individual conformational species of α-syn. Unlike more traditional monoclonal antibodies, these scFVs will not activate or be endocytosed by Fc receptors. For this study, we investigated an scFV directed against oligomeric α-syn fused to the LDL receptor-binding domain from apolipoprotein B (apoB). The modified scFV showed enhanced brain penetration and was imported into neuronal cells through the endosomal sorting complex required for transport (ESCRT) pathway, leading to lysosomal degradation of α-syn aggregates. Further analysis showed that the scFV was effective at ameliorating neurodegenerative pathology and behavioral deficits observed in the mouse model of dementia with Lewy bodies/Parkinsons disease. Thus, the apoB modification had the effect of both increasing accumulation of the scFV in the brain and directing scFV/α-syn complexes for degradation through the ESCRT pathway, leading to improved therapeutic potential of immunotherapy.

Widespread microRNA dysregulation in multiple system atrophy – disease‐related alteration in miR‐96

MicroRNA (miRNA) are short sequences of RNA that function as post‐transcriptional regulators by binding to target mRNA transcripts resulting in translational repression. A number of recent studies have identified miRNA as being involved in neurodegenerative disorders including Alzheimers disease, Parkinsons disease and Huntingtons disease. However, the role of miRNA in multiple system atrophy (MSA), a progressive neurodegenerative disorder characterized by oligodendroglial accumulation of alpha‐synuclein remains unexamined. In this context, this study examined miRNA profiles in MSA cases compared with controls and in transgenic (tg) models of MSA compared with non‐tg mice. The results demonstrate a widespread dysregulation of miRNA in MSA cases, which is recapitulated in the murine models. The study employed a cross‐disease, cross‐species approach to identify miRNA that were either specifically dysregulated in MSA or were commonly dysregulated in neurodegenerative conditions such as Alzheimers disease, dementia with Lewy bodies, progressive supranuclear palsy and corticobasal degeneration or the tg mouse model equivalents of these disorders. Using this approach we identified a number of miRNA that were commonly dysregulated between disorders and those that were disease‐specific. Moreover, we identified miR‐96 as being up‐regulated in MSA. Consistent with the up‐regulation of miR‐96, mRNA and protein levels of members of the solute carrier protein family SLC1A1 and SLC6A6, miR‐96 target genes, were down‐regulated in MSA cases and a tg model of MSA. These results suggest that miR‐96 dysregulation may play a role in MSA and its target genes may be involved in the pathogenesis of MSA.