HIV-1 Tat alters neuronal autophagy by modulating autophagosome fusion to the lysosome: implications for HIV-associated neurocognitive disorders.

Antiretroviral therapy has increased the life span of HIV+ individuals; however, HIV-associated neurocognitive disorder (HAND) occurrence is increasing in aging HIV patients. Previous studies suggest HIV infection alters autophagy function in the aging CNS and HIV-1 proteins affect autophagy in monocyte-derived cells. Despite these findings, the mechanisms leading to dysregulated autophagy in the CNS remain unclear. Here we sought to determine how HIV Tat dysregulates autophagy in neurons. Tat caused a dose-dependent decrease in autophagosome markers, microtubule-associated protein-1 light chain ß II (LC3II), and sequestosome 1(SQSTM1), in a membrane-enriched fraction, suggesting Tat increases autophagic degradation. Bafilomycin A1 increased autophagosome number, LC3II, and SQSTM1 accumulation; Tat cotreatment diminished this effect. Tat had no effect when 3-methyladenine or knockdown of beclin 1 blocked early stages of autophagy. Tat increased numbers of LC3 puncta and resulted in the formation of abnormal autophagosomes in vitro. Likewise, in vivo studies in GFAP-Tat tg mice showed increased autophagosome accumulation in neurons, altered LC3II levels, and neurodegeneration. These effects were reversed by rapamycin treatment. Tat colocalized with autophagosome and lysosomal markers and enhanced the colocalization of autophagosome with lysosome markers. Furthermore, co-IP studies showed that Tat interacts with lysosomal-associated membrane protein 2A (LAMP2A) in vitro and in vivo, and LAMP2A overexpression reduces Tat-induced neurotoxicity. Hence, Tat protein may induce autophagosome and lysosome fusion through interaction with LAMP2A leading to abnormal neuronal autophagy function and dysregulated degradation of critical intracellular components. Therapies targeting Tat-mediated autophagy alterations may decrease neurodegeneration in aging patients with HAND.

Molecular determinants of α-synuclein mutants' oligomerization and membrane interactions.

Parkinson's disease (PD) is associated with the formation of toxic α-synuclein oligomers that can penetrate the cell membrane. Familial forms of PD are caused by the point mutations A53T, A30P, E46K, and H50Q. Artificial point mutations E35K and E57K also increase oligomerization and pore formation. We generated structural conformations of α-synuclein and the above-mentioned mutants using molecular dynamics. We elucidated four main regions in these conformers contacting the membrane and found that the region including residues 39-45 (Zone2) may have maximum membrane penetration. E57K mutant had the highest rate of interaction with the membrane, followed by A53T, E46K, and E35K mutants and wild type (wt) α-synuclein. The mutant A30P had the smallest percentage of conformers that contact the membrane by Zone 2 than all other mutants and wt α-synuclein. These results were confirmed experimentally in vitro. We identified the key amino acids that can interact with the membrane (Y38, E62, and N65 (first hydrophilic layer); E104, E105, and D115 (second hydrophilic layer), and V15 and V26 (central hydrophobic layer)) and the residues that are involved in the interprotein contacts (L38, V48, V49, Q62, and T64). Understanding the molecular interactions of α-synuclein mutants is important for the design of compounds blocking the formation of toxic oligomers.

Parkinson's disease genes VPS35 and EIF4G1 interact genetically and converge on α-synuclein.

Parkinson's disease (PD) is a common neurodegenerative disorder. Functional interactions between some PD genes, like PINK1 and parkin, have been identified, but whether other ones interact remains elusive. Here we report an unexpected genetic interaction between two PD genes, VPS35 and EIF4G1. We provide evidence that EIF4G1 upregulation causes defects associated with protein misfolding. Expression of a sortilin protein rescues these defects, downstream of VPS35, suggesting a potential role for sortilins in PD. We also show interactions between VPS35, EIF4G1, and α-synuclein, a protein with a key role in PD. We extend our findings from yeast to an animal model and show that these interactions are conserved in neurons and in transgenic mice. Our studies reveal unexpected genetic and functional interactions between two seemingly unrelated PD genes and functionally connect them to α-synuclein pathobiology in yeast, worms, and mouse. Finally, we provide a resource of candidate PD genes for future interrogation.

ESCRT-mediated uptake and degradation of brain-targeted α-synuclein single chain antibody attenuates neuronal degeneration in vivo.

Parkinson's 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/Parkinson's 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.

Environmental neurotoxic challenge of conditional alpha-synuclein transgenic mice predicts a dopaminergic olfactory-striatal interplay in early PD.

The olfactory bulb (OB) is one of the first brain regions in Parkinson's disease (PD) to contain alpha-synuclein (α-syn) inclusions, possibly associated with nonmotor symptoms. Mechanisms underlying olfactory synucleinopathy, its contribution to progressive aggregation pathology and nigrostriatal dopaminergic loss observed at later stages, remain unclear. A second hit, such as environmental toxins, is suggestive for α-syn aggregation in olfactory neurons, potentially triggering disease progression. To address the possible pathogenic role of olfactory α-syn accumulation in early PD, we exposed mice with site-specific and inducible overexpression of familial PD-linked mutant α-syn in OB neurons to a low dose of the herbicide paraquat. Here, we found that olfactory α-syn per se elicited structural and behavioral abnormalities, characteristic of an early time point in models with widespread α-syn expression, including hyperactivity and increased striatal dopaminergic marker. Suppression of α-syn reversed the dopaminergic phenotype. In contrast, paraquat treatment synergistically induced degeneration of olfactory dopaminergic cells and opposed the higher reactive phenotype. Neither neurodegeneration nor behavioral abnormalities were detected in paraquat-treated mice with suppressed α-syn expression. By increasing calpain activity, paraquat induced a pathological cascade leading to inhibition of autophagy clearance and accumulation of calpain-cleaved truncated and insoluble α-syn, recapitulating biochemical and structural changes in human PD. Thus our results underscore the primary role of proteolytic failure in aggregation pathology. In addition, we provide novel evidence that olfactory dopaminergic neurons display an increased vulnerability toward neurotoxins in dependence to presence of human α-syn, possibly mediating an olfactory-striatal dopaminergic network dysfunction in mouse models and early PD.

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 Parkinson's 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.

Combined exposure to Maneb and Paraquat alters transcriptional regulation of neurogenesis-related genes in mice models of Parkinson's disease.

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.

α-Synuclein induces alterations in adult neurogenesis in Parkinson disease models via p53-mediated repression of Notch1.

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.

Differential effects of UCHL1 modulation on alpha-synuclein in PD-like models of alpha-synucleinopathy.

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by genetic and environmental factors. Abnormal accumulation and aggregation of alpha-synuclein (a-syn) within neurons, and mutations in the a-syn and UCH-L1 genes have been shown to play a role in the pathogenesis of PD. In light of recent reports suggesting an interaction between a-synuclein and UCH-L1, we investigated the effects of UCH-L1 inhibition on a-syn distribution and expression levels in primary neurons and hippocampal tissues derived from non transgenic (non tg) and a-syn over expressing tg mice. We show that suppression of UCH-L1 activity increased a-syn levels in control, non tg neurons, and resulted in a concomitant accumulation of presynaptic a-syn in these neurons. In contrast, blocking UCH-L1 activity in a-syn over expressing neurons decreased a-syn levels, and enhanced its synaptic clearance. In vitro studies verified the LDN-induced inhibition of UCH-L1 had minimal effect on LC3 (a marker of autophagy) in control cells, in cells over expressing a-syn UCH-L1 inhibition resulted in increased LC3 activity. These findings suggest a possible differential role of UCH-L1 function under normal and pathological conditions. Furthermore, in the context of a-syn-induced pathology, modulation of UCH-L1 activity could serve as a therapeutic tool to enhance the autophagy pathway and induce clearance of the observed accumulated/aggregated a-syn species in the PD brain.