Deregulation of calcium homeostasis mediates secreted aesynuclein - induced neurotoxicity

α-Synuclein (AS) plays a crucial role in Parkinson's disease pathogenesis. AS is normally secreted from neuronal cells and can thus exert paracrine effects. We have previously demonstrated that naturally secreted AS species, derived from SH-SY5Y cells inducibly overexpressing human wild type AS, can be toxic to recipient neuronal cells. In the current study, we show that application of secreted AS alters membrane fluidity and increases calcium (Ca2+) entry. This influx is reduced on pharmacological inhibition of voltage-operated Ca2+ channels. Although no change in free cytosolic Ca2+ levels is observed, a significantly increased mitochondrial Ca2+ sequestration is found in recipient cells. Application of voltage-operated Ca2+ channel blockers or Ca2+ chelators abolishes AS-mediated toxicity. AS-treated cells exhibit increased calpain activation, and calpain inhibition greatly alleviates the observed toxicity. Collectively, our data suggest that secreted AS exerts toxicity through engagement, at least in part, of the Ca2+ homeostatic machinery. Therefore, manipulating Ca2+ signaling pathways might represent a potential therapeutic strategy for Parkinson's disease

Long Non-coding RNAs Associated With Neurodegeneration-Linked Genes Are Reduced in Parkinson’s Disease Patients

Transcriptome analysis has identified a plethora of long non-coding RNAs (lncRNAs) expressed in the human brain and associated with neurological diseases. However, whether lncRNAs expression levels correlate with Parkinson’s disease (PD) pathogenesis remains unknown. Herein, we show that a number of lncRNA genes encompassing transcriptional units in close proximity to PD-linked protein-coding genes, including SNCA, LRRK2, PINK1, DJ-1, UCH-L1, MAPT and GBA1, are expressed in human dopaminergic cells and post-mortem material, such as cortex, Substantia Nigra and cerebellum. Interestingly, these lncRNAs are upregulated during neuronal differentiation of SH-SY5Y cells and of dopaminergic neurons generated from human fibroblast-derived induced pluripotent stem cells. Importantly, six lncRNAs are found under-expressed in the nigra and three in the cerebellum of PD patients compared to controls. Simultaneously, SNCA mRNA levels are increased in the nigra, while LRRK2 and PINK1 mRNA levels are decreased both in the nigra and the cerebellum of PD subjects compared to controls, indicating a possible correlation between the expression profile of the respective lncRNAs with their adjacent coding genes. Interestingly, all dysregulated lncRNAs are also detected in human peripheral blood mononuclear cells and four of them in exosomes derived from human cerebrospinal fluid, providing initial evidence for their potential use as diagnostic tools for PD. Our data raise the intriguing possibility that these lncRNAs may be involved in disease pathogenesis by regulating their neighboring PD-associated genes and may thus represent novel targets for the diagnosis and/or treatment of PD or related diseases

Neuronal autophagy and neurodegenerative diseases

Autophagy is a dynamic cellular pathway involved in the turnover of proteins, protein complexes, and organelles through lysosomal degradation. The integrity of postmitotic neurons is heavily dependent on high basal autophagy compared to non-neuronal cells as misfolded proteins and damaged organelles cannot be diluted through cell division. Moreover, neurons contain the specialized structures for intercellular communication, such as axons, dendrites and synapses, which require the reciprocal transport of proteins, organelles and autophagosomes over significant distances from the soma. Defects in autophagy affect the intercellular communication and subsequently, contributing to neurodegeneration. The presence of abnormal autophagic activity is frequently observed in selective neuronal populations afflicted in common neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis. These observations have provoked controversy regarding whether the increase in autophagosomes observed in the degenerating neurons play a protective role or instead contribute to pathogenic neuronal cell death. It is still unknown what factors may determine whether active autophagy is beneficial or pathogenic during neurodegeneration. In this review, we consider both the normal and pathophysiological roles of neuronal autophagy and its potential therapeutic implications for common neurodegenerative diseases

Passive Immunization Reduces Behavioral and Neuropathological Deficits in an Alpha-Synuclein Transgenic Model of Lewy Body Disease

Dementia with Lewy bodies (DLB) and Parkinson's Disease (PD) are common causes of motor and cognitive deficits and are associated with the abnormal accumulation of alpha-synuclein (α-syn). This study investigated whether passive immunization with a novel monoclonal α-syn antibody (9E4) against the C-terminus (CT) of α-syn was able to cross into the CNS and ameliorate the deficits associated with α-syn accumulation. In this study we demonstrate that 9E4 was effective at reducing behavioral deficits in the water maze, moreover, immunization with 9E4 reduced the accumulation of calpain-cleaved α-syn in axons and synapses and the associated neurodegenerative deficits. In vivo studies demonstrated that 9E4 traffics into the CNS, binds to cells that display α-syn accumulation and promotes α-syn clearance via the lysosomal pathway. These results suggest that passive immunization with monoclonal antibodies against the CT of α-syn may be of therapeutic relevance in patients with PD and DLB

mTOR independent regulation of macroautophagy by Leucine Rich Repeat Kinase 2 via Beclin-1

Leucine rich repeat kinase 2 is a complex enzyme with both kinase and GTPase activities, closely linked to the pathogenesis of several human disorders including Parkinson’s disease, Crohn’s disease, leprosy and cancer. LRRK2 has been implicated in numerous cellular processes; however its physiological function remains unclear. Recent reports suggest that LRRK2 can act to regulate the cellular catabolic process of macroautophagy, although the precise mechanism whereby this occurs has not been identi ed. To investigate the signalling events through which LRRK2 acts to in uence macroautophagy, the mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) and Beclin-1/phosphatidylinositol 3-kinase (PI3K) pathways were evaluated in astrocytic cell models in the presence and absence of LRRK2 kinase inhibitors. Chemical inhibition of LRRK2 kinase activity resulted in the stimulation of macroautophagy in a non-canonical fashion, independent of mTOR and ULK1, but dependent upon the activation of Beclin 1-containing class III PI3-kinase

Progressive Neurodegeneration or Endogenous Compensation in an Animal Model of Parkinson's Disease Produced by Decreasing Doses of Alpha-Synuclein

The pathological hallmarks of Parkinson's disease (PD) are degeneration of dopamine (DA) neurons of the substantia nigra (SN) and the presence of alpha-synuclein (α-syn)-rich Lewy bodies in DA cells that remain. To model these aspects of the disease, we previously showed that high titer (5.1×10exp12 gp/ml) AAV1/2 driven expression of A53T α-syn in the SN of rats caused nigrostriatal pathology including a loss of DA neurons, but also with toxicity in the GFP control group. In the current study, we evaluate the effects of two lower titers by dilution of the vector (1∶3 [1.7×10exp12] and 1∶10 [5.1×10exp11]) to define a concentration that produced pathology specific for α-syn. In GFP and empty vector groups there were no behavioural or post-mortem changes at 3 or 6 weeks post-administration at either vector dose. Dilution of the AAV1/2 A53T α-syn (1∶3) produced significant paw use asymmetry, reductions in striatal tyrosine hydroxylase (TH), and increases in DA turnover at 3 weeks in the absence of overt pathology. By 6 weeks greater evidence of pathology was observed and included, reductions in SN DA neurons, striatal DA, TH and DA-transporter, along with a sustained behavioural deficit. In contrast, the 1∶10 AAV1/2 A53T α-syn treated animals showed normalization between 3 and 6 weeks in paw use asymmetry, reductions in striatal TH, and increased DA turnover. Progression of dopaminergic deficits using the 1∶3 titer of AAV1/2 A53Tα-syn provides a platform for evaluating treatments directed at preventing and/or reversing synucleinopathy. Use of the 1∶10 titer of AAV1/2 A53T α-syn provides an opportunity to study mechanisms of endogenous compensation. Furthermore, these data highlight the need to characterize the titer of vector being utilized, when using AAV to express pathogenic proteins and model disease process, to avoid producing non-specific effects

Impaired mitochondrial biogenesis contributes to depletion of functional mitochondria in chronic MPP+ toxicity: dual roles for ERK1/2

The regulation of mitochondrial quality has emerged as a central issue in neurodegeneration, diabetes, and cancer. We utilized repeated low-dose applications of the complex I inhibitor 1-methyl-4-phenylpyridinium (MPP+) over 2 weeks to study cellular responses to chronic mitochondrial stress. Chronic MPP+ triggered depletion of functional mitochondria resulting in diminished capacities for aerobic respiration. Inhibiting autophagy/mitophagy only partially restored mitochondrial content. In contrast, inhibiting activation of extracellular signal-regulated protein kinases conferred complete cytoprotection with full restoration of mitochondrial functional and morphological parameters, enhancing spare respiratory capacity in MPP+ co-treated cells above that of control cells. Reversal of mitochondrial injury occurred when U0126 was added 1 week after MPP+, implicating enhanced repair mechanisms. Chronic MPP+ caused a >90% decrease in complex I subunits, along with decreases in complex III and IV subunits. Decreases in respiratory complex subunits were reversed by co-treatment with U0126, ERK1/2 RNAi or transfection of dominant-negative MEK1, but only partially restored by degradation inhibitors. Chronic MPP+ also suppressed the de novo synthesis of mitochondrial DNA-encoded proteins, accompanied by decreased expression of the mitochondrial transcription factor TFAM. U0126 completely reversed each of these deficits in mitochondrial translation and protein expression. These data indicate a key, limiting role for mitochondrial biogenesis in determining the outcome of injuries associated with elevated mitophagy

LRRK2 at the interface of autophagosomes, endosomes and lysosomes

Over the past 20 years, substantial progress has been made in identifying the underlying genetics of Parkinson’s disease (PD). Of the known genes, LRRK2 is a major genetic contributor to PD. However, the exact function of LRRK2 remains to be elucidated. In this review, we discuss how familial forms of PD have led us to hypothesize that alterations in endomembrane trafficking play a role in the pathobiology of PD. We will discuss the major observations that have been made to elucidate the role of LRRK2 in particular, including LRRK2 animal models and high-throughput proteomics approaches. Taken together, these studies strongly support a role of LRRK2 in vesicular dynamics. We also propose that targeting these pathways may not only be beneficial for developing therapeutics for LRRK2-driven PD, but also for other familial and sporadic cases