Α-syn Protein Levels Research Articles

Inhibition of 26S proteasome activity by α-synuclein is mediated by the proteasomal chaperone Rpn14/PAAF1.

Parkinson's disease (PD) is characterized by aggregation of α-synuclein (α-syn) into protein inclusions in degenerating brains. Increasing amounts of aggregated α-syn species indicate significant perturbation of cellular proteostasis. Altered proteostasis depends on α-syn protein levels and the impact of α-syn on other components of the proteostasis network. Budding yeast Saccharomyces cerevisiae was used as eukaryotic reference organism to study the consequences of α-syn expression on protein dynamics. To address this, we investigated the impact of overexpression of α-syn and S129A variant on the abundance and stability of most yeast proteins using a genome-wide yeast library and a tandem fluorescent protein timer (tFT) reporter as a measure for protein stability. This revealed that the stability of in total 377 cellular proteins was altered by α-syn expression, and that the impact on protein stability was significantly enhanced by phosphorylation at Ser129 (pS129). The proteasome assembly chaperone Rpn14 was identified as one of the top candidates for increased protein stability by expression of pS129 α-syn. Elevated levels of Rpn14 enhanced the growth inhibition by α-syn and the accumulation of ubiquitin conjugates in the cell. We found that Rpn14 interacts physically with α-syn and stabilizes pS129 α-syn. The expression of α-syn along with elevated levels of Rpn14 or its human counterpart PAAF1 reduced the proteasome activity in yeast and in human cells, supporting that pS129 α-syn negatively affects the 26S proteasome through Rpn14. This comprehensive study into the alternations of protein homeostasis highlights the critical role of the Rpn14/PAAF1 in α-syn-mediated proteasome dysfunction.

Abstract WP314: Post-Stroke Regulation of α-Synuclein by microRNA miR-7 and Circular RNA CDR1as

α-Synuclein (α-Syn) induced after stroke is a known mediator of ischemic brain damage. We currently evaluated the molecular mechanisms that regulate α-Syn expression in the post-ischemic brain. The miR-7 is an abundantly expressed miRNA in brain that targets α-Syn mRNA with high energy. In adult mice subjected to transient middle cerebral artery occlusion (MCAO), miR-7 levels were down-regulated in the cerebral cortex as early as 3h and late as 3 days of reperfusion which leads to derepression of α-Syn mRNA that facilitates α-Syn protein translation. Treating mice after transient MCAO with a miR-7 mimic significantly curtailed ischemic brain damage and promoted better motor function recovery. However, the pri-miR-7a and -7b levels were unaltered indicating that the post-ischemic reduction in miR-7 levels are not due to reduced expression of miR-7 gene. With bioinformatics, we observed that the circRNA CDR1as contains >70 binding sites with good affinity for miR-7. CDR1as is thought to sequester miR-7 to protect it from degradation and/or to transport it to the sites of its necessity. Transient MCAO also led to down-regulation of CDR1as expression between 3h and 3 days of reperfusion. When CDR1as levels were replenished in the post-ischemic mice with a CDR1as AAV9 vector, the miR-7 levels as well as binding of CDR1as to Ago2/miR-7 increased and α-Syn protein levels decreased significantly in the peri-infarct cortex. Furthermore, the CDR1as AAV9 treated mice showed decreased markers of inflammation and apoptosis, better motor function recovery and smaller infarcts. Our studies thus indicate that α-Syn in the post-ischemic brain is controlled by the mutual interaction of a miRNA (miR-7) and a circRNA (CDR1as). Thus, understanding noncoding RNA interactions and mechanisms might help to design better therapies to alleviate post-stroke neuronal damage.

Effects of α7 nicotinic acetylcholine receptor agonist against α-synuclein-induced neurotoxicity

The α7 neuronal nicotinic acetylcholine receptor (α7 nAChR) is a potential target for the development of Parkinson’s disease (PD) therapeutics. α-Synuclein (α-Syn), a principal component of Lewy bodies (cytoplasmic inclusions), is a major contributor to PD pathophysiology. Previous studies have demonstrated that activating α7 nAChR protects against nigrostriatal dopamine degeneration in acute and chronic PD animal models induced by 6-hydroxydopamine and rotenone, respectively. In the present study, we investigated the effects of PNU282987, a selective α7 nAChR agonist, against α-Syn-induced neurotoxicity in α-SynWT-, α-SynA30P-, and α-SynE46K-N2a cells. PNU282987 exhibited substantial neuroprotection against both wild-type and mutant-type α-Syn-induced toxicity. Furthermore, PNU282987 promoted transcription factor EB activity and reduced intracellular α-Syn protein levels through autophagy induction. These results highlight the therapeutic potential of α7 nAChR activation in diseases characterized by α-Syn aggregation, such as PD.

The neuroprotective effects of FG-4592, a hypoxia-inducible factor-prolyl hydroxylase inhibitor, against oxidative stress induced by alpha-synuclein in N2a cells

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra. The pathological hallmark of PD is the appearance of intraneuronal cytoplasmic α-synuclein (α-Syn) aggregation, called Lewy bodies. α-Syn aggregation is deeply involved in the pathogenesis of PD. Oxidative stress is also associated with the progression of PD. In the present study, to investigate whether a hypoxia-inducible factor (HIF)-prolyl hydroxylase (PH) inhibitor, FG-4592 (also called roxadustat), has neuroprotective effects against α-Syn-induced neurotoxicity, we employed a novel α-Syn stably expressing cell line (named α-Syn-N2a cells) utilizing a piggyBac transposon system. In α-Syn-N2a cells, oxidative stress and cell death were induced by α-Syn, and FG-4592 showed significant protection against this neurotoxicity. However, FG-4592 did not affect α-Syn protein levels. FG-4592 triggered heme oxygenase-1 (HO-1) expression downstream of HIF-1α in a concentration-dependent manner. In addition, FG-4592 decreased the production of reactive oxygen species possibly via the activation of HO-1 and subsequently suppressed α-Syn-induced neurotoxicity. Moreover, FG-4592 regulated mitochondrial biogenesis and respiration via the induction of the peroxisome proliferator-activated receptor-γ coactivator-1α. As FG-4592 has various neuroprotective effects against α-Syn and is involved in drug repositioning, it may have novel therapeutic potential for PD.

Honokiol decreases alpha-synuclein mRNA levels and reveals novel targets for modulating alpha-synuclein expression.

Intracytoplasmic inclusions comprised of aggregated alpha-synuclein (αsyn) represent a key histopathological feature of neurological disorders collectively termed "synucleinopathies," which includes Parkinson's disease (PD). Mutations and multiplications in the SNCA gene encoding αsyn cause familial forms of PD and a large body of evidence indicate a correlation between αsyn accumulation and disease. Decreasing αsyn expression is recognized as a valid target for PD therapeutics, with down-regulation of SNCA expression potentially attenuating downstream cascades of pathologic events. Here, we evaluated if Honokiol (HKL), a polyphenolic compound derived from magnolia tree bark with demonstrated neuroprotective properties, can modulate αsyn levels in multiple experimental models. Human neuroglioma cells stably overexpressing αsyn, mouse primary neurons, and human iPSC-derived neurons were exposed to HKL and αsyn protein and SNCA messenger RNA levels were assessed. The effect of HKL on rotenone-induced overexpression of αsyn levels was further assessed and transcriptional profiling of mouse cortical neurons treated with HKL was performed to identify potential targets of HKL. We demonstrate that HKL can successfully reduce αsyn protein levels and SNCA expression in multiple in vitro models of PD with our data supporting a mechanism whereby HKL acts by post-transcriptional modulation of SNCA rather than modulating αsyn protein degradation. Transcriptional profiling of mouse cortical neurons treated with HKL identifies several differentially expressed genes (DEG) as potential targets to modulate SNCA expression. This study supports a HKL-mediated downregulation of SNCA as a viable strategy to modify disease progression in PD and other synucleinopathies. HKL has potential as a powerful tool for investigating SNCA gene modulation and its downstream effects.

Saposin C, Key Regulator in the Alpha-Synuclein Degradation Mediated by Lysosome.

Lysosomal dysfunction has been proposed as one of the most important pathogenic molecular mechanisms in Parkinson disease (PD). The most significant evidence lies in the GBA gene, which encodes for the lysosomal enzyme β-glucocerebrosidase (β-GCase), considered the main genetic risk factor for sporadic PD. The loss of β-GCase activity results in the formation of α-synuclein deposits. The present study was aimed to determine the activity of the main lysosomal enzymes and the cofactors Prosaposin (PSAP) and Saposin C in PD and healthy controls, and their contribution to α-synuclein (α-Syn) aggregation. 42 PD patients and 37 age-matched healthy controls were included in the study. We first analyzed the β-GCase, β-galactosidase (β-gal), β-hexosaminidase (Hex B) and Cathepsin D (CatD) activities in white blood cells. We also measured the GBA, β-GAL, β-HEX, CTSD, PSAP, Saposin C and α-Syn protein levels by Western-blot. We found a 20% reduced β-GCase and β-gal activities in PD patients compared to controls. PSAP and Saposin C protein levels were significantly lower in PD patients and correlated with increased levels of α-synuclein. CatD, in contrast, showed significantly increased activity and protein levels in PD patients compared to controls. Increased CTSD protein levels in PD patients correlated, intriguingly, with a higher concentration of α-Syn. Our findings suggest that lysosomal dysfunction in sporadic PD is due, at least in part, to an alteration in Saposin C derived from reduced PSAP levels. That would lead to a significant decrease in the β-GCase activity, resulting in the accumulation of α-syn. The accumulation of monohexosylceramides might act in favor of CTSD activation and, therefore, increase its enzymatic activity. The evaluation of lysosomal activity in the peripheral blood of patients is expected to be a promising approach to investigate pathological mechanisms and novel therapies aimed to restore the lysosomal function in sporadic PD.

Beta2-adrenoreceptor agonist clenbuterol produces transient decreases in alpha-synuclein mRNA but no long-term reduction in protein

β2-adrenoreceptor (β2AR) agonists have been associated with a decreased risk of developing Parkinson’s disease (PD) and are hypothesized to decrease expression of both alpha-synuclein mRNA (Snca) and protein (α-syn). Effects of β2AR agonist clenbuterol on the levels of Snca mRNA and α-syn protein were evaluated in vivo (rats and mice) and in rat primary cortical neurons by two independent laboratories. A modest decrease in Snca mRNA in the substantia nigra was observed after a single acute dose of clenbuterol in rats, however, this decrease was not maintained after multiple doses. In contrast, α-syn protein levels remained unchanged in both single and multiple dosing paradigms. Furthermore, clenbuterol did not decrease Snca in cultured rat primary cortical neurons, or decrease Snca or α-syn in mice. Additionally, compared to the single-dose paradigm, repeat dosing resulted in substantially lower levels of clenbuterol in plasma and brain tissue in rodents. Based on our observations of a transient decrease in Snca and no effect on α-syn protein in this preclinical study, these data support the conclusion that clenbuterol is not likely a viable disease-modifying strategy for PD.

Manganese-induced alpha-synuclein overexpression aggravates mitochondrial damage by repressing PINK1/Parkin-mediated mitophagy

Chronic manganese (Mn) exposure is related to elevated risks of neurodegenerative diseases, and mitochondrial dysfunction is considered a critical pathophysiological feature of Mn neurotoxicity. Although previous research has demonstrated Mn-induced alpha-synuclein (α-Syn) overexpression, the role of α-Syn in mitochondrial dysfunction remains unclear. Here, we used Wistar rats and human neuroblastoma cells (SH-SY5Y cells) to elucidate the molecular mechanisms underlying how α-Syn overexpression induced by different doses of Mn (15, 30, and 60 mg/kg) results in mitochondrial dysfunction. We found that Mn-induced neural cell injury was associated with mitochondrial damage. Furthermore, Mn upregulated α-Syn protein levels and increased the interaction between α-Syn and mitochondria. We then used a lentivirus vector containing α-Syn shRNA to examine the effect of Mn-induced α-Syn protein on PINK1/Parkin-mediated mitophagy in SH-SY5Y cells. Our data demonstrated that the knockdown of α-Syn decreased the interaction between α-Syn and PINK1. The enhanced level of phosphorylated Parkin (p-Parkin) was due to the decrease of the interaction between α-Syn and PINK1. Moreover, the knockdown of α-Syn increased recruitment of p-Parkin to mitochondria. Collectively, these observations revealed that Mn-induced α-Syn overexpression repressed PINK1/Parkin-mediated mitophagy and exacerbated mitochondrial damage.

Ndfip1 Prevents Rotenone-Induced Neurotoxicity and Upregulation of α-Synuclein in SH-SY5Y Cells.

Nedd4 family interacting protein 1 (Ndfip1) is an adaptor of Nedd4-family ubiquitin ligases. Experimental results showed that Ndfip1 had a potential neuroprotective effect in neurology diseases. However, the neuroprotective effect and the underlying mechanisms of Ndfip1 in Parkinson's disease (PD) have not yet been fully elucidated. Therefore, in this study, we explored the neuroprotective effect of Ndfip1 against mitochondrial complex I inhibitor rotenone in a human dopaminergic neuroblastoma SH-SY5Y cell line and further elucidated its possible underlying mechanisms. Our results showed that rotenone could induce the up-regulation of α-synuclein (α-syn) in both mRNA and protein levels. The expression of Ndfip1 decreased at 24 h after rotenone treatment. Further study showed that high expression of Ndfip1 could protect SH-SY5Y cells against rotenone-induced neurotoxicity and antagonize the rotenone-induced increase in α-syn protein levels. In addition, high expression of Ndfip1 inhibited rotenone-induced increase in the protein levels of caspase-3 and decrease in tyrosine hydroxylase (TH). Further study showed that Ndfip1 did not affect the protein expression of iron regulatory protein 1 (IRP1), transferrin receptor 1 (TfR1), while antagonized the increase in protein levels of P62 and ferritin L caused by rotenone. Our findings provide specific identification of Ndfip1 proteins to inhibit the increase of α-syn in rotenone-induced SH-SY5Y cells. Ndfip1 might be a new theoretical drug target for the prevention and treatment of PD.

Downregulation of α-Synuclein Protein Levels by an Intracellular Single-Chain Antibody.

Accumulation of α-synuclein (αSyn) in the dopaminergic neurons is a common pathology seen in patients with Parkinson's disease (PD). Overproduction of αSyn potentiates the formation of oligomeric αSyn aggregates and enhances dopaminergic neuron degeneration. Downregulating intracellular monomeric αSyn prevents the formation of αSyn oligomers and is a potential therapeutic strategy to attenuate the progression of PD. The purpose of this study is to investigate the efficacy of gene delivery of αSyn-specific single-chain antibodies in vitro and in vivo. The plasmids for αSyn and selective antibodies (NAC32, D10, and VH14) were constructed and were transfected to HEK293 and SH-SY5Y cells. Co-expression of αSyn with NAC32, but not D10 or VH14, profoundly downregulated αSyn protein, but not αSyn mRNA levels in these cells. The interaction of αSyn and NAC32 antibody was next examined in vivo. Adeno-associated virus (AAV)-αSyn combined with AAV-NAC32 or AAV-sc6H4 (a negative control virus) were stereotactically injected into the substantia nigra of adult rats. AAV-NAC32 significantly reduced AAV-encoded αSyn levels in the substantia nigra and striatum and increased tyrosine hydroxylase immunoreactivity in the striatum. Also, in the animals injected with AAV-NAC32 alone, endogenous αSyn protein levels were significantly downregulated in the substantia nigra. Our data suggest that AAV-mediated gene transfer of NAC32 is a feasible approach for reducing the expression of target αSyn protein in brain.

Doublecortin-like Kinase 1 Regulates α-Synuclein Levels and Toxicity.

α-Synuclein (α-Syn) accumulation is a pathological hallmark of Parkinson's disease. Duplications and triplications of SNCA, the gene coding for α-Syn, cause genetic forms of the disease, which suggests that increased α-Syn dosage can drive PD. To identify the proteins that regulate α-Syn, we previously performed a screen of potentially druggable genes that led to the identification of 60 modifiers. Among them, Doublecortin-like kinase 1 (DCLK1), a microtubule binding serine threonine kinase, emerged as a promising target due to its potent effect on α-Syn and potential druggability as a neuron-expressed kinase. In this study, we explore the relationship between DCLK1 and α-Syn in human cellular and mouse models of PD. First, we show that DCLK1 regulates α-Syn levels post-transcriptionally. Second, we demonstrate that knockdown of Dclk1 reduces phosphorylated species of α-Syn and α-Syn-induced neurotoxicity in the SNc in two distinct mouse models of synucleinopathy. Last, silencing DCLK1 in human neurons derived from individuals with SNCA triplications reduces phosphorylated and total α-Syn, thereby highlighting DCLK1 as a potential therapeutic target to reduce pathological α-Syn in disease.SIGNIFICANCE STATEMENT DCLK1 regulates α-Syn protein levels, and Dclk1 knockdown rescues α-Syn toxicity in mice. This study provides evidence for a novel function for DCLK1 in the mature brain, and for its potential as a new therapeutic target for synucleinopathies.

Autophagy\u2013lysosome pathway alterations and alpha-synuclein up-regulation in the subtype of neuronal ceroid lipofuscinosis, CLN5 disease

Neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative lysosomal storage disorders. CLN5 deficiency causes a subtype of NCL, referred to as CLN5 disease. CLN5 is a soluble lysosomal protein with an unclear function in the cell. Increased levels of the autophagy marker protein LC3-II have been reported in several subtypes of NCLs. In this report, we examine whether autophagy is altered in CLN5 disease. We found that the basal level of LC3-II was elevated in both CLN5 disease patient fibroblasts and CLN5-deficient HeLa cells. Further analysis using tandem fluorescent mRFP-GFP-LC3 showed the autophagy flux was increased. We found the alpha-synuclein (α-syn) gene SNCA was highly up-regulated in CLN5 disease patient fibroblasts. The aggregated form of α-syn is well known for its role in the pathogenicity of Parkinson’s disease. Higher α-syn protein levels confirmed the SNCA up-regulation in both patient cells and CLN5 knockdown HeLa cells. Furthermore, α-syn was localized to the vicinity of lysosomes in CLN5 deficient cells, indicating it may have a lysosome-related function. Intriguingly, knocking down SNCA reversed lysosomal perinuclear clustering caused by CLN5 deficiency. These results suggest α-syn may affect lysosomal clustering in non-neuronal cells, similar to its role in presynaptic vesicles in neurons.

Dissecting the Molecular Pathway Involved in PLK2 Kinase-mediated α-Synuclein-selective Autophagic Degradation

Increasing lines of evidence support the causal link between α-synuclein (α-syn) accumulation in the brain and Parkinson's disease (PD) pathogenesis. Therefore, lowering α-syn protein levels may represent a viable therapeutic strategy for the treatment of PD and related disorders. We recently described a novel selective α-syn degradation pathway, catalyzed by the activity of the Polo-like kinase 2 (PLK2), capable of reducing α-syn protein expression and suppressing its toxicity in vivo However, the exact molecular mechanisms underlying this degradation route remain elusive. In the present study we report that among PLK family members, PLK3 is also able to catalyze α-syn phosphorylation and degradation in living cells. Using pharmacological and genetic approaches, we confirmed the implication of the macroautophagy on PLK2-mediated α-syn turnover, and our observations suggest a concomitant co-degradation of these two proteins. Moreover, we showed that the N-terminal region of α-syn is important for PLK2-mediated α-syn phosphorylation and degradation and is implicated in the physical interaction between the two proteins. We also demonstrated that PLK2 polyubiquitination is important for PLK2·α-syn protein complex degradation, and we hypothesize that this post-translational modification may act as a signal for the selective recognition by the macroautophagy machinery. Finally, we observed that the PD-linked mutation E46K enhances PLK2-mediated α-syn degradation, suggesting that this mutated form is a bona fide substrate of this degradation pathway. In conclusion, our study provides a detailed description of the new degradation route of α-syn and offers new opportunities for the development of therapeutic strategies aiming to reduce α-syn protein accumulation and toxicity.

Proteasome subunit and opioid receptor gene expression down-regulation induced by paraquat and maneb in human neuroblastoma SH-SY5Y cells.

Paraquat (PQ) and maneb (MB) are able to induce neurotoxic effects by promoting α-synuclein (α-syn) aggregates and altering tyrosine hydroxylase (TH), thus increasing the risk of Parkinson's disease (PD). These pesticides promote neurotoxic effects also by affecting proteasome function that normally regulate protein turnover. We investigated the effects of the two pesticides exposure on multiple targets involved in PD, using SH-SY5Y cells. First, we evaluated TH and α-syn protein levels following PQ and MB cell exposure and a significant increase of these protein levels was observed. Subsequently, since a relationship between ubiquitin/proteasome and opioid receptors has been proposed, the effects of pesticides on their gene expression have been investigated. A decrease of β1 and Rpt3 proteasome subunit mRNA levels, together with the μ and δ opioid receptor down-regulation, was detected. The reported alterations, here simultaneously observed, help to clarify the involvement of multiple biological markers implicated in PD, often separately evaluated.

Long-term dietary restriction modulates the level of presynaptic proteins in the cortex and hippocampus of the aging rat

Brain aging is related to the numerous structural and functional changes including decreased synaptic plasticity. The beneficial effects of dietary restriction (DR) are well known but insufficiently investigated at the level of plasticity-related markers. Therefore, the aim of this study was to examine the expression profiles of proteins structurally and functionally related to synapses—growth-associated protein 43 (GAP-43), synaptophysin (SPH) and α-synuclein (α-Syn), in the course of aging and in response to long-term DR. The mRNA and protein levels of three presynaptic proteins were assessed by Real Time RT-PCR and Western blotting in the cortex and hippocampus of young (6-month-old), middle-aged (12-month-old), aged (18-month-old) and old (24-month-old) male Wistar rats fed ad libitum and exposed to DR starting from 6 months of age. We observed that long-term DR modulated age-related transcriptional changes by maintaining stable mRNAs levels in the cortex. No major age-related changes of the protein levels were observed in the cortex, while the specific temporal decline was detected in the hippocampus for all three proteins. The SPH levels were decreased across lifespan (0.8-, 0.8- and 0.6-fold change at 12, 18 and 24 months), while the significant decrease of GAP-43 and α-Syn protein was detected at 24 months of age (0.6- and 0.7-fold decrease, respectively). Long-term DR eliminated this decline by increasing GAP-43, SPH and α-Syn protein levels (1.7-, 1.7- and 1.6-fold, respectively) thus reverting protein levels to the values measured in 6-month-old animals.Specific pattern of changes observed in the hippocampus identifies this structure as more vulnerable to the processes of aging and with a more pronounced response to the DR effects. The observed DR-induced stabilization of the levels of three presynaptic proteins indicates the beneficial effect of DR on age-related decline in the capacity for synaptic plasticity.

Endogenous α-Synuclein Is Induced by Valproic Acid through Histone Deacetylase Inhibition and Participates in Neuroprotection against Glutamate-Induced Excitotoxicity

Emerging evidence suggests that alpha-synuclein (alpha-syn), which is traditionally thought to have a pathophysiological role in neurodegenerative diseases, can have neuroprotective effects. This study aimed to investigate whether endogenous alpha-syn in neurons can be induced by valproic acid (VPA), a mood-stabilizer, anticonvulsant and histone deacetylase (HDAC) inhibitor, and if so, whether the alpha-syn induction is neuroprotective. VPA treatment of rat cerebellar granule cells caused a robust dose- and time-dependent increase in levels of alpha-syn protein and mRNA and in the intensity of alpha-syn immunostaining. Knockdown of VPA-induced alpha-syn overexpression with alpha-syn antisense oligonucleotides or siRNA completely blocked VPA-induced neuroprotection. alpha-Syn knockdown also exacerbated glutamate neurotoxicity, stimulated the expression of the proapoptotic gene ubiquitin-conjugating enzyme E2N, and downregulated the expression of the anti-apoptotic gene Bcl-2. Induction of alpha-syn by VPA was associated with inhibition of HDAC activity, resulting in hyperacetylation of histone H3 in the alpha-syn promoter and a marked increase in alpha-syn promoter activity. Moreover, VPA-induced alpha-syn induction and neuroprotection were mimicked by HDAC inhibitors sodium 4-phenylbutyrate and trichostatin A (TSA). alpha-syn was also induced by VPA in rat cerebral cortical neurons. Additionally, treatment of rats with VPA, sodium butyrate, or TSA markedly increased alpha-syn protein levels in the cortex and cerebellum. Together, our results demonstrate for the first time that VPA induces alpha-syn in neurons through inhibition of HDAC and that this alpha-syn induction is critically involved in neuroprotection against glutamate excitotoxicity. Clinically, VPA may represent a suitable treatment for excitotoxicity-related neurodegenerative diseases.