The fine-tuning of gene expression is critical for all cellular processes; aberrations in this activity can lead to pathology, and conversely, resilience. As their role in coordinating organismal ...responses to both internal and external factors have increasingly come into focus, small non-coding RNAs have emerged as an essential component to disease etiology. Using Systemic RNA interference Defective (SID) mutants of the nematode Caenorhabditis elegans, deficient in gene silencing, we examined the potential consequences of dysfunctional epigenomic regulation in the context of Parkinson's disease (PD). Specifically, the loss of either the sid-1 or sid-3 genes, which encode a dsRNA transporter and an endocytic regulatory non-receptor tyrosine kinase, respectively, conferred neuroprotection to dopaminergic (DA) neurons in an established transgenic C. elegans strain wherein overexpression of human α-synuclein (α-syn) from a chromosomally integrated multicopy transgene causes neurodegeneration. We further show that knockout of a specific microRNA, mir-2, attenuates α-syn neurotoxicity; suggesting that the native targets of mir-2-dependent gene silencing represent putative neuroprotective modulators. In support of this, we demonstrated that RNAi knockdown of multiple mir-2 targets enhanced α-syn-induced DA neurodegeneration. Moreover, we demonstrate that mir-2 overexpression originating in the intestine can induce neurodegeneration of DA neurons, an effect that was reversed by pharmacological inhibition of SID-3 activity. Interestingly, sid-1 mutants retained mir-2-induced enhancement of neurodegeneration. Transcriptomic analysis of α-syn animals with and without a sid-1 mutation revealed 27 differentially expressed genes with human orthologs related to a variety of diseases, including PD. Among these was pgp-8, encoding a P-glycoprotein-related ABC transporter. Notably, sid-1; pgp-8 double mutants abolished the neurodegeneration resulting from intestinal mir-2 overexpression. This research positions known regulators of small RNA-dependent gene silencing within a framework that facilitates mechanistic evaluation of epigenetic responses to exogenous and endogenous factors influencing DA neurodegeneration, revealing a path toward new targets for therapeutic intervention of PD.
Parkinson's disease (PD) is defined by the loss of dopaminergic neurons in the substantia nigra and the formation of Lewy body inclusions containing aggregated α-synuclein. Efforts to explain ...dopamine neuron vulnerability are hindered by the lack of dopaminergic cell death in α-synuclein transgenic mice. To address this, we manipulated both dopamine levels and α-synuclein expression. Nigrally targeted expression of mutant tyrosine hydroxylase with enhanced catalytic activity increased dopamine levels without damaging neurons in non-transgenic mice. In contrast, raising dopamine levels in mice expressing human A53T mutant α-synuclein induced progressive nigrostriatal degeneration and reduced locomotion. Dopamine elevation in A53T mice increased levels of potentially toxic α-synuclein oligomers, resulting in conformationally and functionally modified species. Moreover, in genetically tractable Caenorhabditis elegans models, expression of α-synuclein mutated at the site of interaction with dopamine prevented dopamine-induced toxicity. These data suggest that a unique mechanism links two cardinal features of PD: dopaminergic cell death and α-synuclein aggregation.
Abstract
Commonalities and, in some cases, pathological overlap between neurodegenerative diseases have led to speculation that targeting of underlying mechanisms might be of potentially shared ...therapeutic benefit. Alzheimer's disease is characterized by the formation of plaques, composed primarily of the amyloid-β 1-42 (Aβ) peptide in the brain, resulting in neurodegeneration. Previously, we have shown that overexpression of the lysosomal-trafficking protein, human Vps41 (hVps41), is neuroprotective in a transgenic worm model of Parkinson's disease, wherein progressive dopaminergic neurodegeneration is induced by α-synuclein overexpression. Here, we report the results of a systematic comparison of hVps41-mediated neuroprotection between α-synuclein and Aβ in transgenic nematode models of Caenorhabditis elegans. Our results indicate that an ARF-like GTPase gene product, ARL-8, mitigates endocytic Aβ neurodegeneration in a VPS-41-dependent manner, rather than through RAB-7 and AP3 as with α-synuclein. Furthermore, the neuroprotective effect of ARL-8 or hVps41 appears to be dependent on their colocalization and the activity of ARL-8. Additionally, we demonstrate that the LC3 orthologue, LGG-2, plays a critical role in Aβ toxicity with ARL-8. Further analysis of functional effectors of Aβ protein processing via the lysosomal pathway will assist in the elucidation of the underlying mechanism involving VPS-41-mediated neuroprotection. These results reveal functional distinctions in the intracellular management of neurotoxic proteins that serve to better inform the path for development of therapeutic interventions to halt neurodegeneration.
Calcineurin (CN) is a highly conserved Ca ²⁺–calmodulin (CaM)-dependent phosphatase that senses Ca ²⁺ concentrations and transduces that information into cellular responses. Ca ²⁺ homeostasis is ...disrupted by α-synuclein (α-syn), a small lipid binding protein whose misfolding and accumulation is a pathological hallmark of several neurodegenerative diseases. We report that α-syn, from yeast to neurons, leads to sustained highly elevated levels of cytoplasmic Ca ²⁺, thereby activating a CaM-CN cascade that engages substrates that result in toxicity. Surprisingly, complete inhibition of CN also results in toxicity. Limiting the availability of CaM shifts CN's spectrum of substrates toward protective pathways. Modulating CN or CN's substrates with highly selective genetic and pharmacological tools (FK506) does the same. FK506 crosses the blood brain barrier, is well tolerated in humans, and is active in neurons and glia. Thus, a tunable response to CN, which has been conserved for a billion years, can be targeted to rebalance the phosphatase’s activities from toxic toward beneficial substrates. These findings have immediate therapeutic implications for synucleinopathies.
α-Synuclein (α-syn), a protein of unknown function, is the most abundant protein in Lewy bodies, the histological hallmark of Parkinson's disease (PD). In yeast α-syn inhibits endoplasmic reticulum ...(ER)-to-Golgi (ERrightward arrowGolgi) vesicle trafficking, which is rescued by overexpression of a Rab GTPase that regulates ERrightward arrowGolgi trafficking. The homologous Rab1 rescues α-syn toxicity in dopaminergic neuronal models of PD. Here we investigate this conserved feature of α-syn pathobiology. In a cell-free system with purified transport factors α-syn inhibited ERrightward arrowGolgi trafficking in an α-syn dose-dependent manner. Vesicles budded efficiently from the ER, but their docking or fusion to Golgi membranes was inhibited. Thus, the in vivo trafficking problem is due to a direct effect of α-syn on the transport machinery. By ultrastructural analysis the earliest in vivo defect was an accumulation of morphologically undocked vesicles, starting near the plasma membrane and growing into massive intracellular vesicular clusters in a dose-dependent manner. By immunofluorescence/immunoelectron microscopy, these clusters were associated both with α-syn and with diverse vesicle markers, suggesting that α-syn can impair multiple trafficking steps. Other Rabs did not ameliorate α-syn toxicity in yeast, but RAB3A, which is highly expressed in neurons and localized to presynaptic termini, and RAB8A, which is localized to post-Golgi vesicles, suppressed toxicity in neuronal models of PD. Thus, α-syn causes general defects in vesicle trafficking, to which dopaminergic neurons are especially sensitive.
Genomic multiplication of the locus-encoding human α-synuclein (α-syn), a polypeptide with a propensity toward intracellular misfolding, results in Parkinson's disease (PD). Here we report the ...results from systematic screening of nearly 900 candidate genetic targets, prioritized by bioinformatic associations to existing PD genes and pathways, via RNAi knockdown. Depletion of 20 gene products reproducibly enhanced misfolding of α-syn over the course of aging in the nematode Caenorhabditis elegans. Subsequent functional analysis of seven positive targets revealed five previously unreported gene products that significantly protect against age- and dose-dependent α-syn-induced degeneration in the dopamine neurons of transgenic worms. These include two trafficking proteins, a conserved cellular scaffold-type protein that modulates G protein signaling, a protein of unknown function, and one gene reported to cause neurodegeneration in knockout mice. These data represent putative genetic susceptibility loci and potential therapeutic targets for PD, a movement disorder affecting ≈2% of the population over 65 years of age.
Parkinson's Disease (PD) is the second-most common neurodegenerative disease in the world, yet the fundamental and underlying causes of the disease are largely unknown, and treatments remain sparse ...and impotent. Several biological systems have been employed to model the disease but the nematode roundworm
shows unique promise among these to disinter the elusive factors that may prevent, halt, and/or reverse PD phenotypes. Some of the most salient of these
models of PD are those that position the misfolding-prone protein alpha-synuclein (α-syn), a hallmark pathological component of PD, as the primary target for scientific interrogation. By transgenic expression of human α-syn in different tissues, including dopamine neurons and muscle cells, the primary cellular phenotypes of PD in humans have been recapitulated in these
models and have already uncovered multifarious genetic factors and chemical compounds that attenuate dopaminergic neurodegeneration. This review describes the paramount discoveries obtained through the application of different α-syn models of PD in
and highlights their established utility and respective promise to successfully uncover new conserved genetic modifiers, functional mechanisms, therapeutic targets and molecular leads for PD with the potential to translate to humans.
Parkinson's disease (PD), the second most prevalent neurodegenerative disorder, is characterized by the degeneration of dopamine (DA) neurons and age-dependent formation of protein inclusions that ...contain the α-synuclein (α-syn) protein. RNA interference (RNAi) screening using Caenorhabditis elegans identified RTCB-1, an uncharacterized gene product, as one of several significant modifiers of α-syn protein misfolding. RTCB-1 is the worm ortholog of the human HSPC117 protein, a component of RNA trafficking granules in mammalian neurons. Here we show that RTCB-1 protects C. elegans DA neurons from age-dependent degeneration induced by human α-syn. Moreover, neuronal-specific RNAi depletion of rtcb-1 enhanced α-syn-induced degeneration. Similar results were obtained when worms were exposed to the DA neurotoxin 6-hydroxydopamine. HSPC117 has been characterized recently as an essential subunit of the human tRNA splicing ligase complex. tRNA ligases have alternative functions in RNA repair and nonconventional mRNA splicing events. For example, in yeast, unconventional splicing of HAC1, a transcription factor that controls the unfolded protein response (UPR), is mediated by a tRNA ligase. In C. elegans, we demonstrate that RTCB-1 is necessary for xbp-1 (worm homolog of HAC1) mRNA splicing. Moreover, using a RNA ligase-dead mutant, we determine that the ligase activity of worm RTCB-1 is required for its neuroprotective role, which, in turn, is mediated through XBP-1 in the UPR pathway. Collectively, these studies highlight the mechanistic intersection of RNA processing and proteostasis in mediating neuroprotection.
Mutations in leucine-rich repeat kinase 2 (LRRK2) have been identified as a genetic cause of familial Parkinson's disease (PD) and have also been found in the more common sporadic form of PD, thus ...positioning LRRK2 as important in the pathogenesis of PD. Biochemical studies of the disease-causing mutants of LRRK2 implicates an enhancement of kinase activity as the basis of neuronal toxicity and thus possibly the pathogenesis of PD due to LRRK2 mutations. Previously, a chemical library screen identified inhibitors of LRRK2 kinase activity. Here, two of these inhibitors, GW5074 and sorafenib, are shown to protect against G2019S LRRK2-induced neurodegeneration in vivo in Caenorhabditis elegans and in Drosophila. These findings indicate that increased kinase activity of LRRK2 is neurotoxic and that inhibition of LRRK2 activity can have a disease-modifying effect. This suggests that inhibition of LRRK2 holds promise as a treatment for PD.
α-Synuclein (α-syn) is a small lipid-binding protein implicated in several neurodegenerative diseases, including Parkinson's disease, whose pathobiology is conserved from yeast to man. There are no ...therapies targeting these underlying cellular pathologies, or indeed those of any major neurodegenerative disease. Using unbiased phenotypic screens as an alternative to target-based approaches, we discovered an N-aryl benzimidazole (NAB) that strongly and selectively protected diverse cell types from a-syn toxicity. Three chemical genetic screens in wild-type yeast cells established that NAB promoted endosomal transport events dependent on the E3 ubiquitin ligase Rsp5/Nedd4. These same steps were perturbed by α-syn itself. Thus, NAB identifies a druggable node in the biology of α-syn that can correct multiple aspects of its underlying pathology, including dysfunctional endosomal and endoplasmic reticulum-to-Golgi vesicle trafficking.
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