Increases in S-nitrosylation and inactivation of the neuroprotective ubiquitin E3 ligase, parkin, in the brains of patients with Parkinson’s Disease (PD) are thought to be pathogenic and suggest a possible mechanism linking parkin to sporadic PD. Here we demonstrate that physiologic modification of parkin by hydrogen sulfide (H2S), termed sulfhydration, enhances its catalytic activity. Sulfhydration sites are identified by mass spectrometry analysis and investigated by site directed mutagenesis. Parkin sulfhydration is markedly depleted in the brains of patients with PD, suggesting that this loss may be pathologic. This implies that H2S donors may be therapeutic.
Glutamate acting on N-methyl-D-aspartate (NMDA) receptors plays an important role in neurodegenerative diseases and neuronal injury following stroke, through activation of poly(ADP-ribose) polymerase-1 and generation of the death molecule poly(ADP-ribose) (PAR) polymer. Here we identify Iduna, a novel NMDA receptor-induced survival gene that is neuroprotective against glutamate NMDA receptor mediated excitotoxicity both in vitro and in vivo and against stroke through interfering with PAR polymer induced cell death (parthanatos). Iduna’s protective effects are independent and downstream of PARP-1 activity. Iduna is a PAR polymer binding protein and mutations at the PAR polymer binding site abolishes the PAR binding activity of Iduna and attenuates its protective actions. Iduna is protective in vivo against NMDA-induced excitotoxicity and middle cerebral artery occlusion (MCAO)-induced stroke in mice. These results define Iduna as the first endogenous inhibitor of parthanatos. Interfering with PAR polymer signaling offers a new therapeutic strategy for the treatment of neurologic disorders.
Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are the most frequent genetic cause of Parkinson’s disease (PD) and these mutations play important roles in sporadic PD. The LRRK2 protein contains GTPase and kinase domains and several protein-protein interaction domains. The kinase and GTPase activity of LRRK2 seem to be important in regulating LRRK2-dependent cellular signaling pathways. LRRK2’s GTPase and kinase domains may reciprocally regulate each other to direct LRRK2’s ultimate function. While most LRRK2 investigations are centered on LRRK2’s kinase activity, this review focuses on the function of LRRK2’s GTPase in LRRK2 physiology and pathophysiology.
GTPase-activating proteins (GAPs); guanine nucleotide exchange factors (GEFs); GTPase; kinase; LRRK2; Parkinson’s disease
Parkinson’s disease (PD) is caused by the progressive degeneration of dopaminergic neurons in the substantia nigra. Although the etiology for most PD remains elusive, the identification of specific genetic defects in familial cases of PD and the signaling pathways governed by these genes has provided tremendous insight into PD pathogenesis. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are frequently found in familial and sporadic PD. Although current knowledge regarding the regulatory mechanisms of LRRK2 activation is limited, it is becoming increasingly evident that aberrant kinase activity of the pathologic mutants of LRRK2 is associated with neurodegeneration, suggesting that the kinase activity of LRRK2 is a potential therapeutic target. In addition, LRRK2 inhibitors might provide valuable tools to understand the pathophysiological and physiological roles of LRRK2 as well as the etiology of PD. We discuss here the potential and feasibility of targeting LRRK2 as a therapeutic strategy for PD.
DNA damage is a proposed pathogenic factor in neurodegenerative disorders such as Parkinson’s disease. To probe the underpinning mechanism of such neuronal perturbation, we sought to produce an experimental model of DNA damage. We thus first assessed by in situ nick translation and emulsion autoradiography in the mouse brain the effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 4 × 20mg/kg, i.p., every 2 hours), a neurotoxin known to produce a model of Parkinson’s disease, on DNA. Here we show that DNA strand breaks occur in vivo in this mouse model of Parkinson’s disease with kinetics and a topography that parallel the degeneration of substantia nigra neurons, as assessed by FluoroJade-labeling. Previously, nitric oxide synthase (NOS) and cyclooxygenase-2 (Cox-2) were found to modulate MPTP-induced dopaminergic neuronal death. We thus assessed the contribution of these enzymes to DNA damage in mice lacking either neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), or Cox-2. We found that the lack of Cox-2 and of nNOS, but not of iNOS activity, attenuate MPTP-related DNA damage. We also found that not only nuclear, but mitochondrial DNA as well is a target for the MPTP insult. These results suggest that the loss of genomic integrity can be triggered by the concerted actions of nNOS and Cox-2, and provide further support to the view that DNA damage may contribute to the neurodegenerative process in PD.
Cyclooxygenase; DNA; MPTP; neurodegeneration; Nitric Oxide; oxidative damage; PD
Leucine-rich repeat kinase-2 (LRRK2) mutations are the most important cause of familial Parkinson's disease and non-selective inhibitors are protective in rodent disease models. Due to their poor potency and selectivity, the neuroprotective mechanism of these tool compounds has remained elusive so far and it is still unknown whether selective LRRK2 inhibition can attenuate mutant LRRK2-dependent toxicity in human neurons. Here, we employ a chemoproteomics strategy to identify potent, selective and metabolically stable LRRK2 inhibitors. We demonstrate that CZC-25146 prevents mutant LRRK2-induced injury of cultured rodent and human neurons with mid-nanomolar potency. These precise chemical probes further validate this emerging therapeutic strategy. They will enable more detailed studies of LRRK2-dependent signaling and pathogenesis and accelerate drug discovery.
Chemical Proteomics; Drug Discovery; LRRK2; Parkinson's disease; Neurodegeneration; Sunitinib
Mutations in the genes encoding LRRK2 and α-synuclein cause autosomal dominant forms of familial Parkinson's disease (PD). Fibrillar forms of α-synuclein are a major component of Lewy bodies, the intracytoplasmic proteinaceous inclusions that are a pathological hallmark of idiopathic and certain familial forms of PD. LRRK2 mutations cause late-onset familial PD with a clinical, neurochemical and, for the most part, neuropathological phenotype that is indistinguishable from idiopathic PD. Importantly, α-synuclein-positive Lewy bodies are the most common pathology identified in the brains of PD subjects harboring LRRK2 mutations. These observations may suggest that LRRK2 functions in a common pathway with α-synuclein to regulate its aggregation. To explore the potential pathophysiological interaction between LRRK2 and α-synuclein in vivo, we modulated LRRK2 expression in a well-established human A53T α-synuclein transgenic mouse model with transgene expression driven by the hindbrain-selective prion protein promoter. Deletion of LRRK2 or overexpression of human G2019S-LRRK2 has minimal impact on the lethal neurodegenerative phenotype that develops in A53T α-synuclein transgenic mice, including premature lethality, pre-symptomatic behavioral deficits and human α-synuclein or glial neuropathology. We also find that endogenous or human LRRK2 and A53T α-synuclein do not interact together to influence the number of nigrostriatal dopaminergic neurons. Taken together, our data suggest that α-synuclein-related pathology, which occurs predominantly in the hindbrain of this A53T α-synuclein mouse model, occurs largely independently from LRRK2 expression. These observations fail to provide support for a pathophysiological interaction of LRRK2 and α-synuclein in vivo, at least within neurons of the mouse hindbrain.
Parkinson’s disease (PD) is a progressive neurodegenerative disease caused by the interaction of genetic and environmental factors. However, the etiology of PD remains largely unknown. Macroautophagy is known to play an essential role in the degradation of abnormal proteins and organelles. Furthermore, the loss of autophagy-related (Atg) genes results in neurodegeneration and abnormal protein accumulation. Since these are also pathologic features of Parkinson disease, the conditional impairment of autophagy may lead to improved animal models for the study of PD. Using transgenic mice expressing Cre recombinase under the control of either the dopamine transporter or the engrailed-1 promoters, we generated mice with the conditional deletion of Atg7 in the dopamine neurons of the substantia nigra pars compacta, other regions of the midbrain, and also the hindbrain. This conditional impairment of autophagy results in the age-related loss of dopaminergic neurons and corresponding loss of striatal dopamine, the accumulation of low molecular weight α-synuclein, and the presence of ubiquitinated protein aggregates, recapitulating many of the pathologic features of PD. These conditional knockout animals provide insight into the process of autophagy in Parkinson disease pathology.
Regulation of self-renewal and differentiation of neural stem cells is still poorly understood. Here we investigate the role of a developmentally expressed protein, Botch, which blocks Notch, in neocortical development. Downregulation of Botch in vivo leads to cellular retention in the ventricular and subventricular zones, whereas overexpression of Botch drives neural stem cells into the intermediate zone and cortical plate. In vitro neurosphere and differentiation assays indicate that Botch regulates neurogenesis by promoting neuronal differentiation. Botch prevents cell surface presentation of Notch by inhibiting the S1 furin-like cleavage of Notch, maintaining Notch in the immature full-length form. Understanding the function of Botch expands our knowledge regarding both the regulation of Notch signaling and the complex signaling mediating neuronal development.
Genome-wide association (GWAS) methods have identified genes contributing to Parkinson disease (PD); we sought to identify additional genes associated with PD susceptibility.
A two stage design was used. First, individual level genotypic data from five recent PD GWAS (Discovery Sample: 4,238 PD cases and 4,239 controls) were combined. Following imputation, a logistic regression model was employed in each dataset to test for association with PD susceptibility and results from each dataset were meta-analyzed. Second, 768 SNPs were genotyped in an independent Replication Sample (3,738 cases and 2,111 controls).
Genome-wide significance was reached for SNPs in SNCA (rs356165, G: odds ratio (OR)=1.37; p=9.3 × 10−21), MAPT (rs242559, C: OR=0.78; p=1.5 × 10−10), GAK/DGKQ (rs11248051, T:OR=1.35; p=8.2 × 10−9/ rs11248060, T: OR=1.35; p=2.0×10−9), and the HLA region (rs3129882, A: OR=0.83; p=1.2 × 10−8), which were previously reported. The Replication Sample confirmed the associations with SNCA, MAPT, and the HLA region and also with GBA (E326K OR=1.71; p=5 × 10−8 Combined Sample) (N370 OR=3.08; p=7 × 10−5 Replication sample). A novel PD susceptibility locus, RIT2, on chromosome 18 (rs12456492; p=5 × 10−5 Discovery Sample; p=1.52 × 10−7 Replication sample; p=2 × 10−10 Combined Sample) was replicated. Conditional analyses within each of the replicated regions identified distinct SNP associations within GBA and SNCA, suggesting that there may be multiple risk alleles within these genes.
We identified a novel PD susceptibility locus, RIT2, replicated several previously identified loci, and identified more than one risk allele within SNCA and GBA.
Parkinson disease (PD) is a relatively common disorder of the nervous system that afflicts patients later in life with tremor, slowness of movement, gait instability, and rigidity. Treatment of these cardinal features of the disease is a success story of modern science and medicine, as a great deal of disability can be alleviated through the pharmacological correction of brain dopamine deficiency. Unfortunately these therapies only provide temporary, though significant, relief from early symptoms and do not halt disease progression. In addition, pathological changes outside of the motor system leading to cognitive, autonomic, and psychiatric symptoms are not sufficiently treated by current therapies. Much as the discovery of dopamine deficiency led to powerful treatments for motor symptoms, recent discoveries concerning the role of specific genes in PD pathology will lead to the next revolution in disease therapy. Understanding why and how susceptible cells in motor and nonmotor regions of the brain die in PD is the first step toward preventing this cell death and curing or slowing the disease. In this review we discuss recent discoveries in the fields of diagnosis and treatment of PD and focus on how a better understanding of disease mechanisms gained through the study of monogenetic forms of PD has provided novel therapeutic targets.
Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are the most common genetic cause of Parkinson's disease (PD) and cause both autosomal dominant familial and sporadic PD. Currently, the physiological and pathogenic activities of LRRK2 are poorly understood. To decipher the biological functions of LRRK2, including the genes and pathways modulated by LRRK2 kinase activity in vivo, we assayed genome-wide mRNA expression in the brain and peripheral tissues from LRRK2 knockout (KO) and kinase hyperactive G2019S (G2019S) transgenic mice. Subtle but significant differences in mRNA expression were observed relative to wild-type (WT) controls in the cortex, striatum and kidney of KO animals, but only in the striatum in the G2019S model. In contrast, robust, consistent and highly significant differences were identified by the direct comparison of KO and G2019S profiles in the cortex, striatum, kidney and muscle, indicating opposite effects on mRNA expression by the two models relative to WT. Ribosomal and glycolytic biological functions were consistently and significantly up-regulated in LRRK2 G2019S compared with LRRK2 KO tissues. Genes involved in membrane-bound organelles, oxidative phosphorylation, mRNA processing and the endoplasmic reticulum were down-regulated in LRRK2 G2019S mice compared with KO. We confirmed the expression patterns of 35 LRRK2-regulated genes using quantitative reverse transcription polymerase chain reaction. These findings provide the first description of the transcriptional responses to genetically modified LRRK2 activity and provide preclinical target engagement and/or pharmacodynamic biomarker strategies for LRRK2 and may inform future therapeutic strategies for LRRK2-associated PD.
MicroRNAs (miRNAs) are small 19–23 nucleotide regulatory RNAs that function by modulating mRNA translation and/or turnover in a sequence-specific fashion. In the nervous system, miRNAs regulate the production of numerous proteins involved in synaptic transmission. In turn, neuronal activity can regulate the production and turnover of miRNA through a variety of mechanisms. In this way, miRNAs and neuronal activity are in a reciprocal homeostatic relationship that balances neuronal function. The miRNA function is critical in pathological states related to overexcitation such as epilepsy and stroke, suggesting miRNA’s potential as a therapeutic target. We review the current literature relating the interplay of miRNA and neuronal activity and provide future directions for defining miRNA’s role in disease.
microRNA; synaptic plasticity; stroke; epilepsy; neuroprotective agents
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.
MicroRNAs are small non-protein coding RNAs that regulate gene expression through post-transcriptional repression. Recent studies demonstrated the importance of microRNAs in the nervous system development, function and disease. Parkinson’s disease is the second most prevalent neurodegenerative disease with only symptomatic treatment available. Recent success in using small RNAs as therapeutic targets hold a substantial promise for the Parkinson’s disease field. Here we review recent work linking the microRNA pathway to Parkinson’s disease.
microRNA; small noncoding RNA; RNA induced silencing; Parkinson’s disease; neurodegenerative diseases
Both sporadic and autosomal dominant forms of Parkinson’s disease (PD) have been causally linked to mutations in leucine-rich repeat kinase 2 (LRRK2), a large protein with multiple domains. The kinase domain plays an important role in LRRK2 mediated toxicity. While a number of investigations have focused on LRRK2 kinase activity, less is known about the GTPase function of LRRK2. The activity of GTPases is regulated by GTPase activating proteins (GAPs) and GTP exchange factors (GEFs). Here, we identify ArfGAP1 as the first GAP for LRRK2. ArfGAP1 binds LRRK2 predominantly via the WD40 and kinase domain of LRRK2 and it increases LRRK2 GTPase activity and regulates LRRK2 toxicity both in vitro and in vivo in Drosophila melanogaster. Unexpectedly, ArfGAP1 is a LRRK2 kinase substrate whose GAP activity is inhibited by LRRK2, while wild type and G2019S LRRK2 autophosphorylation and kinase activity are significantly reduced in the presence of ArfGAP1. Overexpressed ArfGAP1 exhibits toxicity that is reduced by LRRK2 both in vitro and in vivo. Δ64-ArfGAP1, a dominant negative ArfGAP1, and shRNA knockdown of ArfGAP1 reduce LRRK2 toxicity. Thus, LRRK2 and ArfGAP1 reciprocally regulate the activity of each other. Our results provide insight into the basic pathobiology of LRRK2 and indicate an important role for the GTPase domain and ArfGAP1 in LRRK2 mediated toxicity. These data suggest that agents targeted towards regulation of LRRK2 GTP hydrolysis might be therapeutic agents for the treatment of Parkinson’s disease.
The synaptic insertion or removal of AMPA receptors (AMPAR) plays critical roles in the regulation of synaptic activity reflected in the expression of long-term potentiation (LTP) and long-term depression (LTD). The cellular events underlying this important process in learning and memory are still being revealed. Here we describe and characterize the AAA+ ATPase, Thorase, that regulates the expression of surface AMPAR. In an ATPase-dependent manner Thorase mediates the internalization of AMPAR by disassembling the AMPAR-GRIP1 complex. Following genetic deletion of Thorase, the internalization of AMPAR is substantially reduced, leading to increased amplitudes of miniature excitatory postsynaptic currents, enhancement of LTP and elimination of LTD. These molecular events are expressed as deficits in learning and memory in Thorase null mice. This study identifies an AAA+ ATPase that plays a critical role in regulating the surface expression of AMPAR and thereby regulates synaptic plasticity and learning and memory.
The mitochondrial protein apoptosis-inducing factor (AIF) plays a pivotal role in poly(ADP-ribose) polymerase-1 (PARP-1)-mediated cell death (parthanatos), during which it is released from the mitochondria and translocates to the nucleus. Here, we show that AIF is a high affinity poly(ADP-ribose) (PAR)–binding protein and that PAR binding to AIF is required for parthanatos both in vitro and in vivo. AIF bound PAR at a site distinct from AIF’s DNA binding site and this interaction triggered AIF release from the cytosolic side of the mitochondrial outer membrane. Mutation of the PAR binding site in AIF did not affect its NADH oxidase activity, its ability to bind FAD or DNA, or its ability to induce nuclear condensation. However, this AIF mutant was not released from mitochondria and did not translocate to the nucleus or mediate cell death following PARP-1 activation. These results suggest a mechanism for PARP-1 to initiate AIF-mediated cell death and indicate that AIF’s bioenergetic cell survival-promoting functions are separate from its effects as a mitochondrially-derived death effector. Interference with the PAR-AIF interaction or PAR signaling may provide unique opportunities for preventing cell death following activation of PARP-1.
A hallmark of Parkinson’s disease (PD) is the preferential loss of substantia nigra dopamine neurons. Here we identify a new Parkin Interacting Substrate, PARIS (ZNF746), whose levels are regulated by the ubiquitin proteasome system via binding to and ubiquitination by the E3 ubiquitin ligase, parkin. PARIS is a novel KRAB and zinc finger protein that accumulates in models of parkin inactivation and in human PD brain. PARIS represses the expression of the transcriptional co-activator, PGC-1α and the PGC-1α target gene, NRF-1 by binding to insulin response sequences in the PGC-1α promoter. Conditional knockout of parkin in adult animals leads to progressive loss of dopamine (DA) neurons that is PARIS dependent. Moreover overexpression of PARIS leads to the selective loss of DA neurons in the substantia nigra, which is reversed by either parkin or PGC-1α co-expression. The identification of PARIS provides a molecular mechanism for neurodegeneration due to parkin inactivation.
Methylene blue (MB) is a drug with a long history and good safety profile, and with recently-described features desirable in a treatment for ALS.
We tested oral MB in inbred high-copy number SOD1 G93A mice, at 25 mg/kg/day beginning at 45 days of age. We measured disease onset, progression, and survival. There was no difference in disease onset between MB-treated mice and controls, although subgroup analysis showed a modest but statistically significant delay in disease onset in MB-treated female mice only (control 122±10.2 versus MB 129±10.0 days). MB-treated mice of both sexes spent more time in less severe stages of disease, and less time in later, more severe stages of disease. There was a non-significant trend to longer survival in MB-treated animals (control males reached endpoint at 161±14.1 days, versus 166±10.0 days for MB-treated animals, and control females reached endpoint at 171±6.2 days versus 173±13.4 days for MB-treated animals).
In spite of a strong theoretical rationale, MB had no significant effects on onset or survival in the inbred SOD1 G93A mouse model of ALS.
Neuronal activity regulates a broad range of processes in the hippocampus, including the precise regulation of translation. Disruptions in proper translational control in the nervous system are associated with a variety of disorders that fall in the autistic spectrum. MicroRNA (miRNA) represent a relatively recently discovered player in the regulation of translation in the nervous system. We have conducted an in depth analysis of how neuronal activity regulates miRNA expression in the hippocampus. Using deep sequencing we exhaustively identify all miRNAs, including 15 novel miRNAs, expressed in hippocampus of the adult mouse. We identified 119 miRNAs documented in miRBase but less than half of these miRNA were expressed at a level greater than 0.1% of total miRNA. Expression profiling following induction of neuronal activity by electroconvulsive shock demonstrates that most miRNA show a biphasic pattern of expression: rapid induction of specific mature miRNA expression followed by a decline in expression. These results have important implications into how miRNAs influence activity-dependent translational control.
Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is characterized by the degeneration of dopamine (DA) and non-DA neurons, the almost uniform presence of Lewy bodies, and motor deficits. Although the majority of PD is sporadic, specific genetic defects in rare familial cases have provided unique insights into the pathogenesis of PD. Through the creation of animal and cellular models of mutations in LRRK2 and α-synuclein, which are linked to autosomal dominant PD, and mutations in parkin, DJ-1, and PINK1, which are responsible for autosomal recessive PD, insight into the molecular mechanisms of this disorder are leading to new ideas about the pathogenesis of PD. In this review, we discuss the animal models for these genetic causes of PD, their limitations and value. Moreover, we discuss future directions and potential strategies for optimization of the genetic models.
Genetic alterations in α-synuclein cause autosomal dominant familial Parkinsonism and may contribute to sporadic Parkinson's disease (PD). Synphilin-1 is an α-synuclein-interacting protein, with implications in PD pathogenesis related to protein aggregation. Currently, the in vivo role of synphilin-1 in α-synuclein-linked pathogenesis is not fully understood. Using the mouse prion protein promoter, we generated synphilin-1 transgenic mice, which did not display PD-like phenotypes. However, synphilin-1/A53T α-synuclein double-transgenic mice survived longer than A53T α-synuclein single-transgenic mice. There were attenuated A53T α-synuclein-induced motor abnormalities and decreased astroglial reaction and neuronal degeneration in brains in double-transgenic mice. Overexpression of synphilin-1 decreased caspase-3 activation, increased beclin-1 and LC3 II expression and promoted formation of aggresome-like structures, suggesting that synphilin-1 alters multiple cellular pathways to protect against neuronal degeneration. These studies demonstrate that synphilin-1 can diminish the severity of α-synucleinopathy and play a neuroprotective role against A53T α-synuclein toxicity in vivo.