Parkinson’s Disease (PD) is a chronic progressive neurologic disorder, which affects approximately one million men and women in the U.S. alone. PD represents a heterogeneous disorder with common clinical manifestations and for the most part common neuropathological findings.
This short article reviews the role of the ubiquitin E3 ligase in sporadic PD.
The role of parkin in sporadic PD was reviewed by querying PubMed
Parkin is inactivated in sporadic PD via S-nitrosylation, oxidative and dopaminergic stress, and phosphorylation by the stress activated kinase, c-Abl leading to the accumulation of AIMP2 and PARIS (ZNF746).
Strategies aimed at maintaining parkin in a catalytically active state or interfering with toxicity of AIMP2 and PARIS (ZNF746) offer new therapeutic opportunities.
c-Abl; PARIS; ZNF746; AIMP2; S-nitrosylation; neurodegeneration
There is no proven neuroprotective or neurorestorative therapy for Parkinson’s disease (PD) to date. Several advances in the genetics of PD have created an opportunity to develop mechanistic based therapies that hold particular promise for identifying agents that slow and even halt the progression of PD as well as restore function. Here we review many of the advances in the last decade regarding the identification of new targets for the treatment of PD based on understanding the molecular mechanisms of how mutations in genes linked to PD cause neurodegeneration.
The leucine-rich repeat kinase 2 (LRRK2) gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson’s disease (PD). LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2G2019S pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2G2019S shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells.
Genetic studies have provided valuable insight into the pathological mechanisms underlying Parkinson’s disease (PD). The elucidation of the genetic components to what was once largely considered a non-genetic disease has given rise to a multitude of cell and animal models enabling the dissection of molecular pathways involved in disease etiology. Here, we review advances obtained from models of dominant mutations in α-synuclein and LRRK2 as well as recessive PINK1, Parkin and DJ-1 mutations. Recent genome-wide association studies have implicated genetic variability at two of these loci, α-synuclein and LRRK2, as significant risk factors for developing sporadic PD. This, coupled to the established role of mitochondrial impairment in both familial and sporadic PD highlights the likelihood of common mechanisms fundamental to the etiology of both.
α-synuclein; LRRK2; Parkin; PINK1; DJ-1; PARIS
Finding new therapies for Parkinson’s disease (PD) is a slow process. We assembled an international committee of experts to examine drugs potentially suitable for repurposing to modify PD progression. This committee evaluated multiple drugs currently used, or being developed, in other therapeutic areas, as well as considering several natural, non-pharmaceutical compounds. The committee prioritized which of these putative treatments were most suited to move immediately into pilot clinical trials. Aspects considered included known modes of action, safety, blood-brain-barrier penetration, preclinical data in animal models of PD and the possibility to monitor target engagement in the brain. Of the 26 potential interventions, 10 were considered worth moving forward into small, parallel ‘learning’ clinical trials in PD patients. These trials could be funded in a multitude of ways through support from industry, research grants and directed philanthropic donations. The committee-based approach to select the candidate compounds might help rapidly identify new potential PD treatment strategies for use in clinical trials.
Drug repositioning; disease modification; neuroprotection
To fully use human pluripotent stem cells (PSCs) in regenerative medicine, highly efficient differentiation strategies are required to drive induced PSCs into desired lineages and generate functional cell progenies. Current differentiation protocols for deriving dopaminergic neurons from PSCs involve months of stem cell culture procedures and multiple reagents. This study was motivated by a desire for a rapid and highly efficient system to generate human PSC-derived functional dopaminergic neurons.
Human pluripotent stem cells (PSCs) are a promising cell resource for various applications in regenerative medicine. Highly efficient approaches that differentiate human PSCs into functional lineage-specific neurons are critical for modeling neurological disorders and testing potential therapies. Proneural transcription factors are crucial drivers of neuron development and hold promise for driving highly efficient neuronal conversion in PSCs. Here, we study the functions of proneural transcription factor Atoh1 in the neuronal differentiation of PSCs. We show that Atoh1 is induced during the neuronal conversion of PSCs and that ectopic Atoh1 expression is sufficient to drive PSCs into neurons with high efficiency. Atoh1 induction, in combination with cell extrinsic factors, differentiates PSCs into functional dopaminergic (DA) neurons with >80% purity. Atoh1-induced DA neurons recapitulate key biochemical and electrophysiological features of midbrain DA neurons, the degeneration of which is responsible for clinical symptoms in Parkinson’s disease (PD). Atoh1-induced DA neurons provide a reliable disease model for studying PD pathogenesis, such as neurotoxin-induced neurodegeneration in PD. Overall, our results determine the role of Atoh1 in regulating neuronal differentiation and neuron subtype specification of human PSCs. Our Atoh1-mediated differentiation approach will enable large-scale applications of PD patient-derived midbrain DA neurons in mechanistic studies and drug screening for both familial and sporadic PD.
Induced pluripotent stem cells; Embryonic stem cells; Parkinson’s disease; Basic helix-loop-helix transcription factors; Tet-On
The defining pathogenic feature of Parkinson’s disease is the age dependent loss of dopaminergic neurons. Mutations and inactivation of parkin, an ubiquitin E3 ligase, cause Parkinson’s disease through accumulation of pathogenic substrates. Here we show that transgenic overexpression of the parkin substrate, aminoacyl-tRNA synthetase complex interacting multifunctional protein-2 (AIMP2) leads to a selective, age-dependent progressive loss of dopaminergic neurons via activation of poly(ADP-ribose) polymerase-1 (PARP1). AIMP2 accumulation in vitro and in vivo results in PARP1 overactivation and dopaminergic cell toxicity via direct association of these proteins in the nucleus providing a new path to PARP1 activation other than DNA damage. Inhibition of PARP1 through gene deletion or drug inhibition reverses behavioral deficits and protects in vivo against dopamine neuron death in AIMP2 transgenic mice. These data indicate that brain permeable PARP inhibitors could be effective in delaying or preventing disease progression in Parkinson’s disease.
Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions.
PARP; PARG; Poly(ADP-ribose); WWE; PBZ; Macro domain
Small non-coding microRNA RNA molecules can regulate stem cell function. The role of microRNAs in neural stem/progenitor cells (NS/PCs) differentiation is not entirely clear.
MiRNA profiling, loss and gain of function studies coupled with dendritic tree development morphometric analysis and calcium influx imaging were utilized to investigate the role of micoRNA-223 in differentiating NS/PCs.
MiRNA profiling in human NS/PCs before and after differentiation in vitro reveals modulation of miRNAs following differentiation of NS/PCs. MiR-223, a microRNA well characterized as a hematopoietic-specific miRNA was identified. Cell-autonomous inhibition of miR-223 in the adult mouse dentate gyrus NS/PCs led to a significant increase in immature neurons soma size, dendritic tree total length, branch number per neuron and complexity, while neuronal migration in the dentate gyrus remained unaffected. Overexpression of miR-223 decreased dendritic tree total length, branch number and complexity in neurons differentiated from human embryonic stem cells (hESCs). Inhibition of miR-223 enhanced N-methyl-D-aspartate (NMDA) induced calcium influx in human neurons differentiated from NS/PCs.
Taken together, these findings indicate that miR-223 regulates the differentiation of neurons derived from NS/PCs.
Neural stem cells; microRNA; Dendrite development
Myoclonus-dystonia (M-D) is a movement disorder that is often associated with mutations in epsilon-sarcoglycan (SGCE), a maternally imprinted gene at 7q21.3. We report a 24-year-old male with short stature (<5th percentile) and a movement disorder clinically consistent with M-D. SNP array did not identify significant copy number changes, but revealed three long continuous stretches of homozygosity on chromosome 7 suggestive of uniparental disomy. Parental SNP arrays confirmed that the proband had maternal uniparental disomy (mUPD7) with regions of heterodisomy and isodisomy. mUPD7 is the cause of approximately 5–10% of Silver-Russell syndrome (SRS), a disorder characterized by prenatal and postnatal growth retardation. Although SRS was not suspected in our patient, these findings explain his short stature. SGCE methylation testing showed loss of the unmethylated paternal allele. Our findings provide a unifying diagnosis for his short stature and M-D and help to optimize his medication regimen. In conclusion, we show that M-D is a clinical feature that may be associated with SRS due to mUPD7. Individuals with mUPD7 should be monitored for the development of movement disorders. Conversely, individuals with M-D and short stature should be evaluated for SRS.
Epsilon-sarcoglycan; maternal uniparental disomy of chromosome 7; myoclonus-dystonia; Silver-Russell syndrome
Parkinson's disease (PD) is a complex genetic disorder that is associated with environmental risk factors and aging. Vertebrate genetic models, especially mice, have aided the study of autosomal-dominant and autosomal-recessive PD. Mice are capable of showing a broad range of phenotypes and, coupled with their conserved genetic and anatomical structures, provide unparalleled molecular and pathological tools to model human disease. These models used in combination with aging and PD-associated toxins have expanded our understanding of PD pathogenesis. Attempts to refine PD animal models using conditional approaches have yielded in vivo nigrostriatal degeneration that is instructive in ordering pathogenic signaling and in developing therapeutic strategies to cure or halt the disease. Here, we provide an overview of the generation and characterization of transgenic and knockout mice used to study PD followed by a review of the molecular insights that have been gleaned from current PD mouse models. Finally, potential approaches to refine and improve current models are discussed.
Behavioral impairments, α-synuclein aggregation and degeneration of dopamine neurons have been reproduced in some animal models of PD. Additional models that properly recapitulate dopaminergic neurodegeneration are needed.
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.
The defining anatomical feature of Parkinson’s disease (PD) is the degeneration of substantia nigra pars compacta (SNc) neurons, resulting in striatal dopamine (DA) deficiency and in the subsequent alteration of basal ganglia physiology. Treatments targeting the dopaminergic system alleviate PD symptoms but are not able to slow the neurodegenerative process that underlies PD progression. The nucleus striatum comprises a complex network of projecting neurons and interneurons that integrates different neural signals to modulate the activity of the basal ganglia circuitry. In this review we describe new potential molecular and synaptic striatal targets for the development of both symptomatic and neuroprotective strategies for PD. In particular, we focus on the interaction between adenosine A2A receptors and dopamine D2 receptors, on the role of a correct assembly of NMDA receptors, and on the sGC/cGMP/PKG pathway. Moreover, we also discuss the possibility to target the cell death program parthanatos and the kinase LRRK2 in order to develop new putative neuroprotective agents for PD acting on dopaminergic nigral neurons as well as on other basal ganglia structures.
synaptic plasticity; dopamine receptors; NMDA receptors; LRRK2; Parthanatos
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 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.
The recent explosion of interest in microRNAs (miRNAs) in the nervous system has recently expanded to the investigation of their role in neurodegeneration. These studies have begun to reveal the influence of miRNAs on neuronal survival and the accumulation of toxic proteins associated with neurodegeneration as well as providing clues as to how these toxic proteins can influence miRNA expression.
Mutations in Parkin are the second most common known cause of Parkinson’s disease (PD). Parkin is an ubiquitin E3 ligase that monoubiquitinates and polyubiquitinates proteins to regulate a variety of cellular processes. Loss of parkin’s E3 ligase activity is thought to play a pathogenic role in both inherited and sporadic PD. Here we review parkin biology and pathobiology and its role in the pathogenesis of 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
Congenital toxoplasmosis and toxoplasmic encephalitis can be associated with severe neuropsychiatric symptoms. However, which host cell processes are regulated and how Toxoplasma gondii affects these changes remain unclear. MicroRNAs (miRNAs) are small noncoding RNA sequences critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of over 1000 miRNAs in human neuroepithelioma cells in response to infection with Toxoplasma. MiR-132, a cyclic AMP-responsive element binding (CREB)-regulated miRNA, was the only miRNA that was substantially upregulated by all three prototype Toxoplasma strains. The increased expression of miR-132 was also documented in mice following infection with Toxoplasma. To identify cellular pathways regulated by miR-132, we performed target prediction followed by pathway enrichment analysis in the transcriptome of Toxoplasma-infected mice. This led us to identify 20 genes and dopamine receptor signaling was their strongest associated pathway. We then examined myriad aspects of the dopamine pathway in the striatum of Toxoplasma infected mice 5 days after infection. Here we report decreased expression of D1-like dopamine receptors (DRD1, DRD5), metabolizing enzyme (MAOA) and intracellular proteins associated with the transduction of dopamine-mediated signaling (DARPP-32 phosphorylation at Thr34 and Ser97). Increased concentrations of dopamine and its metabolites, serotonin and 5-hydroxyindoleacetic acid were documented by HPLC analysis; however, the metabolism of dopamine was decreased and serotonin metabolism was unchanged. Our data show that miR-132 is upregulated following infection with Toxoplasma and is associated with changes in dopamine receptor signaling. Our findings provide a possible mechanism for how the parasite contributes to the neuropathology of infection.
Toxoplasma gondii; miR-132; dopamine receptor pathway; alteration in expression; mouse striatum
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.
Botch promotes embryonic neurogenesis through inhibition of Notch-1 signaling through inhibition of the initial S1 furin-like cleavage step of Notch maturation. The biochemical process by which Botch inhibits Notch maturation is not known. Here we show that Botch has γ-glutamyl cyclotransferase (GGCT) activity that deglycinates Notch, which prevents the S1 furin-like cleavage. Moreover, Notch is mono-glycinated on the γ-glutamyl carbon of glutamate 1669. The deglycinase activity of Botch is required for inhibition of Notch signaling, both in vitro and in vivo. When the γ-glutamyl-glycine at position 1669 of Notch is degylcinated it is replaced by 5-oxy-proline. These results reveal that Botch regulates Notch signaling through deglycination and identify a novel posttranslational modification of Notch that plays an important role in neurogenesis.
c-Abl is activated in the brain of Parkinson's disease (PD) patients and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice where it inhibits parkin through tyrosine phosphorylation leading to the accumulation of parkin substrates, and neuronal cell death. In the present study, we evaluated the in vivo efficacy of nilotinib, a brain penetrant c-Abl inhibitor, in the acute MPTP-induced model of PD. Our results show that administration of nilotinib reduces c-Abl activation and the levels of the parkin substrate, PARIS, resulting in prevention of dopamine (DA) neuron loss and behavioral deficits following MPTP intoxication. On the other hand, we observe no reduction in the tyrosine phosphorylation of parkin and the parkin substrate, AIMP2 suggesting that the protective effect of nilotinib may, in part, be parkin-independent or to the pharmacodynamics properties of nilotinib. This study provides a strong rationale for testing other brain permeable c-Abl inhibitors as potential therapeutic agents for the treatment of PD.
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.
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.