ABSTRACT
Haloperidol is used to manage psychotic symptoms in several neurological disorders through mechanisms that involve antagonism of dopamine D2 receptors that are highly expressed in the striatum. Significant side effects of haloperidol, known as extrapyramidal symptoms, lead to motor deficits similar to those seen in Parkinson's disease and present a major challenge in clinical settings. The underlying molecular mechanisms responsible for these side effects remain poorly understood. Parkinson's disease-associated LRRK2 kinase has an important role in striatal physiology and a known link to dopamine D2 receptor signaling. Here, we systematically explore convergent signaling of haloperidol and LRRK2 through pharmacological or genetic inhibition of LRRK2 kinase, as well as knock-in mouse models expressing pathogenic mutant LRRK2 with increased kinase activity. Behavioral assays show that LRRK2 kinase inhibition ameliorates haloperidol-induced motor changes in mice. A combination of electrophysiological and anatomical approaches reveals that LRRK2 kinase inhibition interferes with haloperidol-induced changes, specifically in striatal neurons of the indirect pathway. Proteomic studies and targeted intracellular pathway analyses demonstrate that haloperidol induces a similar pattern of intracellular signaling as increased LRRK2 kinase activity. Our study suggests that LRRK2 kinase plays a key role in striatal dopamine D2 receptor signaling underlying the undesirable motor side effects of haloperidol. This work opens up new therapeutic avenues for dopamine-related disorders, such as psychosis, also furthering our understanding of Parkinson's disease pathophysiology.
ABSTRACT
Several studies have revealed that midbrain dopamine (DA) neurons, even within a single neuroanatomical area, display heterogeneous properties. In parallel, studies using single cell profiling techniques have begun to cluster DA neurons into subtypes based on their molecular signatures. Recent work has shown that molecularly defined DA subtypes within the substantia nigra (SNc) display distinctive anatomic and functional properties, and differential vulnerability in Parkinson's disease (PD). Based on these provocative results, a granular understanding of these putative subtypes and their alterations in PD models, is imperative. We developed an optimized pipeline for single-nuclear RNA sequencing (snRNA-seq) and generated a high-resolution hierarchically organized map revealing 20 molecularly distinct DA neuron subtypes belonging to three main families. We integrated this data with spatial MERFISH technology to map, with high definition, the location of these subtypes in the mouse midbrain, revealing heterogeneity even within neuroanatomical sub-structures. Finally, we demonstrate that in the preclinical LRRK2G2019S knock-in mouse model of PD, subtype organization and proportions are preserved. Transcriptional alterations occur in many subtypes including those localized to the ventral tier SNc, where differential expression is observed in synaptic pathways, which might account for previously described DA release deficits in this model. Our work provides an advancement of current taxonomic schemes of the mouse midbrain DA neuron subtypes, a high-resolution view of their spatial locations, and their alterations in a prodromal mouse model of PD.
ABSTRACT
LRRK2 mutations are closely associated with Parkinson's disease (PD). Convergent evidence suggests that LRRK2 regulates striatal function. Here, by using knock-in mouse lines expressing the two most common LRRK2 pathogenic mutations-G2019S and R1441C-we investigated how LRRK2 mutations altered striatal physiology. While we found that both R1441C and G2019S mice displayed reduced nigrostriatal dopamine release, hypoexcitability in indirect-pathway striatal projection neurons, and alterations associated with an impaired striatal-dependent motor learning were observed only in the R1441C mice. We also showed that increased synaptic PKA activities in the R1441C and not G2019S mice underlie the specific alterations in motor learning deficits in the R1441C mice. In summary, our data argue that LRRK2 mutations' impact on the striatum cannot be simply generalized. Instead, alterations in electrochemical, electrophysiological, molecular, and behavioral levels were distinct between LRRK2 mutations. Our findings offer mechanistic insights for devising and optimizing treatment strategies for PD patients.
Subject(s)
Corpus Striatum , Parkinson Disease , Mice , Animals , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Corpus Striatum/metabolism , Parkinson Disease/pathology , Dopamine , MutationABSTRACT
Mutations in leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease with a similar clinical presentation and progression to idiopathic Parkinson's disease, and common variation is linked to disease risk. Recapitulation of the genotype in rodent models causes abnormal dopamine release and increases the susceptibility of dopaminergic neurons to insults, making LRRK2 a valuable model for understanding the pathobiology of Parkinson's disease. It is also a promising druggable target with targeted therapies currently in development. LRRK2 mRNA and protein expression in the brain is highly variable across regions and cellular identities. A growing body of work has demonstrated that pathogenic LRRK2 mutations disrupt striatal synapses before the onset of overt neurodegeneration. Several substrates and interactors of LRRK2 have been identified to potentially mediate these pre-neurodegenerative changes in a cell-type-specific manner. This review discusses the effects of pathogenic LRRK2 mutations in striatal neurons, including cell-type-specific and pathway-specific alterations. It also highlights several LRRK2 effectors that could mediate the alterations to striatal function, including Rabs and protein kinase A. The lessons learned from improving our understanding of the pathogenic effects of LRRK2 mutations in striatal neurons will be applicable to both dissecting the cell-type specificity of LRRK2 function in the transcriptionally diverse subtypes of dopaminergic neurons and also increasing our understanding of basal ganglia development and biology. Finally, it will inform the development of therapeutics for Parkinson's disease.
Subject(s)
Corpus Striatum/enzymology , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Synapses/enzymology , Amino Acid Sequence , Animals , Disease Models, Animal , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/chemistry , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Models, Biological , Mutation/geneticsABSTRACT
Variations in the LRRK2 gene represent one of the strongest genetic factors for Parkinson's disease (PD). It has become clear that structural knowledge of the encoded large multidomain LRRK2 protein will cast light on its biological function. The new study from Myasnikov, Zhu, et al. provides a high-resolution structure of the full-length LRRK2.
Subject(s)
Parkinson Disease , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/chemistry , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Mutation , Parkinson Disease/genetics , Parkinson Disease/metabolismABSTRACT
OBJECTIVES: Primary nonresponse (PNR) to antitumor necrosis factor-α (TNFα) biologics is a serious concern in patients with inflammatory bowel disease (IBD). We aimed to identify the genetic variants associated with PNR. PATIENTS AND METHODS: Patients were recruited from outpatient GI clinics and PNR was determined using both clinical and endoscopic findings. A case-control genome-wide association study was performed in 589 IBD patients and associations were replicated in an independent cohort of 293 patients. Effect of the associated variant on gene expression and TNFα secretion was assessed by cell-based assays. Pleiotropic effects were investigated by Phenome-wide association study (PheWAS). RESULTS: We identified rs34767465 as associated with PNR to anti-TNFα therapy (odds ratio: 2.07, 95% CI, 1.46-2.94, P = 2.43 × 10-7, [replication odds ratio: 1.8, 95% CI, 1.04-3.16, P = 0.03]). rs34767465 is a multiple-tissue expression quantitative trait loci for FAM114A2. Using RNA-sequencing and protein quantification from HapMap lymphoblastoid cell lines (LCLs), we found a significant decrease in FAM114A2 mRNA and protein expression in both heterozygous and homozygous genotypes when compared to wild type LCLs. TNFα secretion was significantly higher in THP-1 cells [differentiated into macrophages] with FAM114A2 knockdown versus controls. Immunoblotting experiments showed that depletion of FAM114A2 impaired autophagy-related pathway genes suggesting autophagy-mediated TNFα secretion as a potential mechanism. PheWAS showed rs34767465 was associated with comorbid conditions found in IBD patients (derangement of joints [P = 3.7 × 10-4], pigmentary iris degeneration [P = 5.9 × 10-4], diverticulum of esophagus [P = 7 × 10-4]). CONCLUSIONS: We identified a variant rs34767465 associated with PNR to anti-TNFα biologics, which increases TNFα secretion through mechanism related to autophagy. rs34767465 may also explain the comorbidities associated with IBD.
Subject(s)
Genome-Wide Association Study , Inflammatory Bowel Diseases , Case-Control Studies , Cohort Studies , Humans , Inflammatory Bowel Diseases/drug therapy , Inflammatory Bowel Diseases/genetics , Tumor Necrosis Factor-alpha/geneticsABSTRACT
LRRK2 is a kinase expressed in striatal spiny projection neurons (SPNs), cells which lose dopaminergic input in Parkinson's disease (PD). R1441C and G2019S are the most common pathogenic mutations of LRRK2. How these mutations alter the structure and function of individual synapses on direct and indirect pathway SPNs is unknown and may reveal pre-clinical changes in dopamine-recipient neurons that predispose toward disease. Here, R1441C and G2019S knock-in mice enabled thorough evaluation of dendritic spines and synapses on pathway-identified SPNs. Biochemical synaptic preparations and super-resolution imaging revealed increased levels and altered organization of glutamatergic AMPA receptors in LRRK2 mutants. Relatedly, decreased frequency of miniature excitatory post-synaptic currents accompanied changes in dendritic spine nano-architecture, and single-synapse currents, evaluated using two-photon glutamate uncaging. Overall, LRRK2 mutations reshaped synaptic structure and function, an effect exaggerated in R1441C dSPNs. These data open the possibility of new neuroprotective therapies aimed at SPN synapse function, prior to disease onset.
Parkinson's disease is caused by progressive damage to regions of the brain that regulate movement. This leads to a loss in nerve cells that produce a signaling molecule called dopamine, and causes patients to experience shakiness, slow movement and stiffness. When dopamine is released, it travels to a part of the brain known as the striatum, where it is received by cells called spiny projection neurons (SPNs), which are rich in a protein called LRRK2. Mutations in this protein have been shown to cause the motor impairments associated with Parkinson's disease. SPNs send signals to other regions of the brain either via a 'direct' route, which promotes movement, or an 'indirect' route, which suppresses movement. Previous studies suggest that mutations in the gene for LRRK2 influence the activity of these pathways even before dopamine signaling has been lost. Yet, it remained unclear how different mutations independently affected each pathway. To investigate this further, Chen et al. studied two of the mutations most commonly found in the human gene for LRRK2, known as G2019S and R1441C. This involved introducing one of these mutations in to the genetic code of mice, and using fluorescent proteins to mark single SPNs in either the direct or indirect pathway. The experiments showed that both mutations disrupted the connections between SPNs in the direct and indirect pathway, which altered the activity of nerve cells in the striatum. Chen et al. found that individual connections were more strongly affected by the R1441C mutation. Further experiments showed that this was caused by the re-organization of a receptor protein in the nerve cells of the direct pathway, which increased how SPNs responded to inputs from other nerve cells. These findings suggest that LRRK2 mutations disrupt neural activity in the striatum before dopamine levels become depleted. This discovery could help researchers identify new therapies for treating the early stages of Parkinson's disease before the symptoms of dopamine loss arise.
Subject(s)
Corpus Striatum/physiology , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Neural Pathways/physiology , Synapses/metabolism , Animals , Blotting, Western , Dendritic Spines/physiology , Female , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Male , Mice , Mice, Inbred C57BL , Microscopy, Confocal , Synapses/physiologyABSTRACT
Mutations in GBA1 encountered in Gaucher disease are a leading risk factor for Parkinson disease and associated Lewy body disorders. Many GBA1 mutation carriers, especially those with severe or null GBA1 alleles, have earlier and more progressive parkinsonism. To model the effect of partial glucocerebrosidase deficiency on neurological progression in vivo, mice with a human A53T α-synuclein (SNCAA53T) transgene were crossed with heterozygous null gba mice (gba+/-). Survival analysis of 84 mice showed that in gba+/-//SNCAA53T hemizygotes and homozygotes, the symptom onset was significantly earlier than in gba+/+//SNCAA53T mice (p-values 0.023-0.0030), with exacerbated disease progression (p-value <0.0001). Over-expression of SNCAA53T had no effect on glucocerebrosidase levels or activity. Immunoblotting demonstrated that gba haploinsufficiency did not lead to increased levels of either monomeric SNCA or insoluble high molecular weight SNCA in this model. Immunohistochemical analyses demonstrated that the abundance and distribution of SNCA pathology was also unaltered by gba haploinsufficiency. Thus, while the underlying mechanism is not clear, this model shows that gba deficiency impacts the age of onset and disease duration in aged SNCAA53T mice, providing a valuable resource to identify modifiers, pathways and possible moonlighting roles of glucocerebrosidase in Parkinson pathogenesis.
Subject(s)
Gaucher Disease/genetics , Glucosylceramidase/genetics , Haploinsufficiency , Parkinson Disease/genetics , alpha-Synuclein/genetics , Age of Onset , Animals , Brain/metabolism , Disease Models, Animal , Female , Gaucher Disease/complications , Glucosylceramidase/deficiency , Glucosylceramides/analysis , Heterozygote , Humans , Male , Mice , Mice, Knockout , Mice, Transgenic , Mutation , Parkinson Disease/etiology , Psychosine/analogs & derivatives , Psychosine/analysis , Transgenes , alpha-Synuclein/analysis , alpha-Synuclein/deficiency , alpha-Synuclein/metabolism , beta-Glucosidase/deficiency , beta-Glucosidase/geneticsABSTRACT
AIMS: Sirtuins connect energy generation and metabolic stress to the cellular acetylome. Currently, only the mitochondrial sirtuins (SIRT3-5) and SIRT1 have been shown to direct mitochondrial function; however, Aims: NAD-dependent protein deacetylase sirtuin-2 (SIRT2), the primary cytoplasmic sirtuin, is not yet reported to associate with mitochondria. RESULTS: This study revealed a novel physiological function of SIRT2: the regulation of mitochondrial function. First, the acetylation of several metabolic mitochondrial proteins was found to be altered in Sirt2-deficient mice, which was, subsequently, validated by immunoprecipitation experiments in which the acetylated mitochondrial proteins directly interacted with SIRT2. Moreover, immuno-gold electron microscopic images of mouse brains showed that SIRT2 associates with the inner mitochondrial membrane in central nervous system cells. The loss of Sirt2 increased oxidative stress, decreased adenosine triphosphate levels, and altered mitochondrial morphology at the cellular and tissue (i.e., brain) level. Furthermore, the autophagic/mitophagic processes were dysregulated in Sirt2-deficient neurons and mouse embryonic fibroblasts. INNOVATION: For the first time it is shown that SIRT2 directs mitochondrial metabolism. CONCLUSION: Together, these findings support that SIRT2 functions as a mitochondrial sirtuin, as well as a regulator of autophagy/mitophagy to maintain mitochondrial biology, thus facilitating cell survival. Antioxid. Redox Signal. 26, 849-863.
Subject(s)
Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Mitophagy , Sirtuin 2/deficiency , Acetylation , Animals , Cells, Cultured , Humans , Mice , Mice, Knockout , Sirtuin 2/genetics , Sirtuin 2/metabolismABSTRACT
Preferential dysfunction/degeneration of midbrain substantia nigra pars compacta (SNpc) dopaminergic (DA) neurons contributes to the main movement symptoms manifested in Parkinson's disease (PD). Although the Leucine-rich repeat kinase 2 (LRRK2) G2019S missense mutation (LRRK2 G2019S) is the most common causative genetic factor linked to PD, the effects of LRRK2 G2019S on the function and survival of SNpc DA neurons are poorly understood. Using a binary gene expression system, we generated transgenic mice expressing either wild-type human LRRK2 (WT mice) or the LRRK2 G2019S mutation (G2019S mice) selectively in the midbrain DA neurons. Here we show that overexpression of LRRK2 G2019S did not induce overt motor abnormalities or substantial SNpc DA neuron loss. However, the LRRK2 G2019S mutation impaired dopamine homeostasis and release in aged mice. This reduction in dopamine content/release coincided with the degeneration of DA axon terminals and decreased expression of DA neuron-enriched genes tyrosine hydroxylase (TH), vesicular monoamine transporter 2, dopamine transporter and aldehyde dehydrogenase 1. These factors are responsible for dopamine synthesis, transport and degradation, and their expression is regulated by transcription factor paired-like homeodomain 3 (PITX3). Levels of Pitx3 mRNA and protein were similarly decreased in the SNpc DA neurons of aged G2019S mice. Together, these findings suggest that PITX3-dependent transcription regulation could be one of the many potential mechanisms by which LRRK2 G2019S acts in SNpc DA neurons, resulting in downregulation of its downstream target genes critical for dopamine homeostasis and release.
Subject(s)
Dopamine/metabolism , Dopaminergic Neurons/metabolism , Gene Expression Regulation , Mutation, Missense , Parkinson Disease/genetics , Parkinson Disease/metabolism , Protein Serine-Threonine Kinases/genetics , Age Factors , Animals , Behavior, Animal , Disease Models, Animal , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Mesencephalon/metabolism , Mesencephalon/pathology , Mice , Mice, Transgenic , Motor Activity , Nerve Degeneration/genetics , Parkinson Disease/pathology , Substantia Nigra/metabolism , Substantia Nigra/pathology , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Leucine-rich repeat kinase 2 (LRRK2) has been associated with Parkinson's disease (PD) and other disorders. However, its normal physiological functions and pathogenic properties remain elusive. Here we show that LRRK2 regulates the anterograde ER-Golgi transport through anchoring Sec16A at the endoplasmic reticulum exit sites (ERES). LRRK2 interacted and co-localized with Sec16A, a key protein in the formation of ERES. Lrrk2 depletion caused a dispersion of Sec16A from ERES and impaired ER export. In neurons, LRRK2 and Sec16A showed extensive co-localization at the dendritic ERES (dERES) that locally regulate the transport of proteins to the dendritic spines. A loss of Lrrk2 affected the association of Sec16A with dERES and impaired the activity-dependent targeting of glutamate receptors onto the cell/synapse surface. Furthermore, the PD-related LRRK2 R1441C missense mutation in the GTPase domain interfered with the interaction of LRRK2 with Sec16A and also affected ER-Golgi transport, while LRRK2 kinase activity was not required for these functions. Therefore, our findings reveal a new physiological function of LRRK2 in ER-Golgi transport, suggesting ERES dysfunction may contribute to the pathogenesis of PD.
Subject(s)
Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Parkinson Disease/enzymology , Protein Serine-Threonine Kinases/metabolism , Vesicular Transport Proteins/metabolism , Animals , COP-Coated Vesicles/metabolism , Cell Line , Cells, Cultured , Dendritic Spines/metabolism , Gene Expression Regulation , Genes, Reporter , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Mice , Models, Biological , Mutation, Missense , Protein Interaction Mapping , Protein Serine-Threonine Kinases/genetics , Protein Transport , Recombinant Fusion Proteins , Vesicular Transport Proteins/geneticsABSTRACT
Parkinson's disease (PD), the most common degenerative movement disorder, is caused by a preferential loss of midbrain dopaminergic (mDA) neurons. Both α-synuclein (α-syn) missense and multiplication mutations have been linked to PD. However, the underlying intracellular signalling transduction pathways of α-syn-mediated mDA neurodegeneration remain elusive. Here, we show that transgenic expression of PD-related human α-syn A53T missense mutation promoted calcineurin (CN) activity and the subsequent nuclear translocation of nuclear factor of activated T cells (NFATs) in mDA neurons. α-syn enhanced the phosphatase activity of CN in both cell-free assays and cell lines transfected with either human wild-type or A53T α-syn. Furthermore, overexpression of α-syn A53T mutation significantly increased the CN-dependent nuclear import of NFATc3 in the mDA neurons of transgenic mice. More importantly, a pharmacological inhibition of CN by cyclosporine A (CsA) ameliorated the α-syn-induced loss of mDA neurons. These findings demonstrate an active involvement of CN- and NFAT-mediated signalling pathway in α-syn-mediated degeneration of mDA neurons in PD.
Subject(s)
Calcineurin/genetics , Dopaminergic Neurons/metabolism , Mesencephalon/metabolism , NFATC Transcription Factors/genetics , Parkinson Disease/genetics , alpha-Synuclein/genetics , Animals , Apoptosis/drug effects , Calcineurin/metabolism , Calcineurin Inhibitors/pharmacology , Cyclosporine/pharmacology , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/pathology , Gene Expression Regulation , HEK293 Cells , Humans , Mesencephalon/drug effects , Mesencephalon/pathology , Mice , Mice, Transgenic , Mutation , NFATC Transcription Factors/metabolism , Parkinson Disease/drug therapy , Parkinson Disease/metabolism , Parkinson Disease/pathology , Primary Cell Culture , Signal Transduction , alpha-Synuclein/metabolismABSTRACT
Leucine-rich repeat kinase 2 (LRRK2) is enriched in the striatal projection neurons (SPNs). We found that LRRK2 negatively regulates protein kinase A (PKA) activity in the SPNs during synaptogenesis and in response to dopamine receptor Drd1 activation. LRRK2 interacted with PKA regulatory subunit IIß (PKARIIß). A lack of LRRK2 promoted the synaptic translocation of PKA and increased PKA-mediated phosphorylation of actin-disassembling enzyme cofilin and glutamate receptor GluR1, resulting in abnormal synaptogenesis and transmission in the developing SPNs. Furthermore, PKA-dependent phosphorylation of GluR1 was also aberrantly enhanced in the striatum of young and aged Lrrk2(-/-) mice after treatment with a Drd1 agonist. Notably, a Parkinson's disease-related Lrrk2 R1441C missense mutation that impaired the interaction of LRRK2 with PKARIIß also induced excessive PKA activity in the SPNs. Our findings reveal a previously unknown regulatory role for LRRK2 in PKA signaling and suggest a pathogenic mechanism of SPN dysfunction in Parkinson's disease.
Subject(s)
Corpus Striatum/metabolism , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/metabolism , Dendritic Spines/metabolism , Protein Serine-Threonine Kinases/physiology , Receptors, Dopamine D1/metabolism , Synapses/metabolism , Animals , Corpus Striatum/pathology , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/biosynthesis , Dendritic Spines/pathology , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Mice , Mice, Knockout , Mice, Transgenic , Protein Serine-Threonine Kinases/genetics , Receptors, Dopamine D1/agonists , Synapses/pathology , Up-Regulation/geneticsABSTRACT
SIGNIFICANCE: It is a well-established scientific observation that mammalian cells contain fidelity or watchdog proteins that maintain the correct function of cellular organelles. RECENT ADVANCES: Over the past several years, the Sirtuin deacetylase family protein Sirt3 has emerged as a mitochondrial fidelity protein that directs energy generation and regulates reactive oxygen species (ROS) scavenging proteins. Loss of function or genetic mutation of these fidelity proteins has been shown to create a cellular environment that is permissive for the development of cellular damage associated with processes such as aging and carcinogenesis. CRITICAL ISSUES: Mitochondria are the primary organelles that direct oxidative metabolism for the production of ATP; however, this is also a significant source of ROS. Thus, it is reasonable to propose that mitochondria should contain proteins that would signal downstream target molecules and/or ROS scavenger enzymes to maintain mitochondrial and cellular homeostatic poise. It is also reasonable to hypothesize that the mitochondria contain fidelity proteins similar to those found in the nucleus and cytoplasm. We discuss a new role of Sirt3 in the direction of the primary superoxide scavenger protein, manganese superoxide dismutase (MnSOD), and how the acetylation or deacetylation of several specific lysines appears to direct MnSOD enzymatic dismutase activity. FUTURE DIRECTIONS: Aberrant downstream regulation of MnSOD by Sirt3 may be a potential source of cellular damage that accumulates with aging to create a tumor-permissive phenotype. Future studies can explore the role of MnSOD in age-related illness using this new mechanism of enzymatic regulation.
Subject(s)
Aging , Carcinogenesis/metabolism , Sirtuin 3/physiology , Superoxide Dismutase/metabolism , Acetylation , Amino Acid Sequence , Animals , Conserved Sequence , Gene Expression , Gene Expression Regulation , Humans , Oxidative Stress , Protein Processing, Post-Translational , Signal TransductionABSTRACT
Recent genome-wide association studies indicate that a simple alteration of Leucine-rich repeat kinase 2 (LRRK2) gene expression may contribute to the etiology of sporadic Parkinson's disease (PD). However, the expression and regulation of LRRK2 protein in the sporadic PD brains remain to be determined. Here, we found that the expression of LRRK2 protein was enhanced in the sporadic PD patients using the frontal cortex tissue from a set of 16 PD patients and 7 control samples. In contrast, no significant difference was detected in the level of LRRK2 mRNA expression between the control and PD cases, suggesting a potential post-transcriptional modification of the LRRK2 protein expression in the sporadic PD brains. Indeed, it was identified that microRNA-205 (miR-205) suppressed the expression of LRRK2 protein through a conserved-binding site at the 3'-untranslated region (UTR) of LRRK2 gene. Interestingly, miR-205 expression was significantly downregulated in the brains of patients with sporadic PD, showing the enhanced LRRK2 protein levels. Also, in vitro studies in the cell lines and primary neuron cultures further established the role of miR-205 in modulating the expression of LRRK2 protein. In addition, introduction of miR-205 prevented the neurite outgrowth defects in the neurons expressing a PD-related LRRK2 R1441G mutant. Together, these findings suggest that downregulation of miR-205 may contribute to the potential pathogenic elevation of LRRK2 protein in the brains of patients with sporadic PD, while overexpression of miR-205 may provide an applicable therapeutic strategy to suppress the abnormal upregulation of LRRK2 protein in PD.
Subject(s)
MicroRNAs/metabolism , Parkinson Disease/genetics , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Animals , Blotting, Western , Brain/pathology , Dopaminergic Neurons/cytology , Dopaminergic Neurons/metabolism , Down-Regulation , Genetic Markers , Genome-Wide Association Study , HEK293 Cells , HeLa Cells , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Mice , Mice, Transgenic , MicroRNAs/genetics , Molecular Sequence Data , Mutation, Missense , Neurons/cytology , Neurons/metabolism , Parkinson Disease/physiopathology , Prefrontal Cortex/metabolism , Prefrontal Cortex/physiopathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Up-RegulationABSTRACT
α-Synuclein (α-syn) plays a prominent role in the degeneration of midbrain dopaminergic (mDA) neurons in Parkinson's disease (PD). However, only a few studies on α-syn have been performed in the mDA neurons in vivo, which may be attributed to a lack of α-syn transgenic mice that develop PD-like severe degeneration of mDA neurons. To gain mechanistic insights into the α-syn-induced mDA neurodegeneration, we generated a new line of tetracycline-regulated inducible transgenic mice that overexpressed the PD-related α-syn A53T missense mutation in the mDA neurons. Here we show that the mutant mice developed profound motor disabilities and robust mDA neurodegeneration, resembling some key motor and pathological phenotypes of PD. We also systematically examined the subcellular abnormalities that appeared in the mDA neurons of mutant mice and observed a profound decrease of dopamine release, the fragmentation of Golgi apparatus, and the impairments of autophagy/lysosome degradation pathways in these neurons. To further understand the specific molecular events leading to the α-syn-dependent degeneration of mDA neurons, we found that overexpression of α-syn promoted a proteasome-dependent degradation of nuclear receptor-related 1 protein (Nurr1), whereas inhibition of Nurr1 degradation ameliorated the α-syn-induced loss of mDA neurons. Given that Nurr1 plays an essential role in maintaining the normal function and survival of mDA neurons, our studies suggest that the α-syn-mediated suppression of Nurr1 protein expression may contribute to the preferential vulnerability of mDA neurons in the pathogenesis of PD.
Subject(s)
Dopaminergic Neurons/metabolism , Mesencephalon/metabolism , Nerve Degeneration/genetics , Nuclear Receptor Subfamily 4, Group A, Member 2/genetics , Parkinsonian Disorders/genetics , alpha-Synuclein/biosynthesis , alpha-Synuclein/genetics , Animals , Animals, Newborn , Disease Models, Animal , Disease Progression , Dopaminergic Neurons/pathology , Female , HEK293 Cells , Humans , Male , Mesencephalon/pathology , Mesencephalon/physiopathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation, Missense/genetics , Nerve Degeneration/etiology , Nerve Degeneration/pathology , Nuclear Receptor Subfamily 4, Group A, Member 2/antagonists & inhibitors , Parkinsonian Disorders/etiology , Parkinsonian Disorders/pathology , Primary Cell Culture , alpha-Synuclein/physiologyABSTRACT
The mutations in the LRRK2 gene cause clinically typical, late-onset Parkinson disease, strengthening the idea that the familial forms of the disease represent an important tool for the study of the idiopathic forms. Despite the great effort to describe and functionally characterize the LRRK2 gene product, its physiological role remains elusive. In this article, we will discuss along with other references, our recent findings that assigned a critical role of LRRK2 protein on cytosleketal dynamics and how this direction could provide a valuable platform to further appreciate the mechanism underlying LRRK2-mediated pathophysiology of the disease.
ABSTRACT
Leucine-rich repeat kinase 2 (LRRK2) functions as a putative protein kinase of ezrin, radixin, and moesin (ERM) family proteins. A Parkinson's disease-related G2019S substitution in the kinase domain of LRRK2 further enhances the phosphorylation of ERM proteins. The phosphorylated ERM (pERM) proteins are restricted to the filopodia of growing neurites in which they tether filamentous actin (F-actin) to the cytoplasmic membrane and regulate the dynamics of filopodia protrusion. Here, we show that, in cultured neurons derived from LRRK2 G2019S transgenic mice, the number of pERM-positive and F-actin-enriched filopodia was significantly increased, and this correlates with the retardation of neurite outgrowth. Conversely, deletion of LRRK2, which lowered the pERM and F-actin contents in filopodia, promoted neurite outgrowth. Furthermore, inhibition of ERM phosphorylation or actin polymerization rescued the G2019S-dependent neuronal growth defects. These data support a model in which the G2019S mutation of LRRK2 causes a gain-of-function effect that perturbs the homeostasis of pERM and F-actin in sprouting neurites critical for neuronal morphogenesis.
Subject(s)
Actins/metabolism , Cytoskeletal Proteins/metabolism , Cytoskeleton/metabolism , Membrane Proteins/metabolism , Microfilament Proteins/metabolism , Neurogenesis/physiology , Neurons/metabolism , Protein Serine-Threonine Kinases/physiology , Actins/chemistry , Amino Acid Sequence , Animals , Cells, Cultured , Cytoskeletal Proteins/chemistry , Cytoskeleton/chemistry , Enzyme Activation/physiology , Female , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Male , Membrane Proteins/chemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microfilament Proteins/chemistry , Molecular Sequence Data , Neurons/cytology , Phosphorylation/physiology , Substrate Specificity/physiologyABSTRACT
Mutations in alpha-synuclein and Leucine-rich repeat kinase 2 (LRRK2) are linked to autosomal dominant forms of Parkinson's disease (PD). However, little is known about any potential pathophysiological interplay between these two PD-related genes. Here we show in transgenic mice that although overexpression of LRRK2 alone did not cause neurodegeneration, the presence of excess LRRK2 greatly accelerated the progression of neuropathological abnormalities developed in PD-related A53T alpha-synuclein transgenic mice. Moreover, we found that LRRK2 promoted the abnormal aggregation and somatic accumulation of alpha-synuclein in A53T mice, which likely resulted from the impairment of microtubule dynamics, Golgi organization, and the ubiquitin-proteasome pathway. Conversely, genetic ablation of LRRK2 preserved the Golgi structure and suppressed the aggregation and somatic accumulation of alpha-synuclein, and thereby delayed the progression of neuropathology in A53T mice. These findings demonstrate that overexpression of LRRK2 enhances alpha-synuclein-mediated cytotoxicity and suggest inhibition of LRRK2 expression as a potential therapeutic option for ameliorating alpha-synuclein-induced neurodegeneration.
Subject(s)
Brain/metabolism , Nerve Degeneration/metabolism , Parkinson Disease/metabolism , Protein Serine-Threonine Kinases/metabolism , alpha-Synuclein/metabolism , Animals , Brain/physiopathology , Disease Progression , Gene Expression Regulation/genetics , Genetic Predisposition to Disease/genetics , Golgi Apparatus/metabolism , Golgi Apparatus/ultrastructure , Inclusion Bodies/genetics , Inclusion Bodies/metabolism , Inclusion Bodies/pathology , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Mice , Mice, Knockout , Mice, Transgenic , Microtubules/metabolism , Microtubules/ultrastructure , Mutation/genetics , Nerve Degeneration/genetics , Nerve Degeneration/physiopathology , Neurons/metabolism , Neurons/pathology , Parkinson Disease/genetics , Parkinson Disease/physiopathology , Protein Serine-Threonine Kinases/genetics , alpha-Synuclein/geneticsABSTRACT
The study of Amyloid Precursor Protein (APP) processing has been the focus of considerable interest, since it leads to Abeta peptide generation, the main constituent of neuritic plaques found in brains of Alzheimer's disease patients. Therefore, the identification of novel APP binding partners that regulate Abeta peptide production represents a pharmaceutical target aiming at reducing Alphabeta pathology. In this study, we provide evidence that Homer2 and Homer3 but not Homer1 proteins interact specifically with APP. Their expression inhibits APP processing and reduces secretion of Abeta peptides. In addition, they decrease the levels of cell surface APP and inhibit maturation of APP and beta-secretase (BACE1). The effects of Homer2 and Homer3 on APP trafficking to the cell surface and/or on APP and BACE1 maturation could be part of the mechanism by which the expression of these proteins leads to the significant reduction of Abeta peptide production.