Development of phospho-specific Rab protein antibodies to monitor in vivo activity of the LRRK2 Parkinson's disease kinase

Mutations that activate the LRRK2 (leucine-rich repeat protein kinase 2) protein kinase predispose to Parkinson's disease, suggesting that LRRK2 inhibitors might have therapeutic benefit. Recent work has revealed that LRRK2 phosphorylates a subgroup of 14 Rab proteins, including Rab10, at a specific residue located at the centre of its effector-binding switch-II motif. In the present study, we analyse the selectivity and sensitivity of polyclonal and monoclonal phospho-specific antibodies raised against nine different LRRK2-phosphorylated Rab proteins (Rab3A/3B/3C/3D, Rab5A/5B/5C, Rab8A/8B, Rab10, Rab12, Rab29[T71], Rab29[S72], Rab35 and Rab43). We identify rabbit monoclonal phospho-specific antibodies (MJFF-pRAB10) that are exquisitely selective for LRRK2-phosphorylated Rab10, detecting endogenous phosphorylated Rab10 in all analysed cell lines and tissues, including human brain cingulate cortex. We demonstrate that the MJFF-pRAB10 antibodies can be deployed to assess enhanced Rab10 phosphorylation resulting from pathogenic (R1441C/G or G2019S) LRRK2 knock-in mutations as well as the impact of LRRK2 inhibitor treatment. We also identify rabbit monoclonal antibodies displaying broad specificity (MJFF-pRAB8) that can be utilised to assess LRRK2-controlled phosphorylation of a range of endogenous Rab proteins, including Rab8A, Rab10 and Rab35. The antibodies described in the present study will help with the assessment of LRRK2 activity and examination of which Rab proteins are phosphorylated in vivo. These antibodies could also be used to assess the impact of LRRK2 inhibitors in future clinical trials.

Download Full-text

Rab27 GTPases regulate alpha-synuclein uptake, cell-to-cell transmission, and toxicity

10.1101/2020.11.17.387449 ◽

2020 ◽

Author(s):

Rachel Underwood ◽

Bing Wang ◽

Aneesh Pathak ◽

Laura Volpicelli-Daley ◽

Talene A. Yacoubian

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Lewy Bodies ◽

Neuronal Loss ◽

Alpha Synuclein ◽

Rab Gtpases ◽

Double Knockout ◽

The Impact

SUMMARYParkinson’s disease and Dementia with Lewy Bodies are two common neurodegenerative disorders marked by proteinaceous aggregates composed primarily of the protein α-synuclein. α-Synuclein is hypothesized to have prion-like properties, by which misfolded α-synuclein induces the pathological aggregation of endogenous α-synuclein and neuronal loss. Rab27a and Rab27b are two highly homologous Rab GTPases that regulate α-synuclein secretion, clearance, and toxicity in vitro. In this study, we tested the impact of Rab27a/b on the transmission of pathogenic α-synuclein. Double knockout of both Rab27 isoforms eliminated α-synuclein aggregation and neuronal toxicity in primary cultured neurons exposed to fibrillary α-synuclein. In vivo, Rab27 double knockout mice lacked fibril-induced α-synuclein inclusions, dopaminergic neuron loss, and behavioral deficits seen in wildtype mice with fibril-induced inclusions. Studies using AlexaFluor488-labeled α-synuclein fibrils revealed that Rab27a/b knockout prevented α-synuclein internalization without affecting bulk endocytosis. Rab27a/b knockout also blocked the cell-to-cell spread of α-synuclein pathology in multifluidic, multichambered devices. This study provides critical insight into the role of Rab GTPases in Parkinson’s disease and identifies Rab27s as key players in the progression of synucleinopathies.

Download Full-text

Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin

10.1101/235077 ◽

2018 ◽

Cited By ~ 2

Author(s):

Juliette J. Lee ◽

Alvaro Sanchez-Martinez ◽

Aitor Martinez Zarate ◽

Cristiane Benincá ◽

Ugo Mayor ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Dopaminergic Neurons ◽

Flight Muscle ◽

Substantial Impact ◽

Physiological Conditions ◽

The Impact ◽

Dopaminergic Neuron Loss

AbstractParkinson’s disease factors, PINK1 and parkin, are strongly implicated in stress-induced mitophagy in vitro, but little is known about their impact on basal mitophagy in vivo. We generated transgenic Drosophila expressing fluorescent mitophagy reporters to evaluate the impact of Pink1/parkin mutations on basal mitophagy under physiological conditions. We find that mitophagy is readily detectable and abundant in many tissues including Parkinson’s disease relevant dopaminergic neurons. However, we did not detect mitolysosomes in flight muscle. Surprisingly, in Pink1 or parkin null flies we did not observe any substantial impact on basal mitophagy. As these flies exhibit locomotor defects and dopaminergic neuron loss, our findings raise questions about current assumptions of the pathogenic mechanism associated with the PINK1/Parkin pathway. Our findings provide evidence that Pink1 and parkin are not essential for bulk basal mitophagy in Drosophila. They also emphasize that mechanisms underpinning basal mitophagy remain largely obscure.SummaryPINK1/parkin are key mediators of stress-induced mitophagy in vitro but their impact on basal mitophagy in vivo is unclear. Novel Drosophila reporters lines reveal abundant mitophagy in many tissues including dopaminergic neurons but is unaffected by loss of PINK1/parkin.

Download Full-text

Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin

The Journal of Cell Biology ◽

10.1083/jcb.201801044 ◽

2018 ◽

Vol 217 (5) ◽

pp. 1613-1622 ◽

Cited By ~ 94

Author(s):

Juliette J. Lee ◽

Alvaro Sanchez-Martinez ◽

Aitor Martinez Zarate ◽

Cristiane Benincá ◽

Ugo Mayor ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Dopaminergic Neurons ◽

Substantial Impact ◽

Physiological Conditions ◽

The Impact ◽

Dopaminergic Neuron Loss ◽

Transgenic Drosophila

The Parkinson’s disease factors PINK1 and parkin are strongly implicated in stress-induced mitophagy in vitro, but little is known about their impact on basal mitophagy in vivo. We generated transgenic Drosophila melanogaster expressing fluorescent mitophagy reporters to evaluate the impact of Pink1/parkin mutations on basal mitophagy under physiological conditions. We find that mitophagy is readily detectable and abundant in many tissues, including Parkinson’s disease–relevant dopaminergic neurons. However, we did not detect mitolysosomes in flight muscle. Surprisingly, in Pink1 or parkin null flies, we did not observe any substantial impact on basal mitophagy. Because these flies exhibit locomotor defects and dopaminergic neuron loss, our findings raise questions about current assumptions of the pathogenic mechanism associated with the PINK1/parkin pathway. Our findings provide evidence that Pink1 and parkin are not essential for bulk basal mitophagy in Drosophila. They also emphasize that mechanisms underpinning basal mitophagy remain largely obscure.

Download Full-text

The Parkinson's disease VPS35[D620N] mutation enhances LRRK2-mediated Rab protein phosphorylation in mouse and human

Biochemical Journal ◽

10.1042/bcj20180248 ◽

2018 ◽

Vol 475 (11) ◽

pp. 1861-1883 ◽

Cited By ~ 57

Author(s):

Rafeeq Mir ◽

Francesca Tonelli ◽

Pawel Lis ◽

Thomas Macartney ◽

Nicole K. Polinski ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Rab Proteins ◽

Missense Mutations ◽

Knock Out ◽

Rab Protein ◽

Retromer Complex ◽

Pathogenic Mutations ◽

Healthy Donors ◽

Mouse Tissues

Missense mutations in the LRRK2 (Leucine-rich repeat protein kinase-2) and VPS35 genes result in autosomal dominant Parkinson's disease. The VPS35 gene encodes for the cargo-binding component of the retromer complex, while LRRK2 modulates vesicular trafficking by phosphorylating a subgroup of Rab proteins. Pathogenic mutations in LRRK2 increase its kinase activity. It is not known how the only thus far described pathogenic VPS35 mutation, [p.D620N] exerts its effects. We reveal that the VPS35[D620N] knock-in mutation strikingly elevates LRRK2-mediated phosphorylation of Rab8A, Rab10, and Rab12 in mouse embryonic fibroblasts. The VPS35[D620N] mutation also increases Rab10 phosphorylation in mouse tissues (the lung, kidney, spleen, and brain). Furthermore, LRRK2-mediated Rab10 phosphorylation is increased in neutrophils as well as monocytes isolated from three Parkinson's patients with a heterozygous VPS35[D620N] mutation compared with healthy donors and idiopathic Parkinson's patients. LRRK2-mediated Rab10 phosphorylation is significantly suppressed by knock-out or knock-down of VPS35 in wild-type, LRRK2[R1441C], or VPS35[D620N] cells. Finally, VPS35[D620N] mutation promotes Rab10 phosphorylation more potently than LRRK2 pathogenic mutations. Available data suggest that Parkinson's patients with VPS35[D620N] develop the disease at a younger age than those with LRRK2 mutations. Our observations indicate that VPS35 controls LRRK2 activity and that the VPS35[D620N] mutation results in a gain of function, potentially causing PD through hyperactivation of the LRRK2 kinase. Our findings suggest that it may be possible to elaborate compounds that target the retromer complex to suppress LRRK2 activity. Moreover, patients with VPS35[D620N] associated Parkinson's might benefit from LRRK2 inhibitor treatment that have entered clinical trials in humans.

Download Full-text

Nervonic acid amends motor disorder in a mouse model of Parkinson’s disease

Translational Neuroscience ◽

10.1515/tnsci-2020-0171 ◽

2021 ◽

Vol 12 (1) ◽

pp. 237-246

Author(s):

Dandong Hu ◽

Yujuan Cui ◽

Ji Zhang

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Tyrosine Hydroxylase ◽

Mouse Model ◽

Nervonic Acid ◽

Motor Disorder ◽

Behavioral Deficits ◽

Liver And Kidney ◽

The Impact

Abstract Objectives Parkinson’s disease (PD) is a kind of common neurodegenerative disease in the world. Previous studies have proved that nervonic acid (NA), extracted from Xanthoceras sorbifolia Bunge, has the potentials of neuroprotection. However, the effect of NA on the PD remained unknown. This study was designed to investigate the NA’s potential function and relative mechanism on motor disorder. Methods 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used for producing parkinsonism motor disorder on male C57BL/6 mice. Toxicity experiments and behavioral assay were performed to evaluate the effect of NA. Besides, the expression levels of tyrosine hydroxylase and α-synuclein, as well as striatal dopamine (DA), serotonin, and their metabolites were explored through immunoblotting and chromatography after NA treatment in vivo. Results We found that NA could alleviate the MPTP-induced behavioral deficits dose-dependently. Moreover, NA has no toxic effects on the mouse liver and kidney. Of note, we found that NA significantly reduced the impact of MPTP impairment and striatal DA, serotonin, and metabolites were remained unaffected. In addition, tyrosine hydroxylase was upregulated while α-synuclein being downregulated and the oxidative stress was partially repressed evidenced by the upregulation of superoxide dismutase and glutathione activity after NA treatment. Conclusion Our findings unveil NA’s potential for protecting motor system against motor disorder in the PD mouse model without any side effects, indicating NA as an alternative strategy for PD symptom remission.

Download Full-text

MiR-30a-5p regulates GLT-1 function via a PKCα-mediated ubiquitin degradation pathway in a mouse model of Parkinson’s disease

10.21203/rs.2.24022/v1 ◽

2020 ◽

Author(s):

Xingjun Meng ◽

Jianping Zhong ◽

Chong Zeng ◽

Ken Kin Lam Yung ◽

Xiuping Zhang ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Mouse Model ◽

Cell Model ◽

Glutamate Transporters ◽

Acid Uptake ◽

Dependent Manner ◽

The Impact

Abstract Background:Glutamate excitotoxicity caused by dysfunctional glutamate transporters plays an important role in the pathogenesis of Parkinson’s disease (PD); however, the mechanisms that underlie the regulation of glutamate transporters in PD are still not fully elucidated. MicroRNAs have been reported to play key roles in regulating the translation of glutamate-transporter mRNA. Methods: We established model of PD 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice in vivo and 1-methyl-4-phenylpyridinium (MPP+) treated astrocyte in vitro. Stereotaxic injection of shRNA in mouse, and miRNA inhibitor/mimic, or antagonist/agonist treated the cell model, Behavioral experiments, glutamic acid uptake, transport activity of synaptosomes, underlying mechanisms and the impact on neuronal survival were assessed.Results We demonstrated that short-hairpin RNA-mediated knockdown of miR-30a-5p ameliorated motor deficits and pathological changes like astrogliosis and reactive microgliosis in a mouse model of PD. Western blotting and immunofluorescent labeling revealed that miR-30a-5p suppressed the expression and function of GLT-1 in MPTP-treated mice and specifically in astrocytes treated with (cell model of PD). Conclusion Both in vitro and in vivo, we found that miR-30a-5p knockdown promoted glutamate uptake and increased GLT-1 expression by hindering GLT-1 ubiquitination and subsequent degradation in a PKCα-dependent manner. Therefore, miR-30a-5p represents a potential therapeutic target for the treatment of PD.

Download Full-text

Neural stem cells restore cognitive function in Parkinsonian macaques

10.21203/rs.3.rs-137453/v1 ◽

2021 ◽

Author(s):

Julien Vezoli ◽

Florence Wianny ◽

Kwamivi Dzahini ◽

Karim Fifel ◽

Charles Wilson ◽

...

Keyword(s):

Parkinson’S Disease ◽

Stem Cells ◽

Parkinson's Disease ◽

Neural Stem Cells ◽

Functional Recovery ◽

Therapeutic Potential ◽

Motor Symptoms ◽

The Impact ◽

Non Motor Symptoms

Abstract Cognitive deficits as well as disorders of sleep and biological rhythms constitute non-motor symptoms that significantly impact quality of life in Parkinson’s disease patients. Few studies have evaluated the impact of cell replacement therapy on such non-motor symptoms. Here we used a multidisciplinary approach to assess the therapeutic potential of bilateral grafts of neural stem cells in a macaque model of Parkinson’s disease on both motor and non-motor markers of functional recovery. Grafts led to varying degrees of functional recovery while sham experiments did not. We show unprecedented recovery from cognitive symptoms in addition to a clear clinical motor recuperation. Motor and cognitive recovery but not circadian rhythm recovery correlated with the degree of graft integration into the host environment and with in-vivo levels of striatal dopaminergic transporters and function. This study provides empirical evidence that neural stem cells transplantation efficiently restore function at multiple levels in Parkinsonian non-human primates. We demonstrate the promising potential of multiple-sites neural stem cells grafts for Parkinson’s disease but furthermore underline the crucial importance of such multidisciplinary approaches for an effective clinical translation.

Download Full-text

In vivo multimodal (MRI, SPECT) imaging of the 6-OHDA rat model for Parkinson's disease correlated with behavior and histology

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/sj.jcbfm.9591524.0392 ◽

2005 ◽

Vol 25 (1_suppl) ◽

pp. S392-S392

Author(s):

Nadja Van Camp ◽

Koen Van Laere ◽

Ruth Vreys ◽

Marleen Verhoye ◽

Erwin Lauwers ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Rat Model ◽

Spect Imaging ◽

Multimodal Mri

Download Full-text

Coexistent Osteoarthritis and Parkinson’s Disease: Data from the Parkinson’s Foundation Outcomes Project

Journal of Parkinson s Disease ◽

10.3233/jpd-202170 ◽

2020 ◽

Vol 10 (4) ◽

pp. 1601-1610

Author(s):

Jaimie A. Roper ◽

Abigail C. Schmitt ◽

Hanzhi Gao ◽

Ying He ◽

Samuel Wu ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Study Data ◽

Functional Mobility ◽

Quality Improvement Initiative ◽

Timed Up And Go ◽

Mobility Performance ◽

Risk Of Mortality ◽

The Impact

Background: The impact of concurrent osteoarthritis on mobility and mortality in individuals with Parkinson’s disease is unknown. Objective: We sought to understand to what extent osteoarthritis severity influenced mobility across time and how osteoarthritis severity could affect mortality in individuals with Parkinson’s disease. Methods: In a retrospective observational longitudinal study, data from the Parkinson’s Foundation Quality Improvement Initiative was analyzed. We included 2,274 persons with Parkinson’s disease. The main outcomes were the effects of osteoarthritis severity on functional mobility and mortality. The Timed Up and Go test measured functional mobility performance. Mortality was measured as the osteoarthritis group effect on survival time in years. Results: More individuals with symptomatic osteoarthritis reported at least monthly falls compared to the other groups (14.5% vs. 7.2% without reported osteoarthritis and 8.4% asymptomatic/minimal osteoarthritis, p = 0.0004). The symptomatic group contained significantly more individuals with low functional mobility (TUG≥12 seconds) at baseline (51.5% vs. 29.0% and 36.1%, p < 0.0001). The odds of having low functional mobility for individuals with symptomatic osteoarthritis was 1.63 times compared to those without reported osteoarthritis (p < 0.0004); and was 1.57 times compared to those with asymptomatic/minimal osteoarthritis (p = 0.0026) after controlling pre-specified covariates. Similar results hold at the time of follow-up while changes in functional mobility were not significant across groups, suggesting that osteoarthritis likely does not accelerate the changes in functional mobility across time. Coexisting symptomatic osteoarthritis and Parkinson’s disease seem to additively increase the risk of mortality (p = 0.007). Conclusion: Our results highlight the impact and potential additive effects of symptomatic osteoarthritis in persons with Parkinson’s disease.

Download Full-text

Pharmacological treatment with L-DOPA may reduce striatal dopamine transporter binding in in vivo imaging studies

Nuklearmedizin ◽

10.3413/nukmed-0764-15-08 ◽

2016 ◽

Vol 55 (01) ◽

pp. 21-28 ◽

Cited By ~ 4

Author(s):

C. Antke ◽

H. Hautzel ◽

H.-W. Mueller ◽

S. Nikolaus

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Binding Sites ◽

Human Subjects ◽

Synaptic Cleft ◽

Presynaptic Terminal ◽

Imaging Studies ◽

Psychiatric Conditions ◽

Da Release

SummaryNumerous neurologic and psychiatric conditions are treated with pharmacological compounds, which lead to an increase of synaptic dopamine (DA) levels. One example is the DA precursor L-3,4-dihydroxyphenylalanine (L-DOPA), which is converted to DA in the presynaptic terminal. If the increase of DA concentrations in the synaptic cleft leads to competition with exogenous radioligands for presynaptic binding sites, this may have implications for DA transporter (DAT) imaging studies in patients under DAergic medication.This paper gives an overview on those findings, which, so far, have been obtained on DAT binding in human Parkinson’s disease after treatment with L-DOPA. Findings, moreover, are related to results obtained on rats, mice or non-human primates. Results indicate that DAT imaging may be reduced in the striata of healthy animals, in the unlesioned striata of animal models of unilateral Parkinson’s disease and in less severly impaired striata of Parkinsonian patients, if animal or human subjects are under acute or subchronic treatment with L-DOPA. If also striatal DAT binding is susceptible to alterations of synaptic DA levels, this may allow to quantify DA reuptake in analogy to DA release by assessing the competition between endogenous DA and the administered exogenous DAT radioligand.

Download Full-text