scholarly journals Neuron-autonomous susceptibility to induced synuclein aggregation is exacerbated by endogenous Lrrk2 mutations and ameliorated by Lrrk2 genetic knock-out

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Sarah MacIsaac ◽  
Thaiany Quevedo Melo ◽  
Yuting Zhang ◽  
Mattia Volta ◽  
Matthew J Farrer ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Therapeutic Strategy ◽  
Germ Line ◽  
Lewy Bodies ◽  
Neuronal Cultures ◽  
Leucine Rich Repeat ◽  
Kinase Inhibition ◽  
Knock Out

Abstract Neuronal aggregates containing α-synuclein are a pathological hallmark of several degenerative diseases; including Parkinson’s disease, Parkinson’s disease with dementia and dementia with Lewy bodies. Understanding the process of α-synuclein aggregation, and discovering means of preventing it, may help guide therapeutic strategy and drug design. Recent advances provide tools to induce α-synuclein aggregation in neuronal cultures. Application of exogenous pre-formed fibrillar α-synuclein induces pathological phosphorylation and accumulation of endogenous α-synuclein, typical of that seen in disease. Genomic variability and mutations in α-synuclein and leucine-rich repeat kinase 2 proteins are the major genetic risk factors for Parkinson’s disease. Reports demonstrate fibril-induced α-synuclein aggregation is increased in cells from leucine-rich repeat kinase 2 pathogenic mutant (G2019S) overexpressing mice, and variously decreased by leucine-rich repeat kinase 2 inhibitors. Elsewhere in vivo antisense knock-down of leucine-rich repeat kinase 2 protein has been shown to protect mice from fibril-induced α-synuclein aggregation, whereas kinase inhibition did not. To help bring clarity to this issue, we took a purely genetic approach in a standardized neuron-enriched culture, lacking glia. We compared fibril treatment of leucine-rich repeat kinase 2 germ-line knock-out, and G2019S germ-line knock-in, mouse cortical neuron cultures with those from littermates. We found leucine-rich repeat kinase 2 knock-out neurons are resistant to α-synuclein aggregation, which predominantly forms within axons, and may cause axonal fragmentation. Conversely, leucine-rich repeat kinase 2 knock-in neurons are more vulnerable to fibril-induced α-synuclein accumulation. Protection and resistance correlated with basal increases in a lysosome marker in knock-out, and an autophagy marker in knock-in cultures. The data add to a growing number of studies that argue leucine-rich repeat kinase 2 silencing, and potentially kinase inhibition, may be a useful therapeutic strategy against synucleinopathy.

scholarly journals Ablation of RIP3 protects from dopaminergic neurodegeneration in experimental Parkinson’s disease

2019 ◽  
Vol 10 (11) ◽  
Author(s):  
Pedro A. Dionísio ◽  
Sara R. Oliveira ◽  
Maria M. Gaspar ◽  
Maria J. Gama ◽  
Margarida Castro-Caldas ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Death ◽  
Cultured Cells ◽  
Substantia Nigra Pars Compacta ◽  
Neuronal Cultures ◽  
Interacting Protein ◽  
Dopaminergic Neurodegeneration

Abstract Parkinson’s disease (PD) is driven by dopaminergic neurodegeneration in the substantia nigra pars compacta (SN) and striatum. Although apoptosis is considered the main neurodegenerative mechanism, other cell death pathways may be involved. In this regard, necroptosis is a regulated form of cell death dependent on receptor interacting protein 3 (RIP3), a protein also implicated in apoptosis and inflammation independently of its pro-necroptotic activity. Here, we explored the role of RIP3 genetic deletion in in vivo and in vitro PD models. Firstly, wild-type (Wt) and RIP3 knockout (RIP3ko) mice were injected intraperitoneally with MPTP (40 mg/kg, i.p.), and sacrificed after either 6 or 30 days. RIP3ko protected from dopaminergic neurodegeneration in the SN of MPTP-injected mice, but this effect was independent of necroptosis. In keeping with this, necrostatin-1s (10 mg/kg/day, i.p.) did not afford full neuroprotection. Moreover, MPTP led to DNA fragmentation, caspase-3 activation, lipid peroxidation and BAX expression in Wt mice, in the absence of caspase-8 cleavage, suggesting intrinsic apoptosis. This was mimicked in primary cortical neuronal cultures exposed to the active MPTP metabolite. RIP3 deficiency in cultured cells and in mouse brain abrogated all phenotypes. Curiously, astrogliosis was increased in the striatum of MPTP-injected Wt mice and further exacerbated in RIP3ko mice. This was accompanied by absence of microgliosis and reposition of glial cell line-derived neurotrophic factor (GDNF) levels in the striata of MPTP-injected RIP3ko mice when compared to MPTP-injected Wt mice, which in turn showed a massive GDNF decrease. RIP3ko primary mixed glial cultures also presented decreased expression of inflammation-related genes upon inflammatory stimulation. These findings hint at possible undescribed non-necroptotic roles for RIP3 in inflammation and MPTP-driven cell death, which can contribute to PD progression.

Genetic analysis of Parkinson's disease-linked leucine-rich repeat kinase 2

2012 ◽  
Vol 40 (5) ◽  
pp. 1042-1046 ◽  
Author(s):  
Youren Tong ◽  
Jie Shen
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Oxidative Damage ◽  
Inflammatory Responses ◽  
Dopamine D2 Receptor ◽  
Apoptotic Cell ◽  
Protein Carbonyls ◽  
Mutant Form ◽  
Leucine Rich Repeat

Mutations in LRRK2 (leucine-rich repeat kinase 2) are the most common genetic cause of PD (Parkinson's disease). To investigate how mutations in LRRK2 cause PD, we generated LRRK2 mutant mice either lacking its expression or expressing the R1441C mutant form. Homozygous R1441C knockin mice exhibit no dopaminergic neurodegeneration or alterations in steady-state levels of striatal dopamine, but they show impaired dopamine neurotransmission, as was evident from reductions in amphetamine-induced locomotor activity and stimulated catecholamine release in cultured chromaffin cells as well as impaired dopamine D2 receptor-mediated functions. Whereas LRRK2−/− brains are normal, LRRK2−/− kidneys at 20 months of age develop striking accumulation and aggregation of α-synuclein and ubiquitinated proteins, impairment of the autophagy–lysosomal pathway, and increases in apoptotic cell death, inflammatory responses and oxidative damage. Our further analysis of LRRK2−/− kidneys at multiple ages revealed unique age-dependent biphasic alterations of the autophagic activity, which is unchanged at 1 month of age, enhanced at 7 months, but reduced at 20 months. Levels of α-synuclein and protein carbonyls, a general oxidative damage marker, are also decreased in LRRK2−/− kidneys at 7 months of age. Interestingly, this biphasic alteration is associated with increased levels of lysosomal proteins and proteases as well as progressive accumulation of autolysosomes and lipofuscin granules. We conclude that pathogenic mutations in LRRK2 impair the nigrostriatal dopaminergic pathway, and LRRK2 plays an essential role in the dynamic regulation of autophagy function in vivo.

scholarly journals Couldα-Synuclein Amyloid-Like Aggregates Trigger a Prionic Neuronal Invasion?

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Maria Antònia Busquets ◽  
Alba Espargaró ◽  
Joan Estelrich ◽  
Raimon Sabate
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Neurodegenerative Disease ◽  
Prion Protein ◽  
Intracellular Localization ◽  
Lewy Bodies ◽  
Key Factors ◽  
Neuronal Toxicity

Parkinson’s disease (PD), a progressive neurodegenerative disease primarily affecting voluntary and controlled movement, is characterized by abnormal accumulations ofα-synuclein (α-syn) in intraneuronal Lewy bodies. In the last years, the increased number of evidences from both thein vitroandin vivostudies has shown the ability ofα-syn to misfold in amyloid conformations and to spread via neuron-to-neuron transmission, suggesting a prion-like behaviour. However, in contrast to prion protein (PrP),α-syn transmission is far from neuronal invasion. The high neuronal toxicity of both mature fibres and oligomeric species, as well as the intracellular localization of the protein and the difficulty to be secreted, could be key factors impeding the prion ability ofα-syn aggregates.

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

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.

scholarly journals Human loss-of-function variants suggest that partial LRRK2 inhibition is a safe therapeutic strategy for Parkinson’s disease

2019 ◽  
Author(s):  
Nicola Whiffin ◽  
Irina M. Armean ◽  
Aaron Kleinman ◽  
Jamie L. Marshall ◽  
Eric V. Minikel ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Large Scale ◽  
Therapeutic Strategy ◽  
Kinase Activity ◽  
Model Organisms ◽  
Loss Of Function ◽  
Genomic Databases ◽  
Protein Coding

AbstractHuman genetic variants causing loss-of-function (LoF) of protein-coding genes provide natural in vivo models of gene inactivation, which are powerful indicators of gene function and the potential toxicity of therapeutic inhibitors targeting these genes1,2. Gain-of-kinase-function variants in LRRK2 are known to significantly increase the risk of Parkinson’s disease3,4, suggesting that inhibition of LRRK2 kinase activity is a promising therapeutic strategy. Whilst preclinical studies in model organisms have raised some on-target toxicity concerns5–8, the biological consequences of LRRK2 inhibition have not been well characterized in humans. Here we systematically analyse LoF variants in LRRK2 observed across 141,456 individuals sequenced in the Genome Aggregation Database (gnomAD)9 and over 4 million participants in the 23andMe genotyped dataset, to assess their impact at a molecular and phenotypic level. After thorough variant curation, we identify 1,358 individuals with high-confidence predicted LoF variants in LRRK2, several with experimental validation. We show that heterozygous LoF of LRRK2 reduces LRRK2 protein level by ~50% but is not associated with reduced life expectancy, or with any specific phenotype or disease state. These data suggest that therapeutics that downregulate LRRK2 levels or kinase activity by up to 50% are unlikely to have major on-target safety liabilities. Our results demonstrate the value of large scale genomic databases and phenotyping of human LoF carriers for target validation in drug discovery.

scholarly journals Association between CSF alpha-synuclein seeding activity and genetic status in Parkinson’s disease and dementia with Lewy bodies

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Kathrin Brockmann ◽  
Corinne Quadalti ◽  
Stefanie Lerche ◽  
Marcello Rossi ◽  
Isabel Wurster ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Lewy Body ◽  
Dementia With Lewy Bodies ◽  
Lewy Bodies ◽  
High Sensitivity ◽  
Alpha Synuclein ◽  
Genetic Status ◽  
Recessive Genes

AbstractThe clinicopathological heterogeneity in Lewy-body diseases (LBD) highlights the need for pathology-driven biomarkers in-vivo. Misfolded alpha-synuclein (α-Syn) is a lead candidate based on its crucial role in disease pathophysiology. Real-time quaking-induced conversion (RT-QuIC) analysis of CSF has recently shown high sensitivity and specificity for the detection of misfolded α-Syn in patients with Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In this study we performed the CSF RT-QuIC assay in 236 PD and 49 DLB patients enriched for different genetic forms with mutations in GBA, parkin, PINK1, DJ1, and LRRK2. A subgroup of 100 PD patients was also analysed longitudinally. We correlated kinetic seeding parameters of RT-QuIC with genetic status and CSF protein levels of molecular pathways linked to α-Syn proteostasis. Overall, 85% of PD and 86% of DLB patients showed positive RT-QuIC α-Syn seeding activity. Seeding profiles were significantly associated with mutation status across the spectrum of genetic LBD. In PD patients, we detected positive α-Syn seeding in 93% of patients carrying severe GBA mutations, in 78% with LRRK2 mutations, in 59% carrying heterozygous mutations in recessive genes, and in none of those with bi-allelic mutations in recessive genes. Among PD patients, those with severe GBA mutations showed the highest seeding activity based on RT-QuIC kinetic parameters and the highest proportion of samples with 4 out of 4 positive replicates. In DLB patients, 100% with GBA mutations showed positive α-Syn seeding compared to 79% of wildtype DLB. Moreover, we found an association between α-Syn seeding activity and reduced CSF levels of proteins linked to α-Syn proteostasis, specifically lysosome-associated membrane glycoprotein 2 and neurosecretory protein VGF.These findings highlight the value of α-Syn seeding activity as an in-vivo marker of Lewy-body pathology and support its use for patient stratification in clinical trials targeting α-Syn.

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