Biology subjects

Cookson, M. R.

Publications and source records attributed to Cookson, M. R..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Leucine-Rich Repeat Kinase 2 limits dopamine D1 receptor signaling in the striatum and biases against heavy persistent alcohol drinking

The transition from hedonic alcohol drinking to problematic drinking is a hallmark of alcohol use disorder that occurs only in a subset of drinkers. This transition is known to require long-lasting changes in the synaptic drive and the activity of striatal neurons expressing dopamine D1 receptor (D1R). The molecular mechanisms that generate vulnerability in some individuals to undergo the transition are less understood. Here, we report that the Parkinsons-related protein leucine-rich repeat kinase 2 (LRRK2) modulates striatal D1R function to affect the behavioral response to alcohol and the likelihood that mice transition to heavy, persistent alcohol drinking. Deletion of the Lrrk2 gene specifically from D1R-expressing neurons potentiates D1R signaling at the cellular and synaptic level, enhancing alcohol-related behaviors and drinking. Mice with cell-specific deletion of Lrrk2 are more prone to heavy alcohol drinking and consumption is insensitive to punishment. These findings identify a novel role for LRRK2 function in the striatum in promoting resilience against heavy and persistent alcohol drinking.

neuroscience↗

bioRxiv2022

DJ-1 glyoxalase activity makes a modest contribution to cellular defense against methylglyoxal damage in neurons

Human DJ-1 is a cytoprotective protein whose absence causes Parkinsons disease and is also associated with other diseases. DJ-1 has an established role as a redox-regulated protein that defends against oxidative stress and mitochondrial dysfunction. Multiple studies have suggested that DJ-1 is also a protein/nucleic acid deglycase that plays a key role in the repair of glycation damage caused by methylglyoxal (MG), a reactive -keto aldehyde formed by central metabolism. Contradictory reports suggest that DJ-1 is a glyoxalase but not a deglycase and does not play a major role in glycation defense. Resolving this issue is important for understanding how DJ-1 protects cells against insults that can cause disease. We find that DJ-1 reduces levels of reversible adducts of MG with guanine and cysteine in vitro. The steady-state kinetics of DJ-1 acting on reversible hemithioacetal substrates are fitted adequately with a computational kinetic model that requires only a DJ-1 glyoxalase activity, supporting the conclusion that deglycation is an apparent rather than a true activity of DJ-1. Sensitive and quantitative isotope-dilution mass spectrometry shows that DJ-1 modestly reduces the levels of some irreversible guanine and lysine glycation products in primary and cultured neuronal cell lines and whole mouse brain, consistent with a small but measurable effect on total neuronal glycation burden. However, DJ-1 does not improve cultured cell viability in exogenous MG. In total, our results suggest that DJ-1 is not a deglycase and has only a minor role in protecting neurons against methylglyoxal toxicity.

biochemistry↗

bioRxiv2021

Controlling homology-directed repair outcomes in human stem cells with dCas9

Modeling human disease in human stem cells requires precise, scarless editing of single nucleotide variants (SNV) on one or both chromosomes. Here we describe improved conditions for Cas9 RNP editing of SNVs that yield high rates of biallelic homology-directed repair. To recover both heterozygous and homozygous SNV clones, catalytically inactive dCas9was added to moderate high activity Cas9 RNPs. dCas9 can also block re-cutting and damage to SNV alleles engineered with non-overlapping guide RNAs.

genetics↗

bioRxiv2021

A fully automated FAIMS-DIA proteomic pipeline for high-throughput characterization of iPSC-derived neurons

Fully automated proteomic pipelines have the potential to achieve deep coverage of cellular proteomes with high throughput and scalability. However, it is important to evaluate performance, including both reproducibility and ability to provide meaningful levels of biological insight. Here, we present an approach combining high field asymmetric waveform ion mobility spectrometer (FAIMS) interface and data independent acquisition (DIA) proteomics approach developed as part of the induced pluripotent stem cell (iPSC) Neurodegenerative Disease Initiative (iNDI), a large-scale effort to understand how inherited diseases may manifest in neuronal cells. Our FAIMS-DIA approach identified more than 8000 proteins per mass spectrometry (MS) acquisition as well as superior total identification, reproducibility, and accuracy compared to other existing DIA methods. Next, we applied this approach to perform a longitudinal proteomic profiling of the differentiation of iPSC-derived neurons from the KOLF2.1J parental line used in iNDI. This analysis demonstrated a steady increase in expression of mature cortical neuron markers over the course of neuron differentiation. We validated the performance of our proteomics pipeline by comparing it to single cell RNA-Seq datasets obtained in parallel, confirming expression of key markers and cell type annotations. An interactive webapp of this temporal data is available for aligned-UMAP visualization and data browsing (https://share.streamlit.io/anant-droid/singlecellumap). In summary, we report an extensively optimized and validated proteomic pipeline that will be suitable for large-scale studies such as iNDI.

neuroscience↗

bioRxiv2021

Specific Detection of Endogenous S129 Phosphorylated α-Synuclein in Tissue Using Proximity Ligation Assay

BACKGROUNDSynucleinopathies are a group of neurodegenerative disorders that are pathologically characterized by the accumulation of protein aggregates called Lewy Bodies. Lewy bodies are primarily composed of -synuclein (asyn) protein, which is phosphorylated at serine 129 (pS129) when aggregated. Currently available commercial antibodies used to stain for pS129 asyn can cross react with other proteins, thus making it difficult to specifically detect endogenous pS129 asyn and to interpret pS129 asyn staining. OBJECTIVETo develop a staining procedure that detects pS129 asyn with high specificity and low background. METHODSWe use the fluorescent and brightfield in situ Proximity Ligation Assay (PLA) to specifically detect pS129 asyn in cell culture, mouse and human brain sections. RESULTSThe pS129 asyn PLA specifically stained for endogenous, soluble pS129 asyn in cell culture, mouse brain sections, and human brain tissue without significant cross-reactivity or background signal. CONCLUSIONSThis PLA method can be used to specifically detect pS129 asyn in order to further explore and understand its role in health and disease.

neuroscience↗

bioRxiv2021

The activities of LRRK2 and GCase are positively correlated in clinical biospecimens and experimental models of Parkinson's disease

LRRK2 is a kinase involved in different cellular functions, including autophagy, endolysosomal pathways and vesicle trafficking. Mutations in LRRK2 cause autosomal dominant forms of Parkinsons disease (PD). Heterozygous mutations in GBA1, the gene encoding the lysosomal enzyme glucocerebrosidase (GCase), are the most common genetic risk factors for PD. Moreover, GCase function is altered in idiopathic PD and in other genetic forms of the disease. Recent work suggests that LRRK2 kinase activity can regulate GCase function. However, both a positive and a negative correlation have been described. To gain insights into the impact of LRRK2 on GCase, we investigated GCase levels and activity in LRRK2 G2019S knockin mice, in clinical biospecimens from PD patients carrying this mutation and in patient-derived cellular models. In these models we found a positive correlation between the activities of LRRK2 and GCase, which was further confirmed in cell lines with genetic and pharmacological manipulation of LRRK2 kinase activity. Overall, our study indicates that LRRK2 kinase activity affects both the levels and the catalytic activity of GCase.

neuroscience↗

bioRxiv2021

The Foundational data initiative for Parkinsons disease (FOUNDIN-PD): enabling efficient translation from genetic maps to mechanism

The FOUNdational Data INitiative for Parkinsons Disease (FOUNDIN-PD) is an international collaboration producing fundamental resources for Parkinsons disease (PD). FOUNDIN-PD generated a multi-layered molecular dataset in a cohort of induced pluripotent stem cell (iPSC) lines differentiated to dopaminergic (DA) neurons, a major affected cell type in PD. The lines were derived from the Parkinsons Progression Markers Initiative study including participants with PD carrying monogenic PD (SNCA) variants, variants with intermediate effects and variants identified by genome-wide association studies and unaffected individuals. We generated genetic, epigenetic, regulatory, transcriptomic, and longitudinal cellular imaging data from iPSC-derived DA neurons to understand molecular relationships between disease associated genetic variation and proximate molecular events. These data reveal that iPSC-derived DA neurons provide a valuable cellular context and foundational atlas for modelling PD genetic risk. We have integrated these data into a FOUNDIN-PD data browser (https://www.foundinpd.org) as a resource for understanding the molecular pathogenesis of PD.

genomics↗

bioRxiv2021

LAG3 is not expressed in human and murine neurons and does not modulate α-synucleinopathies

While the initial pathology of Parkinsons disease and other -synucleinopathies is often confined to circumscribed brain regions, it can spread and progressively affect adjacent and distant brain locales. This process may be controlled by cellular receptors of -synuclein fibrils, one of which was proposed to be the LAG3 immune checkpoint molecule. Here, we analyzed the expression pattern of LAG3 in human and mouse brains. Using a variety of methods and model systems, we found no evidence for LAG3 expression by neurons. While we confirmed that LAG3 interacts with -synuclein fibrils, the specificity of this interaction appears limited. Moreover, overexpression of LAG3 in cultured human neural cells did not cause any worsening of -synuclein pathology ex vivo. The overall survival of A53T -synuclein transgenic mice was unaffected by LAG3 depletion and the seeded induction of -synuclein lesions in hippocampal slice cultures was unaffected by LAG3 knockout. These data suggest that the proposed role of LAG3 in the spreading of -synucleinopathies is not universally valid.

neuroscience↗

bioRxiv2021

Association of a common genetic variant with Parkinson's disease is propagated through microglia

Studies of the genetic basis of Parkinsons disease (PD) have identified many disease-associated genetic variants, but the mechanisms linking variants to pathogenicity are largely unknown. PD risk is attributed to both coding mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene and to common non-coding variation upstream of the LRRK2 locus. Here we show that the influence of genotype at non-coding variant rs76904798 on LRRK2 expression is propagated specifically through microglia, in contrast to evaluations based on general rather than genotype-dependent expression. We find evidence of microglia-specific regulatory regions that may modulate LRRK2 expression using single nuclei sequencing analyses of human frontal cortex and confirm these results in a human induced pluripotent stem cell-derived microglia model. Our study demonstrates that cell type is an important consideration in interrogation of the role of non-coding variation in disease pathogenesis.

neuroscience↗

bioRxiv2020

Membrane targeting activates Leucine-rich repeat kinase 2 with differential effects on downstream Rab activation

Genetic variation at the Leucine-rich repeat kinase 2 (LRRK2) locus contributes to enhanced lifetime risk of familial and sporadic Parkinsons disease. Previous data have demonstrated that recruitment to various membranes of the endolysosomal system results in LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes and the cellular consequences of these events are still poorly understood. Here, we directed LRRK2 to lysosomes and early endosomes, triggering both LRRK2 autophosphorylation and phosphorylation of the direct LRRK2 substrates Rab10 and Rab12. However, when directed to the lysosomal membrane, pRab10 was restricted to perinuclear lysosomes, whereas pRab12 was visualized on both peripheral and perinuclear LRRK2-positive lysosomes, suggesting that lysosomal positioning provides additional regulation of LRRK2-dependent Rab phosphorylation. Anterograde transport of lysosomes to the cell periphery by increasing expression of ARL8B and SKIP or by knockdown of the motor adaptor protein JIP4 blocked recruitment and phosphorylation of Rab10 by LRRK2. Conversely, overexpression of the Rab7 effector protein RILP resulted in lysosomal clustering within the perinuclear area and increased LRRK2-dependent Rab10 recruitment and phosphorylation. The regulation of Rab10 phosphorylation in the perinuclear area depends on counteracting phosphatases, as knockdown of phosphatase PPM1H significantly increased pRab10 signal and lysosomal tubulation in the perinuclear region. Our novel findings suggest LRRK2 can be activated at multiple cellular membranes including lysosomes, and that lysosomal positioning further provides regulation of some Rab substrates likely via differential phosphatase activity in nearby cellular compartments.

cell biology↗

bioRxiv2020

Pathogenic mutations in Leucine-rich repeat kinase 2 sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinsons disease (PD) while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a are poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a into lysosomes in cells while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive accumulation of endocytosed transferrin into Rab8a-positive lysosomes leading to a dysregulation of iron transport. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in post-mortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.

cell biology↗

bioRxiv2020

Genetic analysis of amyotrophic lateral sclerosis identifies contributing pathways and cell types.

Despite the considerable progress in unraveling the genetic causes of amyotrophic lateral sclerosis (ALS), we do not fully understand the molecular mechanisms underlying the disease. We analyzed genome-wide data involving 78,500 individuals using a polygenic risk score approach to identify the biological pathways and cell types involved in ALS. This data-driven approach identified multiple aspects of the biology underlying the disease that resolved into broader themes, namely neuron projection morphogenesis, membrane trafficking, and signal transduction mediated by ribonucleotides. We also found that genomic risk in ALS maps consistently to GABAergic cortical interneurons and oligodendrocytes, as confirmed in human single-nucleus RNA-seq data. Using two-sample Mendelian randomization, we nominated five differentially expressed genes (ATG16L2, ACSL5, MAP1LC3A, PLXNB2, and SCFD1) within the significant pathways as relevant to ALS. We conclude that the disparate genetic etiologies of this fatal neurological disease converge on a smaller number of final common pathways and cell types.

genetics↗

bioRxiv2020

CoExp Web, a web tool for the exploitation of co-expression networks

SummaryThis paper presents the CoExp Web tool. CoExp is a software tool and a Web resource that fosters research on co-expression network models by providing a highly usable means to exploit and share multiple network models. Availability and Implementationthe Web resource is accessible at https://snca.atica.um.es/coexp/. The software at the front and back-ends is accessible at https://github.com/SoniaRuiz/CoExp_Web. Contactskgtsg0@ucl.ac.uk (Sonia Garcia-Ruiz)

bioinformatics↗

bioRxiv2020

Combined knockout of Lrrk2 and Rab29 does not result in behavioral abnormalities in vivo

Coding mutations in the LRRK2 gene, encoding for a large protein kinase, have been shown to cause familial Parkinsons disease (PD). The immediate biological consequence of LRRK2 mutations is to increase kinase activity, leading to the suggestion that inhibition of this enzyme might be useful therapeutically to slow disease progression. Genome-wide association studies have identified the chromosomal loci around LRRK2 and one of its proposed substrates, RAB29, as contributors towards the lifetime risk of sporadic PD. Considering the evidence for interactions between LRRK2 and RAB29 on the genetic and protein levels, here we generated a double knockout mouse model and determined whether there are any consequences on brain function with aging. From a battery of motor and non-motor behavioral tests, we noted only that 18-24 month Rab29-/- and double (Lrrk2-/-/Rab29-/-) knockout mice had diminished locomotor behavior in open field compared to wildtype mice. However, no genotype differences were seen in number of substantia nigra pars compacta (SNc) dopamine neurons or in tyrosine hydroxylase levels in the SNc and striatum, which might reflect a PD-like pathology. These results suggest that depletion of both Lrrk2 and Rab29 is tolerated, at least in mice, and support that this pathway might be able to be safely targeted for therapeutics in humans. Significance statementGenetic variation in LRRK2 that result in elevated kinase activity can cause Parkinsons disease (PD), suggesting LRRK2 inhibition as a therapeutic strategy. RAB29, a substrate of LRRK2, has also been associated with increased PD risk. Evidence exists for an interactive relationship between LRRK2 and RAB29. Mouse models lacking either LRRK2 or RAB29 do not show brain pathologies. We hypothesized that the loss of both targets would result in additive effects across in vivo and post-mortem assessments in aging mice. We found that loss of both LRRK2 and RAB29 did not result in significant behavioral deficits or dopamine neuron loss. This evidence suggests that chronic inhibition of this pathway should be tolerated clinically.

animal behavior and cognition↗

bioRxiv2020

Large-scale pathway-specific polygenic risk, transcriptomic community networks and functional inferences in Parkinson disease

Polygenic inheritance plays a central role in Parkinson disease (PD). A priority in elucidating PD etiology lies in defining the biological basis of genetic risk. Unraveling how risk leads to disruption will yield disease-modifying therapeutic targets that may be effective. Here, we utilized a high-throughput and hypothesis-free approach to determine biological pathways underlying PD using the largest currently available cohorts of genetic data and gene expression data from International Parkinsons Disease Genetics Consortium (IPDGC) and the Accelerating Medicines Partnership - Parkinsons disease initiative (AMP-PD), among other sources. We placed these insights into a cellular context. We applied large-scale pathway-specific polygenic risk score (PRS) analyses to assess the role of common variation on PD risk in a cohort of 457,110 individuals by focusing on a compilation of 2,199 publicly annotated gene sets representative of curated pathways, of which we nominate 46 pathways associated with PD risk. We assessed the impact of rare variation on PD risk in an independent cohort of whole-genome sequencing data, including 4,331 individuals. We explored enrichment linked to expression cell specificity patterns using single-cell gene expression data and demonstrated a significant risk pattern for adult dopaminergic neurons, serotonergic neurons, and radial glia. Subsequently, we created a novel way of building de novo pathways by constructing a network expression community map using transcriptomic data derived from the blood of 1,612 PD patients, which revealed 54 connecting networks associated with PD. Our analyses highlight several promising pathways and genes for functional prioritization and provide a cellular context in which such work should be done.

genetics↗

bioRxiv2020

Sequential screening nominates the Parkinson's disease associated kinase LRRK2 as a regulator of Clathrin-mediated endocytosis

Mutations in leucine-rich repeat kinase 2 (LRRK2) are an established cause of inherited Parkinsons disease (PD). LRRK2 is expressed in both neurons and glia in the central nervous system, but its physiological function(s) in each of these cell types is uncertain. Through sequential screens, we report a functional interaction between LRRK2 and Clathrin adaptor protein complex 2 (AP2). Analysis of LRRK2 KO tissue revealed a significant dysregulation of AP2 complex components, suggesting LRRK2 may act upstream of AP2. In line with this hypothesis, expression of LRRK2 was found to modify recruitment and phosphorylation of AP2. Furthermore, expression of LRRK2 containing the R1441C pathogenic mutation resulted in impaired clathrin-mediated endocytosis (CME). A decrease in activity-dependent synaptic vesicle endocytosis was also observed in neurons harboring an endogenous R1441C LRRK2 mutation. Alongside LRRK2, several PD-associated genes intersect with membrane-trafficking pathways. To investigate the genetic association between Clathrin-trafficking and PD, we used polygenetic risk profiling from IPDGC genome wide association studies (GWAS) datasets. Clathrin-dependent endocytosis genes were found to be associated with PD across multiple cohorts, suggesting common variants at these loci represent a cumulative risk factor for disease. Taken together, these findings suggest CME is a LRRK2-mediated, PD relevant pathway.

molecular biology↗

bioRxiv2020

Common variation at the LRRK2 locus is associated with survival in the primary tauopathy progressive supranuclear palsy

The genetic basis of variation in the rate of disease progression of primary tauopathies has not been determined. In two independent progressive supranuclear palsy cohorts, we show that common variation at the LRRK2 locus determines survival from motor symptom onset to death, possibly through regulation of gene expression. This links together genetic risk in alpha-synuclein and tau disorders, and suggests that modulation of proteostasis and neuro-inflammation by LRRK2 inhibitors may have a therapeutic role across disorders.

genetics↗

bioRxiv2020

LRRK2 mediates tubulation and vesicle sorting from membrane damaged lysosomes

Mutations in the leucine rich repeat kinase 2 (LRRK2) gene are a cause of familial and sporadic Parkinsons disease (PD). Nonetheless, the biological functions of LRRK2 remain incompletely understood. Here, we observed that LRRK2 is recruited to lysosomes that have a ruptured membrane. Using unbiased proteomics, we observed that LRRK2 is able to recruit the motor adaptor protein JIP4 to permeabilized lysosomes in a kinase-dependent manner through the phosphorylation of RAB35 and RAB10. Super-resolution live cell imaging microscopy and FIB-SEM revealed that once at the lysosomal membrane, JIP4 promotes the formation of LAMP1-negative lysosomal tubules that release membranous content from ruptured lysosomes. Released vesicular structures are able to interact with other lysosomes. Thus, we described a new process that uses lysosomal tubulation to release vesicular structures from permeabilized lysosomes. LRRK2 orchestrates this process that we name LYTL (LYsosomal Tubulation/sorting driven by LRRK2) that, given the central role of the lysosome in PD, is likely to be disease relevant.

cell biology↗