Biology subjects

Durcan, T. M.

Publications and source records attributed to Durcan, T. M..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

Parkinsons disease (PD) is a neurodegenerative disorder mainly characterized by the loss of dopaminergic neurons from the substantia nigra. Affected neurons exhibit intracellular aggregates primarily composed of misfolded and phosphorylated alpha-synuclein (aSyn). In pathological conditions, this presynaptic protein has been shown to be transmitted from cell to cell in a prion-like manner, which contributes to the progression of the disease. Single-chain variable fragments (scFvs) are small polypeptides derived from the binding domains of antibodies that are less immunogenic and have better tissue penetration compared to full antibodies. In this work, we aimed to demonstrate the potential of extracellular scFvs to slow down the propagation of pathological aSyn in an in vivo model of synucleinopathy. We generated scFvs that target aSyn, and tested two of them in a PD mouse model consisting of transgenic M83 mice injected with human aSyn pre-formed fibrils (PFFs). The sequence encoding each anti-aSyn scFv was cloned in a self-complementary AAV2 viral vector, and purified particles were administered intravenously. CNS expression of either scFv protected against the development of paralysis and limb weakness, in addition to significantly reducing pathologic aggregates of phosphorylated aSyn in the brain. Moreover, in vitro results in human iPSCs-derived dopaminergic neurons suggest that the scFvs can mitigate aSyn spreading by preventing its internalization. Overall, our findings demonstrate that single-chain antibody fragments exhibit strong therapeutic potential in a preclinical mouse model. Thus, our minimally invasive, gene-mediated immunotherapy approach has the potential to serve as an effective treatment for halting the progression of Lewy body diseases.

neuroscience↗

bioRxiv2024

The functional and pathogenic consequences of fibrinogen on human oligodendroglia

Fibrinogen is a blood-derived protein involved in coagulation, and can make its way into the central nervous system (CNS) following breakdown of the blood-brain barrier. This molecule has been implicated in multiple sclerosis (MS), a disease marked by inflammation and demyelination in the CNS, as well as other neurological disorders. However, the effect of this molecule has not been studied on human myelinating cells. This study examines how fibrinogen influences human oligodendrocyte (OL) lineage cells at various stages of development. Using induced pluripotent stem cell-derived (iPSC) OL precursors and human primary OLs, we examined the effects of fibrinogen on cell differentiation, viability and myelination-related function. Here we show that fibrinogen induces an aberrant differentiation of early lineage OLs, by inhibiting their maturation and inducing an astrocytic phenotype, as seen in previous studies. On mature OLs, fibrinogen was found to promote myelination capacity as shown by ensheathment assays as well as on the RNA level. These effects were associated with the activation of BMP signalling, both in early and mature OLs. Transcriptomic analysis of human MS brain tissue shows similar pro-myelination changes in a subset of OLs, suggesting in vivo relevance. These findings indicate that fibrinogen has a lineage-dependent effect, where it may be inhibitory earlier in the lineage while promoting OL function in later stages. Understanding this dual role will provide insight into remyelination failure in MS and highlights the importance of timing and target in future therapeutic strategies. Significance StatementIn multiple sclerosis (MS), the blood protein fibrinogen leaks into the brain and has been shown to interfere with myelin repair. This study demonstrates that fibrinogen has opposite effects on human oligodendrocyte-lineage cells depending on their stage of maturation. While it blocks the differentiation of early-stage cells, it enhances the functional capacity of mature oligodendrocytes. These findings help explain why remyelination may fail in MS and suggest that fibrinogen could both hinder and support repair, depending on the cell context. This dual role has important implications for developing stage-specific therapies for MS.

neuroscience↗

bioRxiv2024

Sex-focused analyses of M83 A53T hemizygous mouse model with recombinant human alpha-synuclein preformed fibril injection identifies female resilience to disease progression: A combined magnetic resonance imaging and behavioural study.

Alpha-synuclein (aSyn) pathology has been extensively studied in mouse models harbouring human mutations. In spite of the known sex differences in age of onset, prevalence and disease presentation in human synucleinopathies, the impact of sex on aSyn propagation has received very little attention. To address this need, we examined sex differences in whole brain signatures of neurodegeneration due to aSyn toxicity in the M83 mouse model using longitudinal magnetic resonance imaging (MRI; T1-weighted; 100 m3 isotropic voxel; acquired -7, 30, 90 and 120 days post-injection [dpi]; n[≥]8 mice/group/sex/time point). To initiate aSyn spreading, M83 mice were inoculated with recombinant human aSyn preformed fibrils (Hu-PFF) or phosphate buffered saline (PBS) injected in the right dorsal striatum. We observed more aggressive neurodegenerative profiles over time for male M83 Hu-PFF-injected mice when examining voxel-wise trajectories. However, at 90 dpi, we observed widespread patterns of neurodegeneration in the female Hu-PFF-injected mice. These differences were not accompanied with any differences in motor symptom onset between the male and female Hu-PFF-injected mice. However, male Hu-PFF-injected mice reached their humane endpoint sooner. These findings suggest that post-motor symptom onset, even though more accelerated disease trajectories were observed for male Hu-PFF-injected mice, neurodegeneration may appear sooner in female Hu-PFF-injected mice (prior to motor symptomatology). These findings suggest that sex-specific synucleinopathy phenotypes urgently need to be considered to improve our understanding of neuroprotective and neurodegenerative mechanisms.

neuroscience↗

bioRxiv2024

Generation of human iPSC-derived phrenic-like motor neurons to model respiratory motor neuron degeneration in ALS

The fatal motor neuron (MN) disease Amyotrophic Lateral Sclerosis (ALS) is characterized by progressive MN degeneration. Phrenic MNs (phMNs) controlling the activity of the diaphragm are prone to degeneration in ALS, leading to death by respiratory failure. Understanding of the mechanisms of phMN degeneration in ALS is limited, mainly because human experimental models to study phMNs are lacking. Here we describe a method enabling the derivation of phrenic-like MNs from human iPSCs (hiPSC-phMNs) within 30 days. This protocol uses an optimized combination of small molecules followed by cell-sorting based on a cell-surface protein enriched in hiPSC-phMNs, and is highly reproducible using several hiPSC lines. We show further that hiPSC-phMNs harbouring ALS-associated amplification of the C9orf72 gene progressively lose their activity and undergo increased death compared to isogenic controls. These studies establish a previously unavailable protocol to generate human phMNs, offering a disease-relevant system to study mechanisms of respiratory MN dysfunction.

neuroscience↗

bioRxiv2023

A deep learning convolutional neural network distinguishes neuronal models of Parkinson's disease from matched controls

Parkinsons disease (PD) is a neurodegenerative disorder that results in the loss of dopaminergic neurons in the substantia nigra pars compacta. Despite advances in understanding PD, there is a critical need for novel therapeutics that can slow or halt its progression. Induced pluripotent stem cell (iPSC)-derived dopaminergic neurons have been used to model PD but measuring differences between PD and control cells in a robust, reproducible, and scalable manner remains a challenge. In this study, we developed a binary classifier convolutional neural network (CNN) to accurately classify microscopy images of PD models and matched control cells. We acquired images of iPSC-derived neural precursor cells (NPCs) and dopaminergic (DANs) and trained multiple CNN models comparing control cells to genetic and chemical models of PD. Our CNN accurately predicted whether control NPC cells were treated with the PD-inducing pesticide rotenone with 97.60% accuracy. We also compared control to a genetic model of PD (deletion of the Parkin gene) and found a predictive accuracy of 86.77% and 95.47% for NPC and DAN CNNs, respectively. Our cells were stained for nuclei, mitochondria, and plasma membrane, and we compared the contribution of each to the CNNs accuracy. Using all three features together produced the best accuracy, but nuclear staining alone produced a highly predictive CNN. Our study demonstrates the power of deep learning and computer vision for analyzing complex PD-related phenotypes in DANs and suggests that these tools hold promise for identifying new targets for therapy and improving our understanding of PD.

neuroscience↗

bioRxiv2023

The Parkinson's disease risk gene cathepsin B promotes fibrillar alpha-synuclein clearance, lysosomal function and glucocerebrosidase activity in dopaminergic neurons

Variants in the CTSB gene encoding the lysosomal hydrolase cathepsin B (catB) are associated with increased risk of Parkinsons disease (PD). However, neither the specific CTSB variants driving these associations nor the functional pathways that link catB to PD pathogenesis have been characterized. CatB activity contributes to lysosomal protein degradation and regulates signaling processes involved in autophagy and lysosome biogenesis. Previous in vitro studies have found that catB can cleave monomeric and fibrillar alpha-synuclein, a key protein involved in the pathogenesis of PD that accumulates in the brains of PD patients. However, truncated synuclein isoforms generated by catB cleavage have an increased propensity to aggregate. Thus, catB activity could potentially contribute to lysosomal degradation and clearance of pathogenic alpha synuclein from the cell, but also has the potential of enhancing synuclein pathology by generating aggregation-prone truncations. Therefore, the mechanisms linking catB to PD pathophysiology remain to be clarified. Here, we conducted genetic analyses of the association between common and rare CTSB variants and risk of PD. We then used genetic and pharmacological approaches to manipulate catB expression and function in cell lines and induced pluripotent stem cell-derived dopaminergic neurons and assessed lysosomal activity and the handling of aggregated synuclein fibrils. We find that catB inhibition impairs autophagy, reduces glucocerebrosidase (encoded by GBA1) activity, and leads to an accumulation of lysosomal content. In cell lines, reduction of CTSB gene expression impairs the degradation of pre-formed alpha-synuclein fibrils, whereas CTSB gene activation enhances fibril clearance. In midbrain organoids and dopaminergic neurons treated with alpha-synuclein fibrils, catB inhibition potentiates the formation of inclusions which stain positively for phosphorylated alpha-synuclein. These results indicate that the reduction of catB function negatively impacts lysosomal pathways associated with PD pathogenesis, while conversely catB activation could promote the clearance of pathogenic alpha-synuclein.

neuroscience↗

bioRxiv2023

An adapted stem cell-derived microglia protocol for the study of microgliopathies and other neurological disorders

BackgroundAdult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a primary microgliopathy caused by pathogenic variants in the colony-stimulating factor 1 receptor (CSF1R) gene. Since CSF1R signaling is crucial for microglia development, survival and function, induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial defects caused by ALSP patient-specific CSF1R variants. MethodsSerial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an ALSP patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL. ResultsThe newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to primary human microglia and with higher phagocytic and inflammatory competence at [~]3-fold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 (P2RY12) expression. Finally, ALSP patient-derived cells showed surprisingly high phagocytic capacity, which was associated with higher lysosomal content. ConclusionsWe optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory and phagocytic activities.

neuroscience↗

bioRxiv2023

Assessing the performance of commercial reagent antibodies

Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, demonstrates that: i) more than 50% of all antibodies failed in one or more tests, ii) yet, [~]50-75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first such study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts.

molecular biology↗

bioRxiv2023

Excitatory neuron-specific suppression of the integrated stress response pathway contributes to autism-related phenotypes in a mouse model of fragile X syndrome

Dysregulation of protein synthesis is one of the key mechanisms underlying autism spectrum disorder (ASD). However, the role of a major pathway controlling protein synthesis, the integrated stress response (ISR), in ASD remains poorly understood. Here, we demonstrate that the main arm of the ISR, eIF2 phosphorylation (p-eIF2), is suppressed in excitatory but not inhibitory neurons in a mouse model of fragile X syndrome (FXS; Fmr1-/y). We further show that the decrease in p-eIF2 is mediated via activation of the mTORC1. Genetic reduction of p-eIF2 only in excitatory neurons is sufficient to increase general protein synthesis and cause autism-like behavior. In Fmr1-/y mice, genetic restoration of p-eIF2 solely in excitatory neurons reverses elevated protein synthesis and rescues autism-related phenotypes. Thus, we reveal a previously unknown causal relationship between excitatory neuron-specific translational control via the ISR pathway, general protein synthesis and core phenotypes reminiscent of autism in a mouse model of FXS.

neuroscience↗

bioRxiv2023

Homozygous ALS-linked mutations in TARDBP/TDP-43 lead to progressive synaptic dysfunction in human iPSC-derived motor neurons

Cytoplasmic mislocalization and aggregation of the RNA-binding protein TDP-43 is a pathological hallmark of the motor neuron (MN) disease amyotrophic lateral sclerosis (ALS). Furthermore, while mutations in the TARDBP gene (encoding TDP-43) have been associated with ALS, the pathogenic consequences of these mutations remain poorly understood. Using CRISPR/Cas9, we engineered two homozygous knock-in iPSC lines carrying mutations in TARDBP encoding TDP-43A382T and TDP-43G348C, two common yet understudied ALS TDP-43 variants. MNs differentiated from knock-in iPSCs had normal viability and displayed no significant changes in TDP-43 subcellular localization, phosphorylation, solubility, or aggregation compared with isogenic control MNs. However, our results highlight synaptic impairments in both TDP-43A382T and TDP-43G348C MN cultures, as reflected in synapse abnormalities and alterations in spontaneous neuronal activity. Collectively, our findings suggest that MN dysfunction may precede the occurrence of TDP-43 pathology and neurodegeneration in ALS and further implicate synaptic and excitability defects in the pathobiology of this disease.

neuroscience↗

bioRxiv2023

MerTK mediates the immunologically silent uptake of alpha-synuclein fibrils by human microglia

MerTK is a receptor tyrosine kinase that mediates the immunologically silent phagocytic uptake of diverse types of cellular debris. Highly expressed on the surface of microglial cell, MerTK is of importance in brain development, homeostasis, plasticity, and disease. Yet, involvement of this receptor in the clearance of protein aggregates that accumulate with aging and in neurodegenerative diseases has yet to be defined. The current study explored the function of MerTK in the microglial uptake of alpha-synuclein fibrils which play a causative role in the pathobiology of synucleinopathies. Using human primary and induced pluripotent stem cell-derived microglia, the MerTK- dependence of alpha-synuclein fibril internalization was investigated in vitro. Relevance of this pathway to synucleinopathies was assessed by analyzing MerTK expression in patient-derived cells and tissues. Pharmacological inhibition of MerTK and siRNA-mediated MERTK knockdown both caused a decreased rate of alpha-synuclein fibril internalization by human microglia. Consistent with the immunologically silent nature of MerTK-mediated phagocytosis, alpha-synuclein fibril internalization did not induce secretion of pro-inflammatory cytokines from microglia. In addition, burden analysis in two independent patient cohorts revealed a significant association between rare functionally deleterious MERTK variants and Parkinsons disease in one of the cohorts (p = 0.002). Accordingly, MERTK expression was significantly upregulated in nigral microglia from Parkinsons disease/Lewy body dementia patients compared to those from non-neurological control donors in a single-nuclei RNA-sequencing dataset (p = 5.08x10-21), and MerTK protein expression positively correlated with alpha-synuclein level in human cortex lysates (p = 0.0029). Taken together, our findings define a novel role for MerTK in mediating the uptake of alpha-synuclein aggregates by human microglia, with possible involvement in limiting alpha-synuclein spread in synucleinopathies such as Parkinsons disease.

cell biology↗

bioRxiv2023

α-synuclein preformed fibrils bind to β-neurexins and impair β-neurexin-mediated presynaptic organization

Synucleinopathies form a group of neurodegenerative diseases defined by misfolding and aggregation of alpha-synuclein (-syn). Abnormal accumulation and spreading of -syn aggregates lead to synapse dysfunction and neuronal cell death. Yet, little is known about synaptic mechanisms underlying -syn pathology. Here we identified {beta}-isoforms of neurexins ({beta}-NRXs) as presynaptic organizing proteins that interact with -syn preformed fibrils (-syn PFFs), toxic -syn aggregates, but not -syn monomers. Our cell surface protein binding assays and surface plasmon resonance assays reveal that -syn PFFs bind directly to {beta}-NRX through their N-terminal histidine-rich domain (HRD) at nanomolar range (Kd: ~500 nM monomer equivalent). Furthermore, our artificial synapse formation assays show that -syn PFFs diminish excitatory and inhibitory presynaptic organization induced by a specific isoform of neuroligin 1 that binds only {beta}-NRXs, but not -isoforms of neurexins. Thus, our data suggest that -syn PFFs interact with {beta}-NRXs to inhibit {beta}-NRX-mediated presynaptic organization, providing novel molecular insight into how -syn PFFs induce synaptic pathology in synucleinopathies such as Parkinsons disease and dementia with Lewy bodies.

neuroscience↗

bioRxiv2022

CelltypeR: A framework to identify and characterize cell types in human midbrain organoids using flow cytometry

Motivated by the growing number of single cell RNA sequencing datasets (scRNAseq) revealing the cellular heterogeneity in complex tissues, particularly in brain and induced pluripotent stem cell (iPSC)-derived brain models, we developed a high-throughput, standardized approach for reproducibly characterizing cell types in complex neuronal tissues based on protein expression levels. Our approach combines a flow cytometry (FC) antibody panel targeting brain cells with a computational pipeline called CelltypeR, with functions for aligning and transforming datasets, optimizing unsupervised clustering, annotating and quantifying cell types, and statistical comparisons. We applied this workflow to human iPSC-derived midbrain organoids and identified the expected brain cell types, including neurons, astrocytes, radial glia, and oligodendrocytes. Defining gates based on the expression levels of our protein markers, we performed Fluorescence-Activated Cell Sorting of astrocytes, radial glia, and neurons, cell types were then confirmed by scRNAseq. Among the sorted neurons, we identified three subgroups of dopamine (DA) neurons; one reminiscent of substantia nigra DA neurons, the cell type most vulnerable in Parkinsons disease. Finally, we use our workflow to track cell types across a time course of organoid differentiation. Overall, our adaptable analysis framework provides a generalizable method for reproducibly identifying cell types across FC datasets.

neuroscience↗

bioRxiv2022

Presymptomatic neuroanatomical and cognitive biomarkers of alpha-synuclein propagation in a mouse model of synucleinopathy

There is significant evidence suggesting aggregated misfolded alpha-synuclein, a major component of Lewy bodies, propagates in a prion-like manner contributing to disease progression in Parkinsons disease (PD) and other synucleinopathies. Animal models are essential for understanding and developing treatments for these diseases. However, despite modelling human pathology, most endpoints studied in mice do not translate to humans. Furthermore, the progression by which alpha-synuclein misfolding affects human-relevant measures such as brain volume and underlying subtle, high-level cognitive deficits is poorly understood. Here we used a mouse model of synucleinopathy; hemizygous M83 human A53T alpha-synuclein transgenic mice inoculated with recombinant human alpha-synuclein preformed fibrils (PFF) injected in the right striatum to initiate alpha-synuclein misfolding and aggregation. We examined alpha-synuclein-induced atrophy at 90 days post-injection using ex vivo magnetic resonance imaging as well as high-level cognition and motor function, as biomarkers of alpha-synuclein toxicity. We observed widespread atrophy in bilateral regions that project to or receive input from the injection site, highlighting a network of regions that are consistent with structural changes observed in humans with PD. Moreover, we detected early deficits in reversal learning with touchscreen testing in PFF-injected mice prior to motor dysfunction, consistent with the pathology observed in cortical-striatal and thalamic loops. We show, using translational approaches in mice, that progression of prion-like spreading of alpha-synuclein causes selective atrophy via connected brain regions leading to high-level cognitive deficits. We propose that precise imaging and cognitive biomarkers can provide a more direct and human-relevant measurement of alpha-synuclein-induced toxicity in pre-clinical testing. Significance StatementThe work described in this manuscript showcases the utility of state-of-the-art methodologies (magnetic resonance imaging and touchscreen behavioural tasks) to examine endophenotypes, both in terms of symptomatology and neuroanatomy, of alpha-synuclein propagation in a mouse model of synucleinopathy. Our work further validates the M83-Hu-PFF mouse model of synucleinopathy-associated pathogenesis of neurodegenerative diseases while highlighting precise imaging and cognitive biomarkers of protein misfolding toxicity. Specifically, we identified rapid and translational biomarkers that can serve as a proxy for the direct examination of cellular levels for pathology. We anticipate that these biomarkers can measure progression of toxicity, specifically in the early phases, and may be more reliable than end stage pathology and more useful as endpoints in the examination of novel therapeutics.

neuroscience↗

bioRxiv2022

An optimized workflow to generate and characterize iPSC-derived motor neuron (MN) spheroids

Motor neuron diseases (MNDs) are characterized by the progressive degeneration of motor neurons (MNs) from the cortex, brainstem and/or the spinal cord. In an effort to understand the underlying causes of this selective degeneration, a multitude of in vitro models based on induced pluripotent stem cell (iPSC)-derived MNs have been developed. Moreover, different groups have started to use advanced 3D structures, composed of MNs and other cell types to increase the physiological relevance of such in vitro models. For instance, spheroids are simple 3D models that have the potential to be generated in large numbers that can be used across different assays. In this study, we generated MN spheroids and developed a workflow to analyze them. We confirmed the expression of different MN markers as the MN spheroids differentiate, at both the transcript and protein level, as well as their capacity to display functional activity in the form of action potentials (APs) and bursts. We also identified the presence of other cell types, namely interneurons and oligodendrocytes, which share the same neural progenitor pool with MNs. In summary, we successfully developed a MN 3D model, and we optimized a workflow that can be applied to their characterization and analysis. In the future, we will apply this model and workflow to the study of MNDs by generating MN spheroids from patient-derived iPSC lines, aiming to contribute to the development of more advance and physiological in vitro disease models.

neuroscience↗

bioRxiv2022

Systematic comparison of culture media uncovers phenotypic shift of human microglia defined by reduced reliance to CSF1R signaling

Efforts to understand microglia function in health and diseases have been hindered by the lack of culture models that recapitulate in situ cellular properties. In recent years, the use of serum-free media with brain-derived growth factors (CSF1R ligands and TGF-{beta}1/2) have been favored for the maintenance of rodent microglia as they promote morphological features observed in situ. Here we study the functional and transcriptomic impacts of such media on human microglia. Media formulation had little impact on microglia transcriptome assessed by RNA sequencing which was sufficient to significantly alter microglia capacity to phagocytose myelin debris and to elicit an inflammatory response to lipopolysaccharide. When compared to immediately ex vivo microglia from the same donors, the addition of fetal bovine serum to culture media, but not growth factors, was found to aid in the maintenance of key signature genes including those involved in phagocytic processes. A phenotypic shift characterized by CSF1R downregulation in culture correlated with a lack of reliance on CSF1R signaling for survival. Consequently, no improvement in cell survival was observed following culture supplementation with CSF1R ligands. Our study provides better understanding of human microglia in culture, with observations that diverge from those previously made in rodent microglia. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/500101v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@16ea011org.highwire.dtl.DTLVardef@1cef01dorg.highwire.dtl.DTLVardef@f621aborg.highwire.dtl.DTLVardef@11c9f97_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

Structure-based design and characterization of Parkin activating mutations

Human autosomal recessive mutations in the Parkin gene are causal for Parkinsons disease (PD). Parkin encodes a ubiquitin E3 ligase that functions together with the PD associated kinase, PINK1, in a mitochondrial quality control pathway. Structural studies reveal that Parkin exists in an inactive conformation mediated by multiple autoinhibitory domain interfaces. Here we have performed comprehensive mutational analysis of both human and rat Parkin to unbiasedly determine Parkin activating mutations across all major autoinhibitory interfaces. Out of 31 mutations tested, we identify 11 activating mutations clustered near the RING0:RING2 or REP:RING1 interfaces, which reduce the thermal stability of Parkin. Of these, we demonstrate that three mutations, V393D, A401D, and W403A located at the REP:RING1 interface were able to completely rescue a Parkin S65A mutant, defective in mitophagy, in cell-based studies. Overall our data extends previous analysis of Parkin activation mutants and suggests that small molecules that mimic REP:RING1 destabilisation offer therapeutic potential for PD patients harbouring select Parkin mutations. Summary blurbParkin, an E3 ubiquitin ligase involved in Parkinsons disease, is inactive in the basal state and is activated by PINK1 to mediate mitophagy. Here we characterized 31 mutations and discovered three that activate Parkin and rescue loss of PINK1 phosphorylation.

biochemistry↗

bioRxiv2021

Loss of C9orf72 perturbs the Ran-GTPase gradient and generates compositionally diverse cytoplasmic Importin β-1 granules in motor and cortical neurons in vivo

Repeat expansions in C9orf72 cause Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) eliciting toxic effects through generation of RNA foci, dipeptide repeat proteins and/or loss of C9orf72 protein. Defects in nucleocytoplasmic transport (NCT) have been implicated as a pathogenic mechanism underlying repeat expansion toxicity. Here, we show that loss of C9orf72 causes neuronal specific phenotypes, disrupting the Ran-GTPase gradient both in vitro and in vivo. We describe compositionally different types of cytoplasmic Importin {beta}-1 granules that exhibit neuronal subtype-specific properties in vivo. We show that the abundance of Importin {beta}-1 granules is increased in the context of C9orf72 deficiency, disrupting interactions with nuclear pore complex proteins. These granules appear to bud from the nuclear envelope and are co-immunoreactive for G3BP1 and K63-ubiquitin. These findings link loss of C9orf72 protein to gain-of-function mechanisms and defects in NCT.

neuroscience↗