Biology subjects

Luk, K.

Publications and source records attributed to Luk, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Blood-brain barrier model architecture shapes peripheral immune cell trafficking in Parkinson's disease

Parkinsons disease (PD) is a neurodegenerative disorder traditionally characterized by dopaminergic neuron loss in the substantial nigra pars compacta, but peripheral immune dysregulation and blood-brain barrier (BBB) dysfunction have been increasingly implicated in disease etiology. However, how circulating immune cells interact with the human BBB and how these interactions are captured across experimental models remains poorly understood. In particular, human BBB models offer multiple platforms to interrogate these biological questions, with organ-on-chip approaches attracting significant interest. In this context, it is essential to determine whether model architecture influences the assessment of immune-endothelial interactions in PD, and if it may lead to fundamentally different interpretations of immune cell trafficking at the human BBB. Here, we compared peripheral blood mononuclear cells (PBMCs) from control donors or individuals with PD in human induced pluripotent stem cell (iPSC)-derived BBB models to determine how static and dynamic BBB systems influence immune cell behavior. To do so, we leveraged our brain chip platform to establish a model based on expression of the PD-associated SNCA triplication mutation. Using a two-dimensional transwell system and a three-dimensional (3D) microfluidic BBB chip, we evaluated PBMC attachment and transmigration under conditions of PBMC disease status, endothelial genotype associated with SNCA triplication, and exposure to -synuclein (-Syn) monomers or preformed fibrils (PFFs). PBMCs from PD donors showed increased baseline reactivity and altered endothelial interactions compared with controls. In transwell models, SNCA triplication increased PBMC attachment and selectively enhanced PD PBMC transmigration, while PFF increased attachment without affecting transmigration. In contrast, in the microfluidic BBB chip, attachment was largely unchanged by endothelial genotype or -Syn exposure, whereas transmigration increased following -Syn monomer pre-treatment. Together, PBMC-BBB interactions in PD appear to be shaped by immune cell status, endothelial genotype, and -Syn exposure, but are strongly influenced by BBB model dimensionality and flow. This study underscores the importance of physiologically relevant multicellular and flow-based BBB systems and provides a human-focused framework for studying peripheral immune cell trafficking across the diseased BBB. These findings also emphasize that biological insights into BBB function are inherently shaped by the experimental model used, underscoring the need for complementary human BBB platforms.

neuroscience↗

bioRxiv2026

Fully Modified SpyCas9 Guide RNAs Enable Robust Genome Editing In Cells and In Vivo

Precision engineering of CRISPR/Cas9 components has advanced genome editing toward therapeutic applications. Completely chemically stabilized guide RNAs (gRNAs) have the potential to improve in vivo editing efficacy while enabling greater flexibility in delivery strategies. However, previous generations of fully modified guides have been associated with reduced Cas9 activity. Here, we employed an iterative, structure-guided optimization strategy to systematically introduce chemical modifications at each position of SpyCas9 gRNAs. Extending beyond commonly used nucleotide modifications, we incorporated 2-amino-RNA, 4-thio-RNA, and extended nucleic acid (exNA) to generate gRNA designs in which 90-100% of the nucleotides are sugar- or backbone-modified. Although certain modification patterns exhibit sequence-dependent variability, we have established a growing repertoire of guides that consistently maintain or enhance editing efficacy when applied both in vitro and in vivo. Collectively, our heavily and fully modified gRNAs hold potential for applications in nuclease editing, base editing, and other genome editing tools. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/725424v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1118f08org.highwire.dtl.DTLVardef@1c57069org.highwire.dtl.DTLVardef@1572148org.highwire.dtl.DTLVardef@14a111e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Pharmacologic NLRP3 Inhibition Modulates Parkinson's Disease-Associated Microglial Transcriptomic Signatures and Mitigates α-Synuclein-Triggered Neurodegeneration

BackgroundParkinsons disease (PD), the second most common neurodegenerative disorder after Alzheimers disease, and the rare disorder multiple system atrophy (MSA), are both characterized by intracellular accumulation of -synuclein fibrils and early, sustained microglial reactivity in parallel to the neurodegeneration. Activation of the NLRP3 inflammasome in disease-associated reactive microglia is increasingly recognized as a key pathogenic driver and a promising therapeutic target in synucleinopathies. Dapansutrile (OLT1177(R)) is a selective, orally bioavailable NLRP3 inhibitor with a favorable safety profile in clinical trials for non-neurological indications. Here, we evaluated the therapeutic potential of dapansutrile in preclinical models of PD and MSA and explored the predictive and translational value of its effects. MethodsTwo established mouse models of synucleinopathy with nigral neurodegeneration were employed: the -synuclein preformed fibril (PFF) propagation model and the transgenic PLP--syn model expressing human wild-type -synuclein in oligodendrocytes. Pharmacokinetic analyses assessed plasma and brain exposure after oral administration. The efficacy of six-month dapansutrile treatment was examined in both preventive (post-PFF injection) and therapeutic (PLP--syn mice) paradigms, using behavioral, histopathological, and molecular readouts. Transcriptomic profiling of striatal and midbrain microglia identified differentially expressed genes (DEGs) associated with treatment and compared them with post-mortem transcriptomic signatures of disease-associated microglia in PD patients. Plasma IL-18 and neurofilament light chain (NfL) levels were evaluated as translational biomarkers. ResultsChronic oral dapansutrile treatment at clinically relevant doses improved motor performance, reduced -synuclein inclusions, attenuated gliosis, and mitigated nigral neurodegeneration in both models. Microglial transcriptomic analyses revealed that dapansutrile reversed key transcriptional signatures characteristic of PD-associated reactive microglia. Moreover, plasma IL-18 and NfL levels correlated with neuropathological and functional outcomes, supporting their potential as biomarkers of target engagement and treatment efficacy. ConclusionsThese data identify chronic NLRP3 activation as a shared and targetable mechanism in PD and MSA and highlight dapansutrile as a CNS-penetrant, clinically advanced candidate for disease modification in -synucleinopathies. The observed transcriptomic reprogramming of microglia and the parallel changes in blood biomarkers provide a strong translational bridge to clinical development.

neuroscience↗

bioRxiv2025

A Novel Mouse Model of Parkinson's Disease for Investigating Progressive Pathology and Neuroprotection

Developing animal models that successfully recapitulate the features of progressive Parkinsons disease (PD) is crucial for understanding disease progression mechanisms and creating effective therapeutic interventions. In this study, we created a mouse model of PD by overexpressing -synuclein through a combined injection of AAV6--synuclein and preformed fibrils (PFFs) into the medial and lateral substantia nigra (SN). We also demonstrated that chronic administration of the c-Abl inhibitor PD180970 provides neuroprotection in this model. Mice injected with the AAV6--synuclein and PFF combination showed a progressive loss of dopaminergic (DA) neurons in the SN and their projections in the striatum over 24 weeks. This neuronal loss coincided with a time-dependent accumulation of phosphorylated -synuclein (p-syn) in the SN. The p-syn aggregates spread to synaptically connected DARPP-32-positive neurons in the striatum and further extended to the cortex. We also observed a contralateral spread of p-syn aggregates. Additionally, -synuclein overexpression led to a significant increase in activated microglia and astrocytes at all timepoints, with the strongest activation occurring early and gradually diminishing over time. Daily administration of PD180970 significantly reduced the loss of DA neurons caused by -synuclein injection and decreased the accumulation of p-syn in the SN. PD180970 treatment also reduced the neuroinflammation significantly. Overall, the combined injection of AAV6--synuclein and preformed fibrils into the mouse brain establishes a robust PD model, enabling detailed mechanistic studies of the disease. We further demonstrate the models utility for chronic neuroprotection studies using the potential drug PD180970, highlighting its broad applicability. Significance StatementThis study establishes a robust mouse model of Parkinsons disease (PD) by combining AAV6-mediated -synuclein overexpression and preformed fibrils (PFFs) to replicate key features of PD, such as progressive dopaminergic neuron loss, phosphorylated -synuclein accumulation, and neuroinflammation. The model captures the spread of pathological aggregates to synaptically connected brain regions, closely mimicking the human disease. By testing the c-Abl inhibitor PD180970, we demonstrate its neuroprotective effects, including reduced neuronal loss, decreased -synuclein accumulation, and neuroinflammation highlighting its therapeutic potential. This model offers a valuable platform for investigating PD mechanisms and evaluating novel interventions, bridging the gap between preclinical and clinical applications.

neuroscience↗

bioRxiv2025

NERINE reveals rare variant associations in gene networks across multiple phenotypes and implicates an SNCA-PRL-LRRK2 subnetwork in Parkinson's disease

There are two primary approaches to study the genetic basis of human phenotypes. Experiments in model systems generate interpretable gene networks but, in isolation, do not establish relevance to the human condition. Statistical genetics identifies relevant association signals at the variant or gene level but lacks tools to test specific mechanistic models, as existing methods do not incorporate the topology of gene-gene interactions. We bridge these two strategies by introducing a method that competitively tests network hypotheses with rare variant associations. A hierarchical model-based association test NERINE for the first time incorporates gene network topology while remaining resilient to network inaccuracies. We demonstrate NERINEs ability to test network hypotheses derived from both canonical pathway databases and model system screens. Comprehensive database-wide search of pathway networks with NERINE uncovers compelling associations for breast cancer, cardiovascular diseases, and type II diabetes, which are undetected by single-gene tests. Testing bespoke networks from experimental screens targeting key PD pathologies: dopaminergic neuron survival and -synuclein pathobiology, NERINE highlights rare variant burden in gene modules related to autophagy, vesicle traiicking, and protein homeostasis. Genome-scale CRISPRi-screening of -synuclein toxicity modifiers in human neurons and NERINE converge on PRL, revealing an intraneuronal -synuclein/prolactin stress response that may impact resilience to PD pathologies.

genetics↗

bioRxiv2024

α-Synuclein Conformations in Plasma Distinguish Parkinson's Disease from Dementia with Lewy Bodies

AbstractSpread and aggregation of misfolded -synuclein (aSyn) within the brain is the pathologic hallmark of Lewy body diseases (LBD), including Parkinsons disease (PD) and dementia with Lewy bodies (DLB). While evidence exists for multiple aSyn protein conformations, often termed "strains" for their distinct biological properties, it is unclear whether PD and DLB result from aSyn strain differences, and biomarkers that differentiate PD and DLB are lacking. Moreover, while pathological forms of aSyn have been detected outside the brain (e.g., in skin, gut, blood), the functional significance of these peripheral aSyn species is unclear. Here, we developed assays using monoclonal antibodies selective for two different aSyn species generated in vitro - termed Strain A and Strain B - and used them to evaluate human brain tissue, cerebrospinal fluid (CSF), and plasma, through immunohistochemistry, enzyme-linked immunoassay, and immunoblotting. Surprisingly, we found that plasma aSyn species detected by these antibodies differentiated individuals with PD vs. DLB in a discovery cohort (UPenn, n=235, AUC 0.83) and a multi-site replication cohort (Parkinsons Disease Biomarker Program, or PDBP, n=200, AUC 0.72). aSyn plasma species detected by the Strain A antibody also predicted rate of cognitive decline in PD. We found no evidence for aSyn strains in CSF, and ability to template aSyn fibrillization differed for species isolated from plasma vs. brain, and in PD vs. DLB. Taken together, our findings suggest that aSyn conformational differences may impact clinical presentation and cortical spread of pathological aSyn. Moreover, the enrichment of these aSyn strains in plasma implicates a non-central nervous system source.

neuroscience↗

bioRxiv2023

G51D mutation of the endogenous rat Snca gene disrupts synaptic localisation of α-synuclein priming for Lewy-like pathology

Point mutations in the SNCA gene, encoding -synuclein (Syn), are a known cause of familial Parkinsons disease. The G51D mutation causes early onset neurodegeneration with complex pathology. We used CRISPR/Cas9 in rats to introduce the G51D mutation into the endogenous Snca gene. Co-localisation immunostaining studies with synaptic proteins showed that SynG51D protein is no longer efficiently localised to synapses. Furthermore, biochemical isolation of synaptosomes from rat cortex demonstrated a significant depletion of Syn in SncaG51D/+ and SncaG51D/G51D rats. Unbiased proteomic investigation of the cortex identified significant synaptic dysregulation in SncaG51D/G51D animals. Finally, we compared the propensity for Lewy-like pathology of Snca+/+ and SncaG51D/G51D rats by stereotaxically delivering Syn pre-formed fibrils (PFFs) into the pre-frontal cortex. At an early time-point, 6 weeks post-injection, we observed discrete Lewy-like structures positive for phosphoserine-129-Syn (pS129-Syn) only in SncaG51D/G51D brains. At 26 weeks post-injection of PFFs SncaG51D/G51D brains exhibited intense, discrete pS129-Syn-positive structures, while Snca+/+ brains exhibited diffuse pS129-Syn immunostaining. Quantification of discrete pS129-Syn-positive structures revealed the striatum of SncaG51D/G51D rats had significantly more Lewy-like pathology than Snca+/+ rats. In summary, this novel SncaG51D rat model exhibits molecular characteristics of early synaptic dysfunction and is primed for Syn pathology.

neuroscience↗

bioRxiv2022

The MHC class II transactivator affects local and systemic immune responses in an α-synuclein seeded rat model for Parkinson's disease

BACKGROUNDParkinsons disease (PD) is characterized by alpha-synuclein (-Syn) pathology, neurodegeneration and neuroinflammation. HLA variants associated with PD and -Syn specific circulating CD4+ T lymphocytes in PD patients highlight the importance of antigen presentation in PD etiology. The class II transactivator (CIITA) is the major regulator of MHCII expression. Reduced Ciita levels significantly increase -Syn pathology, nigrostriatal neurodegeneration and behavioral deficits in -Syn seed-induced rat PD models. OBJECTIVETo characterize immune profiles associated with enhanced PD-like pathology observed in rats expressing lower Ciita levels (DA.VRA4) compared to the background (DA) strain. METHODSTo model PD, we combined rAAV-mediated -Syn overexpression in the substantia nigra with striatal injection of -Syn pre-formed fibrils (PFF). Immune profiles in brain and blood were analyzed by flow cytometry and multiplexed ELISA in naive rats, 4- and 8 weeks post rAAV injection. RESULTSFlow cytometry showed Ciita-dependent regulation of MHCII on microglia, brain macrophages and circulating myeloid cells. The MHCII-dependent microglial response peaked at 4 weeks post rAAV injection, whereas the MHCII levels in circulating myeloid cells peaked at 8 weeks. There was no major infiltration of macrophages or T lymphocytes into the CNS in response to -Syn and only subtle Ciita- and/or -Syn-dependent changes in the T lymphocyte compartment. Lower Ciita levels were consistently associated with higher TNF levels in serum. CONCLUSIONSThese results suggest that Ciita regulates susceptibility to PD-like pathology through minor but detectable changes in resident and peripheral immune cells and TNF levels, and indicate that mild immunomodulatory therapies could have therapeutic effects in PD.

neuroscience↗

bioRxiv2022

PKB/AKT attenuates Lewy Body-like pathology in primary neurons via Cathepsins B and D

Motor symptoms of Parkinsons disease (PD) are caused by the loss of dopamine neurons in the substantia nigra pars compacta. Current PD therapies offer only symptomatic relief, and the research on novel ways to stop or slow down the degeneration of dopamine neurons in PD attracts considerable efforts. A characteristic feature of PD is the abnormal accumulation and spread of misfolded -synuclein (-syn) protein leading to the formation of Lewy neurites and bodies in different neuronal populations, including dopamine neurons. The process of Lewy body formation compromises neuronal functions and contributes to the progression of the disease and neurodegeneration in PD patients. Treatments preventing -syn misfolding, aggregation and/or spread may therefore provide much-needed disease-modifying therapies for PD. We and others have demonstrated that -syn aggregation and Lewy body formation can be modelled in primary neuronal cultures treated with -syn pre-formed fibrils (PFFs). Using this approach, we have identified several factors preventing the accumulation of phosphorylated -syn in dopamine neurons in vitro and in vivo. In particular, we and others have demonstrated a prominent role of PKB/AKT pathway activation in promoting the survival of dopamine neurons. Here, we utilize lentiviral vectors (LVs) to express constitutively active myristoylated serine-threonine protein kinase AKT1 (mAKT1) in primary dopamine neurons. We show that lentivirally-delivered mAKT1 promoted survival of dopamine neurons after thapsigargin-induced endoplasmic reticulum (ER) stress and prevented fibril-induced accumulation of phosphorylated -syn. Furthermore, using pharmacological tools, we demonstrate that this activity of mAKT1 is dependent on the functional Cathepsins B and D. These proof-of-principle results demonstrate the critical importance of lysosomal enzymes for processing misfolded -syn and establish a basis for the use of LVs to develop neuroprotective gene therapy strategies for dopamine neurons in PD.

neuroscience↗

bioRxiv2022

Generation of floxed alleles for cell-specific knockout in zebrafish

A benefit of the zebrafish as a model is its amenability to genetic approaches. However, a lack of conditional deletion alleles makes it a challenge to perform stage- or cell-specific knockout. Here, we initially applied an existing protocol to establish a floxed allele for gata2a and encountered several issues, including off-target integration and incomplete knock-in. To address these problems, we developed a protocol incorporating simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counter-screening to identify off-target insertions. We applied this protocol to establish a floxed allele of foxc1a in a single generation. We demonstrate the utility of our floxed alleles by verifying Cre-dependent deletion, which yielded expected phenotypes in each case. Finally, we used the floxed gata2a allele to demonstrate an endothelial autonomous requirement in lymphatic valve development. Together, our results provide a framework for straightforward generation and application of floxed alleles in zebrafish.

genetics↗

bioRxiv2022

Optimization of NLS Composition Improves CRISPR-Cas12a Editing Rates in Human Primary Cells

Type V CRISPR-Cas12a systems are an attractive alternative nuclease platform for specific genome editing applications. However, previous studies demonstrate that there is a gap in overall activity between Cas12a and Cas9 in primary cells. Here we describe optimization to the nuclear localization signal composition and architecture of Cas12a to facilitate highly efficient targeted mutagenesis in mammalian cell lines (HEK293T, Jurkat, and K562 cells) and primary cells (NK cells and CD34+ HSPCs), regardless of Cas12a ortholog. A 3xNLS Cas12a architecture resulted in the most robust editing platform. The improved editing activity of Cas12a in both NK cells and CD34+ HSPCs resulted in pronounced phenotypic changes associated with target gene editing. Lastly, we demonstrated that optimization of the NLS composition and architecture of Cas12a did not decrease the specificity of editing in HEK293T and CD34+ HSPCs. Our new Cas12a NLS variant provides an improved nuclease platform for therapeutic genome editing.

molecular biology↗

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

Global mRNA and chromatin accessibility profiling elucidate how HIV-1 perturbs ILC and NK cell subsets

The interrelatedness of human blood innate lymphoid cell (ILC) subsets, and how they are perturbed by HIV-1, remains unclear. Transcriptional and chromatin profiling separated blood ILCs into ILC2s, ILCPs, one cluster that included CD56dim and CD56-NK cells, and CD56hiNK cells that have features of both CD56dim/-NK cells and ILCs. In contrast to mice, human NK cells expressed tissue repair protein amphiregulin (AREG), with greater production by CD56hiNK cells than by ILCs. AREG was induced by TCF7/WNT signaling, IL-2, or IL-15, but not by inflammatory cytokines, and was inhibited by TGFB1, a cytokine elevated in people living with HIV-1. NK cell knockout of the TGFB1-stimulated WNT antagonist RUNX3 increased AREG production. In people living with HIV-1, AREG+NK cell percentage correlated with numbers of ILCs and CD4+T cells, and correlated inversely with inflammatory cytokine IL-6. RNA-Seq showed increased antiviral gene expression in all ILC subsets from people who were HIV-1 viremic, and increased expression of anti-inflammatory gene MYDGF in CD56hiNK cells from elite controllers. Functionally-defective CD56-NK cells were increased in people living with HIV-1 in inverse correlation with CD56dimNK cells, ILCs, and CD4+T cells. Experiments with human PBMCs ex vivo and in humanized mice revealed that CD4+T cells and their production of IL-2 prevented CD56dim transition to CD56-NK cells by activating mTOR, and, in people living with HIV-1, plasma IL-2 correlated with CD4+T cell number but not with CD8+T cells. These studies clarify how ILC subsets are interrelated and provide insight into how HIV-1 infection disrupts NK cells, including homeostatic functions of NK cells discovered here. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/440368v3_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@9d6be2org.highwire.dtl.DTLVardef@115f0e8org.highwire.dtl.DTLVardef@17b1a28org.highwire.dtl.DTLVardef@f262e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2020

CRISPR-enhanced human adipocyte 'browning' as cell therapy for metabolic disease

Obesity and type 2 diabetes (T2D) are associated with poor tissue responses to insulin1,2, disturbances in glucose and lipid fluxes3-5 and comorbidities including steatohepatitis6 and cardiovascular disease7,8. Despite extensive efforts at prevention and treatment9,10, diabetes afflicts over 400 million people worldwide11. Whole body metabolism is regulated by adipose tissue depots12-14, which include both lipid-storing white adipocytes and less abundant "brown" and "brite/beige" adipocytes that express thermogenic uncoupling protein UCP1 and secrete factors favorable to metabolic health15-18. Application of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing19,20 to enhance "browning" of white adipose tissue is an attractive therapeutic approach to T2D. However, the problems of cell-selective delivery, immunogenicity of CRISPR reagents and long term stability of the modified adipocytes are formidable. To overcome these issues, we developed methods that deliver complexes of SpyCas9 protein and sgRNA ex vivo to disrupt the thermogenesis suppressor gene NRIP121,22 with near 100% efficiency in human or mouse adipocytes. NRIP1 gene disruption at discrete loci strongly ablated NRIP1 protein and upregulated expression of UCP1 and beneficial secreted factors, while residual Cas9 protein and sgRNA were rapidly degraded. Implantation of the CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreased adiposity and liver triglycerides while enhancing glucose tolerance compared to mice implanted with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic modification of human adipocytes without exposure of the recipient to immunogenic Cas9 or delivery vectors.

bioengineering↗

bioRxiv2020

Degradation of ATFS-1 by LONP-1 promotes deleterious mitochondrial genome heteroplasmy

The accumulation of deleterious mitochondrial genomes ({Delta}mtDNAs) underlies inherited mitochondrial diseases and contributes to the aging-associated decline in mitochondrial function. In heteroplasmic cells, oxidative phosphorylation (OXPHOS) function declines as the population of {Delta}mtDNAs increase relative to wildtype mtDNAs. In response to mitochondrial perturbations, the bZIP protein ATFS-1 induces a transcription program to promote the recovery of mitochondrial function. Paradoxically, ATFS-1 is also required to maintain {Delta}mtDNAs in heteroplasmic worms. However, the mechanism(s) by which ATFS-1 promotes {Delta}mtDNA accumulation relative to wildtype mtDNAs is unclear. Here, we show that mitochondrial-localized ATFS-1 binds almost exclusively to {Delta}mtDNAs in heteroplasmic worms. Moreover, we demonstrate that mitochondrial ATFS-1 promotes the preferential binding of the mtDNA replicative polymerase (POLG) to {Delta}mtDNAs. Interestingly, inhibition of the mtDNA-bound protease LONP-1 increased ATFS-1 and POLG binding to wildtype mtDNAs. Furthermore, LONP-1 inhibition in C. elegans and human cybrid cells improved the heteroplasmy ratio and restored OXPHOS function. Our findings suggest that ATFS-1 promotes mtDNA replication by recruiting POLG to mtDNA in a manner that is antagonized by LONP-1. We speculate that this mechanism promotes the repair and expansion of the mitochondrial network by synchronizing mtDNA replication with UPRmt activation driven by nuclear ATFS-1 activity. However, this repair mechanism cannot resolve OXPHOS defects in mitochondria harboring {Delta}mtDNAs, resulting in an accumulation of ATFS-1 in dysfunctional mitochondria and constitutive replication of {Delta}mtDNAs.

cell biology↗

bioRxiv2020

ZNF410 represses fetal globin by devoted control of CHD4/NuRD

Major effectors of adult-stage fetal globin silencing include the transcription factors (TFs) BCL11A and ZBTB7A/LRF and the NuRD chromatin complex, although each has potential on-target liabilities for rational {beta}-hemoglobinopathy therapeutic inhibition. Here through CRISPR screening we discover ZNF410 to be a novel fetal hemoglobin (HbF) repressing TF. ZNF410 does not bind directly to the{gamma} -globin genes but rather its chromatin occupancy is solely concentrated at CHD4, encoding the NuRD nucleosome remodeler, itself required for HbF repression. CHD4 has two ZNF410-bound regulatory elements with 27 combined ZNF410 binding motifs constituting unparalleled genomic clusters. These elements completely account for ZNF410s effects on{gamma} -globin repression. Knockout of ZNF410 reduces CHD4 by 60%, enough to substantially de-repress HbF while avoiding the cellular toxicity of complete CHD4 loss. Mice with constitutive deficiency of the homolog Zfp410 are born at expected Mendelian ratios with unremarkable hematology. ZNF410 is dispensable for human hematopoietic engraftment potential and erythroid maturation unlike known HbF repressors. These studies identify a new rational target for HbF induction for the {beta}-hemoglobin disorders with a wide therapeutic index. More broadly, ZNF410 represents a special class of gene regulator, a conserved transcription factor with singular devotion to regulation of a chromatin subcomplex.

genetics↗

bioRxiv2020

Snca-GFP knock-in mice reflect patterns of endogenous expression and pathological seeding

Alpha-synuclein (aSyn) participates in synaptic vesicle trafficking and synaptic transmission, but its misfolding is also strongly implicated in Parkinsons disease (PD) and other neurodegenerative disorders known as synucleinopathies where misfolded aSyn accumulates in different regions of the central and peripheral nervous systems. Although increased aSyn expression levels or altered aggregation propensities likely underlie familial PD with SNCA amplification or mutations, the majority of synucleinopathies arise sporadically, indicating that disease can develop under normal levels of wildtype aSyn. We report here the development and characterization of a mouse line expressing an aSyn-GFP fusion protein under the control of native Snca regulatory elements. Regional and subcellular localization of the aSyn-GFP fusion protein in brains and peripheral tissues of knock-in (KI) mice are indistinguishable from that of wildtype littermates. Importantly, similar to wildtype aSyn, aSyn-GFP disperses from synaptic vesicles upon membrane depolarization, indicating that the tag does not alter normal aSyn dynamics at synapses. In addition, intracerebral injection of aSyn pre-formed fibrils into KI mice induced the formation of aSyn-GFP inclusions with a distribution pattern similar to that observed in wildtype mice, albeit with attenuated kinetics due to the GFP tag. We anticipate that this new mouse model will facilitate in vitro and in vivo studies requiring in situ detection of endogenous aSyn, therefore providing new insights into aSyn function in health and disease. Significance StatementAlpha-synuclein (aSyn) participates in synaptic vesicle function and represents a major component of the Lewy pathology found in Parkinsons and related neurodegenerative diseases. The function of aSyn and the sequence of events leading to its aggregation and neurotoxicity are not fully understood. Here we present a new mouse model in which Enhanced Green Fluorescence Protein (GFP) has been knocked-in at the C-terminal of the Snca gene. The resulting fusion protein shows identical expression and localization to that of wildtype animals, is functional, and is incorporated into pathological aggregates in vitro and in vivo. This new tool allows for monitoring aSyn under a variety of physiological and pathological conditions, and may uncover additional insights into its function and dysfunction.

neuroscience↗

bioRxiv2020

Developmental exposure to the organochlorine pesticide dieldrin causes male-specific exacerbation of α-synuclein-preformed fibril-induced toxicity and motor deficits

Human and animal studies have shown that exposure to the organochlorine pesticide dieldrin is associated with increased risk of Parkinsons disease (PD). Previous work showed that developmental dieldrin exposure increased neuronal susceptibility to MPTP toxicity in male C57BL/6 mice, possibly via changes in dopamine (DA) packaging and turnover. However, the relevance of the MPTP model to PD pathophysiology has been questioned. We therefore studied dieldrin-induced neurotoxicity in the -synuclein (-syn)-preformed fibril (PFF) model, which better reflects the -syn pathology and toxicity observed in PD pathogenesis. Specifically, we used a "two-hit" model to determine whether developmental dieldrin exposure increases susceptibility to -syn PFF-induced synucleinopathy. Dams were fed either dieldrin (0.3 mg/kg, every 3-4 days) or vehicle corn oil starting 1 month prior to breeding and continuing through weaning of pups at postnatal day 22. At 12 weeks of age, male and female offspring received intrastriatal PFF or control saline injections. Consistent with the male-specific increased susceptibility to MPTP, our results demonstrate that developmental dieldrin exposure exacerbates PFF-induced toxicity in male mice only. Specifically, in male offspring, dieldrin exacerbated PFF-induced motor deficits on the challenging beam and increased DA turnover in the striatum 6 months after PFF injection. However, male offspring showed neither exacerbation of phosphorylated -syn (pSyn) aggregation in the substantia nigra (SN) at 1 or 2 months post-PFF injection, nor exacerbation of PFF-induced TH and NeuN loss in the SN 6 months post-PFF injection. Collectively, these data indicate that developmental dieldrin exposure produces a male-specific increase in neuronal vulnerability to synucleinopathy. This sex-specific result is consistent with both previous work in the MPTP model, our previously reported sex-specific effects of this exposure paradigm on the male and female epigenome, and the higher prevalence and more severe course of PD in males. The novel two-hit environmental toxicant/PFF exposure paradigm established in this project can be used to explore the mechanisms by which other PD-related exposures alter neuronal vulnerability to synucleinopathy in sporadic PD. HighlightsO_LIDevelopmental dieldrin exposure increases -syn-PFF-induced motor deficits C_LIO_LIDevelopmental dieldrin exposure increases PFF-induced deficits in DA handling C_LIO_LIDevelopmental dieldrin exposure does not affect PFF-induced loss of nigral neurons C_LIO_LIThis is a novel paradigm modeling how environmental factors increase risk of PD C_LIO_LIFemale mice show PFF-induced pathology, but no PFF-induced motor deficits. C_LI

neuroscience↗