Biology subjects

McPherson, P. S.

Publications and source records attributed to McPherson, P. S..

13 recordsLinked to original sources

bioRxiv2026

Myeloid PINK1 represses mtDNA release and immune signaling that impacts neuronal pathology in patient-derived idiopathic PD models

Parkinsons disease (PD) is a neurodegenerative disorder marked by the development of cardinal motor deficits preceded by a protracted prodromal period of non-motor symptoms often involving the gastrointestinal (GI) tract. There is an emerging consensus that both the peripheral immune system and local neuroinflammation play key roles in the etiology of PD. We previously demonstrated a critical function for the Parkinsons related proteins PINK1 and Parkin as repressors of the innate to adaptive immune response in cultured cells and mouse models of infection. However, it remained unclear whether these processes were conserved in patient-derived models, and precisely how immune signaling may ultimately drive the death of dopaminergic neurons. Here we show that GI infection of PINK1 knockout (KO) mice triggered acute neurodegeneration which was evident early in the enteric nervous system. Treating wild type enteric or dopaminergic neurons with conditioned medium from immune-stimulated PINK1 KO macrophages was sufficient to promote neuronal disruption in both mouse and human neurons in vitro. Within immune-activated macrophages, we reveal that loss of PINK1 led to an enhanced release of mitochondrial DNA (mtDNA) within mitochondrial derived vesicles, leading to the activation of cGAS/STING pathways. These changes were seen in both mouse/human in vitro models and in PD patient-derived primary macrophages. Notably, pharmacological modulation using a PINK1 activator with high therapeutic potential attenuated pro-inflammatory profiles elicited by the mtDNA-dependent STING/NF-{kappa}B pathway in idiopathic patient-derived macrophages. Ultimately, our study lays the foundation for understanding PINK1-related peripheral macrophage mechanisms in idiopathic PD and provides a target for further development to treat the disease at early stages. Graphical abstractPINK1-related immune mechanisms of Parkinsons disease and associations with early neurodegenerative events. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/694713v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@6f6488org.highwire.dtl.DTLVardef@13139aaorg.highwire.dtl.DTLVardef@c0d4d1org.highwire.dtl.DTLVardef@1d41e25_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Cell type specific analysis of ALS associated proteins reveals immune-regulated targets

A substantial fraction of amyotrophic lateral sclerosis (ALS)-associated proteins remain poorly characterized, in part because of the limited availability of validated research antibodies. We established knockout (KO)-based antibody characterization workflows and demonstrated that widely used antibodies against the major ALS-associated protein C9orf72 lacked specificity (Laflamme et al., 2019). We subsequently scaled this framework to systematically benchmark research antibodies, revealing that up to 61% fail to perform as recommended by manufacturers (Ayoubi et al., 2023). Here, we extend this approach by establishing the ALS-Reproducible Antibody Platform (ALS-RAP), a comprehensive effort to generate a publicly available dataset of KO-validated antibodies targeting proteins encoded by ALS risk genes. In total, we characterized 303 antibodies against 33 ALS-associated proteins to identify high-quality reagents for use in western blot, immunoprecipitation, and immunofluorescence. Using these antibodies, we profiled protein levels across human induced pluripotent stem cell (iPSC)-derived and primary neurological cell types. These analyses revealed diverse cellular distributions and higher levels of several ALS-associated proteins in glial populations, consistent with emerging evidence for immune contributions to ALS. Together, ALS-RAP provides a validated antibody toolbox and protein expression resource to support the study of ALS-associated proteins.

neuroscience↗

bioRxiv2023

DENND6A couples Arl8b to a Rab34/RILP/dynein complex regulating retrograde lysosomal trafficking and autophagy

Lysosomes help maintain cellular proteostasis, and defects in lysosomal positioning and function lead to diseases including neurodegenerative disorders. The spatiotemporal distribution of lysosomes is regulated by small GTPases, including Rabs, which are activated by guanine nucleotide exchange factors (GEFs). DENN domain-bearing proteins are the largest family of Rab GEFs. Using a cell-based assay, we screened the DENN domain protein family member DENND6A for potential GEF activity toward all 60 Rabs and identified 20 potential DENND6A substrates, including Rab34. Here, we demonstrate that DENND6A activates Rab34, which recruits a RILP/dynein complex to the lysosomes, promoting lysosomal retrograde trafficking from the cell periphery to the minus ends of microtubules. Further, we identify DENND6A as an effector of Arl8b, a major regulatory GTPase on lysosomes. Initially known for promoting anterograde transport of lysosomes, Arl8b also facilitates lysosomal retrograde transport. We demonstrate that Arl8b recruits DENND6A to peripheral lysosomes to activate Rab34 and initiate retrograde transport, regulating nutrient-dependent lysosomal juxtanuclear repositioning and loss of DENND6A impairs autophagic flux. Our findings support a model whereby Arl8b/DENND6A/Rab34-dependent lysosomal retrograde trafficking controls autophagy.

cell biology↗

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

A dual hit of α-synuclein internalization and immune challenge leads to formation and maintenance of Lewy body-like inclusions in human dopaminergic neurons

Lewy bodies (LBs), rich in -synuclein, are a hallmark of Parkinsons disease (PD). Understanding their biogenesis is likely to provide insight into the pathophysiology of PD, yet a cellular model for LB formation remains elusive. The realization that the immune challenge is a trigger for neurodegenerative diseases has been a breakthrough in the understanding of PD. Here, iPSC-derived human dopaminergic (DA) neurons from multiple healthy donors were found to form LB-like inclusions following treatment with - synuclein preformed fibrils, but only when coupled to an immune challenge (interferon-gamma or interleukin-1 beta) or when co-cultured with activated microglia. Human cortical neurons derived from the same iPSC lines did not form LB-like inclusions. Exposure to interferon-gamma impairs autophagy in a lysosomal-specific manner in vitro, similar to the disruption of proteostasis pathways that contribute to PD. We find that lysosomal membrane proteins LAMP1 and LAMP2 and transcription factors regulating lysosomal biogenesis and function are downregulated in DA but not cortical neurons. Finally, due to the excellent sample preservation afforded by cells compared to post-mortem PD brain tissue, we conclude that the LB-like inclusions in DA neurons are membrane-bound, suggesting they are not limited to the cytoplasmic compartment. In Brief O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/542776v3_figabs.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1f41855org.highwire.dtl.DTLVardef@69798dorg.highwire.dtl.DTLVardef@ab55bdorg.highwire.dtl.DTLVardef@8a16b8_HPS_FORMAT_FIGEXP M_FIG C_FIG Bayati et al. identify that iPSC-derived dopaminergic neurons undergoing a dual hit treatment of exogenous -synuclein fibrils and proinflammatory cytokines form Lewy body-like inclusions. The dual hit treatment also led to the downregulation of lysosomal proteins. Characterization of inclusions revealed that inclusions were membrane-bound and LC3B-positive, suggesting they are dysfunctional autophagosomes. HighlightsO_LI-synuclein preformed fibril administration coupled with Interferon-gamma exposure leads dopaminergic neurons to form Lewy body-like inclusions C_LIO_LIInclusions are filamentous, membranous, and filled with aberrant organelles C_LIO_LIImpaired autophagic flux and downregulation of TFEB, NRF2, LAMP1, and LAMP2 correlated with inclusion formation C_LIO_LIActivation of NRF2 through the treatment of neurons with the antioxidant perillaldehyde, prevents inclusion formation C_LI

neuroscience↗

bioRxiv2023

DENND2B activates Rab35 at the intercellular bridge regulating cytokinetic abscission and tetraploidy

Cytokinesis is the final stage of cell division. Successful cytokinesis requires membrane trafficking pathways regulated by Rabs, molecular switches activated by guanine nucleotide exchange factors (GEFs). Late in cytokinesis, an intercellular cytokinetic bridge (ICB) connecting the two daughter cells undergoes abscission, which requires depolymerization of actin. Rab35 recruits MICAL1 to oxidate and depolymerize actin filaments. We report that DENND2B, a protein previously implicated in cancer, mental retardation and multiple congenital disorders functions as a GEF for Rab35 and recruits and activates the GTPase at the ICB. Unexpectedly, the N-terminal region of DENND2B interacts with an active mutant of Rab35, suggesting that DENND2B is both a Rab35 GEF and effector. Knockdown of DENND2B delays abscission resulting in increased multinucleated cells and overaccumulation of F-actin at the ICB. F-actin accumulation leads to formation of a chromatin bridge, a process known to activate the NoCut/abscission checkpoint, and DENND2B knockdown actives Aurora B kinase, a hallmark of checkpoint activation. This study identifies DENND2B as a crucial player in cytokinetic abscission and provides insight into the multisystem disorder associated with DENND2B mutation.

cell biology↗

bioRxiv2022

The identification of potent and selective antibodies for Serine/threonine-protein kinase TBK1, for use in immunoblot, immunofluorescence and immunoprecipitation

TBK1 is a serine-threonine kinase that has been linked to a number of diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. Reproducible research on TBK1 has been hampered by the lack of well characterized antibodies. In this study, we characterized 11 commercial antibodies for immunoblot, immunofluorescence and immunoprecipitation, using a knock-out cell line as the control. For each application, we identified several potent and selective antibodies that will facilitate studies on TBK1.

biochemistry↗

bioRxiv2022

A novel form of macropinocytosis mediates ultra-rapid transfer of pathological alpha- synuclein to lysosomes

The nervous system spread of alpha-synuclein fibrils is thought to cause Parkinsons disease (PD) and other synucleinopathies, yet the mechanisms underlying internalization and cellular spread are enigmatic. Here we use confocal and super-resolution microscopy, subcellular fractionation and electron microscopy (EM) of immunogold labelled alpha-synuclein preformed fibrils (PFF) to demonstrate that this fibril form of alpha-synuclein undergoes rapid internalization and is targeted directly to lysosomes in as little as 2 minutes. Uptake of PFF is disrupted by macropinocytic inhibitors and circumvents classical endosomal pathways. Immunogold-labelled PFF are seen at the highly curved inward edge of membrane ruffles, in newly formed macropinosomes, in multivesicular bodies and in lysosomes. While most fibrils remain in lysosomes, a portion is transferred to neighboring naive cells along with markers of exosomes. These data indicate that PFF use a unique internalization mechanism as a component of cell-to-cell propagation.

cell biology↗

bioRxiv2022

Host kinase CSNK2 is a target for inhibition of pathogenic β-coronaviruses including SARS-CoV-2

Inhibition of the protein kinase CSNK2 with any of 30 specific and selective inhibitors representing different chemotypes, blocked replication of pathogenic human and murine {beta}-coronaviruses. The potency of in-cell CSNK2A target engagement across the set of inhibitors correlated with antiviral activity and genetic knockdown confirmed the essential role of the CSNK2 holoenzyme in {beta}-coronavirus replication. Spike protein uptake was blocked by CSNK2A inhibition, indicating that antiviral activity was due in part to a suppression of viral entry. CSNK2A inhibition may be a viable target for development of new broad spectrum anti-{beta}-coronavirus drugs. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/474779v3_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@5d2799org.highwire.dtl.DTLVardef@1d2de35org.highwire.dtl.DTLVardef@fa852eorg.highwire.dtl.DTLVardef@13da300_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Role of the ubiquitin ligase ITCH in clathrin-mediated endocytosis of the epidermal growth factor receptor

Once activated by ligand, epidermal growth factor receptor (EGFR) is endocytosed in clathrin-coated pits. ITCH is an E3 ubiquitin ligase that interacts with and ubiquitinates several proteins involved in clathrin-mediated endocytosis (CME) including endophilin. To further investigate the function of ITCH in EGFR endocytosis, the internalization of fluorescent EGF was measured in ITCH-/- HeLa cells. In the absence of ITCH, there was a significant decrease in the CME of EGF. Rescue experiments using wild-type ITCH confirmed the importance of the protein for normal EGF uptake. ITCH point mutations that disrupt the interaction of ITCH with endophilin failed to rescue the defects in EGFR uptake, as did a non-catalytic form of ITCH. ITCH-/- cells also displayed a delay in the rate of phospho-EGFR degradation as well as prolonged ERK1/2 signaling. Our study uncovers a pathway regulating EGFR trafficking and reveals for the first time that the protein ITCH is required for CME of EGFR.

cell biology↗

bioRxiv2020

SARS-CoV-2 uses clathrin-mediated endocytosis to gain access into cells

With more than 51 million cases and 1.3 million deaths, and with the resulting social upheaval, the COVID-19 pandemic presents one of the greatest challenges ever to human society. It is thus vital to fully understand the biology of SARS-CoV-2, the causative agent of COVID-19. SARS-CoV-2 uses its spike glycoprotein to interact with the cell surface as a first step in the infection process. Using purified spike glycoprotein and lentivirus pseudotyped with spike glycoprotein, we now demonstrate that following engagement with the plasma membrane, SARS-CoV-2 undergoes rapid clathrin-mediated endocytosis. This suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system, and importantly clathrin-heavy chain knockdown, which blocks clathrin-mediated endocytosis, reduces viral infectivity. This discovery reveals important new information about the basic biology of SARS-CoV-2 infectivity.

cell biology↗

bioRxiv2020

Early defects in lysosomal storage diseases disrupt excitatory synaptic transmission

At least two thirds of patients affected with lysosomal storage disorders (LSD) exhibit neurological symptoms. For mucopolysaccharidosis type IIIC (MPS IIIC, Sanfilippo disease type C) caused by mutations in the HGSNAT gene and lysosomal storage of heparan sulfate the major burden is progressive and severe neuropsychiatric problems, mental retardation, and dementia though to be mainly mediated by neurodegeneration. HGSNAT knockout mice match human clinical phenotype and develop hyperactivity followed by memory impairment and death. In order to understand whether early clinical symptoms in MPS IIIC mice occurring before the onset of massive neurodegeneration are caused by neuronal dysfunction we studied synaptic transmission and morphology in cultured hippocampal and CA1 pyramidal neurons of MPSIIIC mice. Synaptic spines were also studied in other mouse LSD models and postsynaptic densities in post-mortem cortices of human neurological MPS patients. Cultured hippocampal and CA1 pyramidal neurons of MPS IIIC mice showed a drastic decrease or abnormal distribution of multiple pre- and postsynaptic proteins that could be rescued in vitro and in vivo by virus-mediated gene correction. Dendritic spine densities were immature in cultured hippocampal MPS IIIC mouse neurons and reduced in pyramidal neurons of mouse models of MPS IIIC and other (Tay-Sachs, sialidosis) LSD starting from postnatal day 10. MPS IIIC neurons presented alterations in frequency and amplitude of miniature excitatory and inhibitory postsynaptic currents, sparse synaptic vesicles, reduced postsynaptic densities, disorganised microtubule network and partially impaired axonal transport of synaptic proteins. Postsynaptic densities were also reduced in post-mortem cotrees of human MPS I, II, IIIA, C and D patients suggesting that the pathology is common for these neurological LSD. Together, our results demonstrate that lysosomal storage causes alterations in synaptic structure and abnormalities in neurotransmission originating from disrupted vesicular transport and preceding the first cognitive symptoms and suggest drugs known to affect synaptic transmission can be potentially applied to treat behavioral and cognitive defects in neurological LSD patients.

neuroscience↗

bioRxiv2020

An Arf/Rab cascade controls the growth and invasiveness of glioblastoma

Glioblastoma is the most common and deadly malignant brain cancer. We now demonstrate that loss of function of the endosomal GTPase Rab35 in human brain tumor initiating cells (BTICs) increases glioblastoma growth and decreases animal survival following BTIC implantation in mouse brain. Mechanistically, we identify that the GTPase Arf5 interacts with the guanine nucleotide exchange factor (GEF) for Rab35, DENND1/connecdenn and allosterically enhances its GEF activity towards Rab35. Knockdown of either Rab35 or Arf5 increases cell migration, invasiveness and self-renewal in culture and enhances the growth and invasiveness of BTIC-initiated brain tumors in mice. RNAseq of the tumors reveals upregulation of the tumor-promoting transcription factor SPOCD1, and disruption of the Arf5/Rab35 axis in glioblastoma cells leads to strong activation of the epidermal growth factor receptor with resulting enhancement of SPOCD1 levels. These discoveries reveal an unexpected cascade between an Arf and a Rab and indicate a role for the cascade, and thus endosomal trafficking, in brain tumors.

cancer biology↗