Biology subjects

Mandell, M. A.

Publications and source records attributed to Mandell, M. A..

6 recordsLinked to original sources

bioRxiv2025

A role for CASM in the repair of damaged Golgi architecture

The term CASM describes a process in which MAP1LC3B/LC3B and other Atg8-family proteins are covalently ligated to lipids in damaged endomembranes. While CASM is commonly described as a cytoprotective response to multiple types of membrane damage, how CASM helps cells maintain homeostasis is still unclear. Here, we show that CASM maintains Golgi apparatus architecture following the loss of TRIM46, a ubiquitin ligase with roles in microtubule organization. TRIM46 deficient cells were notable for enhanced TFEB-driven lysosomal biogenesis and Golgi ribbon fragmentation, with colocalization of the trans-Golgi marker TGOLN2 and the Atg8-family proteins LC3B and GABARAP. Further studies revealed that the Golgi Atg8ylation seen in TRIM46 knockout cells was not degradative and mechanistically resembled CASM. Genetic inhibition of CASM in TRIM46 deficient cells reduced TFEB activation and exacerbated the Golgi morphology defects, suggesting that CASM contributes to Golgi repair. Accordingly, Golgi reformation after drug-induced fragmentation was impaired upon knockdown of CASM genes. Together, these studies identify lysosomal biogenesis and CASM as coordinated features of a Golgi damage response, with CASM acting to preserve Golgi integrity.

cell biology↗

bioRxiv2025

Pathological tau activates inflammatory nuclear factor-kappa B (NF-κB) and pT181-Qβ vaccine attenuates NF-κB in PS19 tauopathy mice

Tau regulates neuronal integrity. In tauopathy, phosphorylated tau detaches from microtubules and aggregates, and is released into the extracellular space. Microglia are the first responders to the extracellular tau, a danger/damage-associated molecular pattern (DAMP), which can be cleared by proteostasis and activate innate immune response gene expression by nuclear factor-kappa B (NF-{kappa}B). However, longitudinal NF-{kappa}B activation in tauopathies and whether pathological tau (pTau) contributes to NF-{kappa}B activity is unknown. Here, we tau oligomers from human Alzheimers disease brain (AD-TO) activate NF-{kappa}B in mouse microglia and macrophages reducing the I{kappa}B via promoting its secretion in the extracellular space. NF-{kappa}B activity peaks at 9- and 11-months age in PS19Luc+ and hTauLuc+ mice, respectively. Reducing pTau via pharmacological (DOX), genetic (Mapt-/-) or antibody-mediated neutralization (immunization with pT181-Q{beta} vaccine) reduces NF-{kappa}B activity, and together suggest pTau is a driver of NF-{kappa}B and chronic neuroinflammation tauopathies. SummaryNeuronal tau activates microglial NF-{kappa}B constitutively by secreting its inhibitor I{kappa}B. NF-{kappa}B activation in PS19Luc+ and hTauLuc+ mice peaks at 9- and 11-months of age, respectively. Neutralizing pTau with pT181-Q{beta} vaccine (targeting phosphorylated threonine 181 tau) alleviates NF-{kappa}B activity in tauopathy mice.

neuroscience↗

bioRxiv2023

TBK1 is ubiquitinated by TRIM5alpha to assemble mitophagy machinery

Ubiquitination of mitochondrial proteins provides a basis for the downstream recruitment of mitophagy machinery, yet whether ubiquitination of the machinery itself contributes to mitophagy is unknown. Here, we show that K63-linked polyubiquitination of the key mitophagy regulator TBK1 is essential for its mitophagy functions. This modification is catalyzed by the ubiquitin ligase TRIM5. Mitochondrial damage triggers TRIM5s auto-ubiquitination and its interaction with ubiquitin-binding autophagy adaptors including NDP52, optineurin, and NBR1. Autophagy adaptors, along with TRIM27, enable TRIM5 to engage with TBK1. TRIM5 with intact ubiquitination function is required for the proper accumulation of active TBK1 on damaged mitochondria in Parkin-dependent and Parkin-independent mitophagy pathways. Additionally, we show that TRIM5 can directly recruit autophagy initiation machinery to damaged mitochondria. Our data support a model in which TRIM5 provides a self-amplifying, mitochondria-localized, ubiquitin-based, assembly platform for TBK1 and mitophagy adaptors that is ultimately required to recruit the core autophagy machinery.

cell biology↗

bioRxiv2022

Quantitative single-cell analysis of Leishmania major amastigote differentiation demonstrates variably extended expression of the lipophosphoglycan (LPG) virulence factor in different host cell types

Immediately following their deposition into the mammalian host by an infected sand fly vector, Leishmania parasites encounter and are engulfed by a variety of cell types. From there, parasites may transit to other cell types, primarily macrophages or dendritic cells, where they replicate and induce pathology. During this time, Leishmania cells undergo a dramatic transformation from the motile non-replicating metacyclic stage to the non-motile replicative amastigote stage, a differentiative process that can be termed amastigogenesis. To follow this at the single cell level, we identified a suite of experimental landmarks delineating different stages of amastigogenesis qualitatively or quantitatively, including new uses of amastigote-specific markers that showed interesting cellular localizations at the anterior or posterior ends. We compared amastigogenesis in synchronous infections of peritoneal and bone-marrow derived macrophages (PEM, BMM) or dendritic cells (BMDC). Overall, the marker suite expression showed an orderly transition post-infection with similar kinetics between host cell types, with the emergence of several amastigote traits within 12 hours, followed by parasite replication after 24 hours, with parasites in BMM or BMDC initiating DNA replication more slowly. Lipophosphoglycan (LPG) is a Leishmania virulence factor that facilitates metacyclic establishment in host cells but declines in amastigotes. Whereas LPG expression was lost by parasites within PEM by 48 hours, >40% of the parasites infecting BMM or BMDC retained metacyclic-level LPG expression at 72 hr. Thus L. major may prolong LPG expression in different intracellular environments, thereby extending its efficacy in promoting infectivity in situ and during cell-to-cell transfer of parasites expressing this key virulence factor. Author SummaryLeishmaniasis caused by species of the trypanosomatid protozoan parasite Leishmania is an important widespread global health problem. Humans become infected by Leishmania following the bite of sand flies bearing the flagellated metacyclic promastigote stage, and the first 24-48 hours thereafter are critical to the outcome of infection. During this time, parasites are engulfed by several host cell types, where they differentiate into the rounded amastigote stage, adapted for intracellular survival and proliferation, with concomitant changes in metabolism and virulence factor expression. We developed a suite of markers that allowed us to monitor promastigote-to-amastigote differentiation (amastigogenesis) on a single-cell level. Two showed amastigote-specific expression and localized to the anterior or posterior regions. Our marker suite allowed us to chart the course of amastigogenesis in different host cell environments and to determine the timing of amastigote development relative to the initiation of parasite replication and the expression of the virulence factor lipophosphoglycan (LPG). We report that the amastigogenesis process follows a determined sequence of events that occurs prior to parasite replication. In contrast, parasites may respond to different host cell environments by prolonging LPG expression, thereby extending the duration of its pro-infectivity functions within or in transit between host cell destinations.

microbiology↗

bioRxiv2021

Interactomic analysis reveals a new homeostatic role for the HIV restriction factor TRIM5α in mitophagy

The protein TRIM5 has multiple roles in anti-retroviral defense, but the mechanisms underlying TRIM5 action are unclear. Here, we used an APEX2-based proteomics approach to identify TRIM5-interacting proteins. Analysis of the TRIM5 interactome found proteins participating in a wide variety of cellular functions including regulating antiviral signaling pathways. We used this data set to uncover a novel role for TRIM5 in mitophagy, an autophagy-based mode of mitochondrial quality control that is compromised in multiple human diseases. Mitochondrial damage triggered the relocalization of TRIM5 to ER-mitochondria contact sites where TRIM5 colocalized with markers of autophagy initiation and autophagosome biogenesis. Furthermore, we found that TRIM5 knockout attenuated both Parkin-dependent and Parkin-independent mitophagy by preventing the recruitment of autophagy regulators FIP200 and ATG13 to unhealthy mitochondria. Finally, TRIM5 knockout cells showed reduced mitochondrial function under basal conditions and were more susceptible to uncontrolled immune activation and cell death in response to mitochondrial damage than were wild type cells. Taken together, our studies have identified a homeostatic role for a protein previously recognized exclusively for its antiviral actions.

cell biology↗

bioRxiv2020

A non-canonical role for the autophagy machinery in anti-retroviral signaling mediated by TRIM5α.

TRIM5 is a key cross-species barrier to retroviral infection, with certain TRIM5 alleles conferring increased risk of HIV-1 infection in humans. TRIM5 is best known as a species-specific restriction factor that directly inhibits the viral life cycle. Additionally, it is also a pattern-recognition receptor (PRR) that activates inflammatory signaling. How TRIM5 carries out its multi-faceted actions in antiviral defense remains incompletely understood. Here, we show that proteins required for autophagy, a cellular self-digestion pathway, play an important role in TRIM5s function as a PRR. Genetic depletion of proteins involved in all stages of the autophagy pathway prevented TRIM5-driven expression of NF-{kappa}B and AP1 responsive genes. One of these genes is the preeminent antiviral cytokine interferon {beta} (IFN-{beta}), whose TRIM5-dependent expression was lost in cells lacking the autophagy proteins ATG7, BECN1, and ULK1. Moreover, we found that the ability of TRIM5 to stimulate IFN-{beta} expression in response to recognition of a TRIM5-restricted HIV-1 capsid mutant (P90A) was abrogated in cells lacking autophagy factors. Stimulation of human macrophage-like cells with the P90A virus protected them against subsequent infection with an otherwise resistant wild type HIV-1 in a manner requiring TRIM5, BECN1, and ULK1. Mechanistically, TRIM5 was attenuated in its ability to activate the kinase TAK1 in autophagy deficient cells, and both BECN1 and ATG7 contributed to the assembly of TRIM5-TAK1 complexes. These data demonstrate a non-canonical role for the autophagy machinery in assembling antiviral signaling complexes and demonstrate a role for autophagy in the establishment of a TRIM5-dependent antiviral state. SIGNIFICANCE STATEMENTTRIM5 is an antiretroviral protein that employs multiple mechanisms to protect cells against infection. Previous studies have linked TRIM5 to autophagy, a cytoplasmic quality control pathway with numerous roles in immunity, raising the possibility that TRIM5 engages autophagy in antiviral defense. This concept has been controversial, since TRIM5s best-known role as a directly acting antiretroviral effector is autophagy independent. However, retroviral restriction is only one aspect of TRIM5 function. We demonstrate that autophagy is crucial to another TRIM5 action: its role as a pattern-recognition receptor. We show that autophagy machinery is required for TRIM5 to transduce antiviral signaling and to establish an antiretroviral state. Our data indicate that autophagy provides TRIM5 with a platform upon which to activate antiviral responses.

cell biology↗