Biology subjects

Freeman, M.

Publications and source records attributed to Freeman, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Structural polymorphism and population-variable coding capacity of HERV-K(HML-2) in human pangenomes

Approximately 8% of the human genome is derived from ancient retroviral infections. The most recently integrated of these endogenous retroviruses is the HERV-K(HML-2) clade, whose expression has been associated with cancer, amyotrophic lateral sclerosis, and embryogenesis. Studies of HERV expression, particularly HML-2, have relied predominantly on short-read sequencing. However, the high similarity among HML-2 proviruses prevents many short reads from being assigned uniquely to individual loci. We therefore compared haplotype-resolved long-read genome assemblies from 292 donors to resolve variation in proviral structure and coding capacity. Several loci previously thought to be fixed were structurally polymorphic. Tandem arrays occurred at 13 loci and contained up to six proviral copies in a single array. At 8q11.23, we identified a previously undescribed full-length provirus in one haplotype. All 583 other haplotypes carried a solo-LTR. We found that standard reference genomes failed to represent the coding capacity retained in many individuals, whose proviruses contained intact open reading frames despite disruptive mutations in the reference sequences. Short-read genotypes left 32.5% of the tested donor-variant pairs unresolved at sites associated with viral reading frames. These findings show why HML-2 expression must be interpreted in the context of the structural and coding alleles each individual carries.

genomics↗

bioRxiv2026

The rhomboid-like pseudoprotease TMEM115 defines a two-factor mechanism for Rab6A effector recruitment

Protein trafficking and organelle identity depends on precise localisation of membrane proteins, anchored by small GTPases that bind to specific cellular compartments. Here we show that the rhomboid-like pseudoprotease TMEM115 defines an unexpected two-factor system with Rab6A to target the golgin TMF1. In the absence of TMEM115, TMF1 is delocalised from the rim of the Golgi apparatus; instead, it is retained in Golgi-targeting vesicles. TMEM115, TMF1, and Rab6A assemble into a ternary complex which, in striking contrast to canonical rhomboid-like mechanisms, is independent of transmembrane domain interactions. Disruption of the TMEM115-Rab6A-TMF1 complex affects the efficiency of trafficking of proteins from the Golgi; moreover, human disease-associated mutations target the interfaces of this ternary complex, thereby delocalising TMF1 from the Golgi. Finally, loss of TMEM115 in Drosophila causes abnormal Golgi morphology, and mouse knockouts undergo perinatal death. We propose that two-factor effector recruitment defines a mechanism for precise Rab GTPase-mediated membrane targeting.

cell biology↗

bioRxiv2026

Diet-dependent sleep modulation by the Drosophila amino acid transporter ANIDRA

Sleep is a conserved animal behavior necessary for survival. It is under tight circadian and homeostatic control, and modulated by diet. Here, we identify the amino acid transporter ANIDRA (ANID) as an important sleep regulator in Drosophila. Flies lacking ANID show decreased and poorly consolidated daytime and nighttime sleep. Contrary to wild-type controls, anid mutant flies are unable to adjust their sleep to their diet, behaving as if they were constantly on a complete diet rich in amino acids. ANID is expressed in ensheathing and cortex glia, where it inhibits mTOR activity in a diet-dependent manner. Moreover, pharmacological inhibition of mTOR attenuates the anid mutant sleep phenotypes. Interestingly, DH44-expressing brain neurons, which promote arousal and sense amino acids, are constantly active in ANIDs absence. We therefore propose that ANID mediates detection of dietary amino acids by ensheathing and cortex glia to regulate the activity of arousal-promoting neurons.

neuroscience↗

bioRxiv2026

A sterol-binding pocket in iRhom1 underlies paralog-specific regulation of the sheddase ADAM17

ADAM17, the major sheddase in mammalian cells, releases membrane-tethered EGFR ligands and inflammatory cytokines, and is a central regulator of cell signalling. The rhomboid pseudoproteases, iRhom1 and iRhom2, function as essential cofactors of ADAM17, controlling its maturation and activation. In contrast to the well-characterized iRhom2, the mechanism and regulation of its ubiquitously expressed paralog iRhom1 remain undefined. Here, we present a 2.5 [A] cryo-EM structure of the full-length human iRhom1/ADAM17 complex, revealing a previously unrecognized sterol-binding pocket located between TMD2 and TMD5. Structure-guided mutagenesis and pharmacological perturbation of sterol binding demonstrate that sterol binding is required to stabilize the iRhom1/ADAM17 complex and sustain its shedding activity. Strikingly, this regulation is paralog-specific: iRhom2 precludes sterol binding and instead stabilizes ADAM17 through direct intramolecular interactions. Furthermore, two human iRhom1 variants associated with cardiac disease localize adjacent to the sterol-binding pocket and disrupt ADAM17 maturation and activity. Together, these findings uncover mechanistic divergence between iRhom paralogs and establish a sterol-binding pocket in iRhom1 as a critical determinant of ADAM17 stability, revealing a potential avenue for paralog-selective therapeutic targeting.

cell biology↗

bioRxiv2025

A dNmnat-sensitized in vivo platform for unbiased discovery of regulators of neurodegeneration

Axon degeneration drives nervous system dysfunction in diverse neurological diseases and injuries. While key regulators of injury-induced Wallerian degeneration have been identified, approaches have largely relied on axotomy, a particularly extreme injury for a neuron. Here, we developed a forward genetic screening platform in the adult Drosophila wing that sensitizes neurons to degeneration through depletion of nicotinamide mononucleotide adenylyltransferase (dNmnat), the essential NAD+ biosynthetic enzyme. We screened 9,393 mutagenized chromosomes and recovered 59 mutations that suppress neurodegeneration induced by dNmant depletion, including the known pro-degenerative molecule dSarm, thereby validating our approach. We show loss of CG4098, the Drosophila homolog of the mammalian enzyme NUDT9, robustly preserved axons and induced widespread remodeling of NAD+-related metabolites, which provides a new link between cADP-ribose hydrolase activity and neuronal survival. We further show mutations in the transcription factor Abrupt resulted in elevated dNmnat protein levels and blocked axon degeneration, suggesting Abrupt normally tunes dNmnat levels and neuronal NAD+ homeostasis. dNmnat depletion is thus a versatile approach for unbiased discovery of axon death pathway components, each of which may provide new entry points for therapeutic strategies to preserve axons in neurodegenerative diseases.

neuroscience↗

bioRxiv2025

Better antibodies engineered with a GLIMPSE of human data

GLIMPSE-1 is a protein language model trained solely on paired human antibody sequences. It captures immunological features and achieves best-in-class performance in humanization benchmarks. We demonstrate the utility of GLIMPSE-1 in humanization; engineering of antibodies for affinity, species cross-reactivity, and key developability parameters; and the creation of highly divergent functional variants with <90% sequence identity to a marketed antibody. Learning exclusively from human antibody data enables GLIMPSE-1 to enhance therapeutics and native antibodies based on patterns in the human repertoire. DisclaimerWhile we provide detailed descriptions of experimental methods and success metrics, certain methodological details of GLIMPSE-1 remain proprietary and/or redacted in this work for commercial considerations. We warmly invite researchers and potential collaborators interested in accessing GLIMPSE-1 to connect with our team via partnerships@infinimmune.com.

bioinformatics↗

bioRxiv2025

Hypoxia-inducible factor 1 protects neurons from Sarm1-mediated neurodegeneration

The Sarm1 NAD+ hydrolase drives neurodegeneration in many contexts, but how Sarm1 activity is regulated remains poorly defined. Using CRISPR/Cas9 screening, we found loss of VHL suppressed Sarm1-mediated cellular degeneration. VHL normally promotes O2-dependent constitutive ubiquitination and degradation of hypoxia-inducible factor 1 (HIF-1), but during hypoxia, HIF-1 is stabilized and regulates gene expression. We observed neuroprotection after depletion of VHL or other factors required for HIF-1 degradation, and expression of a non-ubiquitinated HIF-1 variant led to even stronger blockade of axon degeneration in mammals and Drosophila. Neuroprotection required HIF-1 DNA binding, prolonged expression, and resulted in broad gene expression changes. Unexpectedly, stabilized HIF-1 prevented the precipitous NAD+ loss driven by Sarm1 activation in neurons, despite NAD+ hydrolase activity being intrinsic to the Sarm1 TIR domain. Our work argues hypoxia inhibits Sarm1 activity through HIF-1 driven transcriptional changes, rendering neurons less sensitive to Sarm1-mediated neurodegeneration when in a hypoxic state. Competing interestsMarc Freeman is co-founder of Nura Bio, a biotech startup pursuing novel neuroprotective therapies including SARM1 inhibition. The remaining authors declare no competing interests.

neuroscience↗

bioRxiv2024

The gut microbiome promotes mitochondrial respiration in the brain of a Parkinson's disease mouse model

The pathophysiology of Parkinsons disease (PD) involves gene-environment interactions that impair various cellular processes such as autophagy, lysosomal function, or mitochondrial dysfunction. Specifically, mitochondria-associated gene mutations increase PD risk, mitochondrial respiration is altered in the PD brain, and mitochondrial-damaging toxins cause PD-like motor and gastrointestinal symptoms in animal models. The gut microbiome is altered in PD patients and represents an environmental risk, however a relationship between mitochondrial function and the microbiome in PD has not been previously established. Herein, we report that striatal mitochondria are functionally overactive in -synuclein-overexpressing (ASO) mice, a model of PD, and that microbiome depletion restores respiration and mitochondria-associated gene expression patterns to wild-type levels. ASO mice harboring a complex microbiome produce increased reactive oxygen species in the striatum whereas germ-free counterparts express elevated levels of antioxidant proteins that may buffer against oxidative damage. Indeed, antioxidant treatment improves motor performance in ASO mice and, remarkably, blocking oxidant scavenging in germ-free mice induces -synuclein-dependent motor deficits. Thus, the gut microbiome increases mitochondrial respiration and oxidative stress in the brain, which enhances motor symptoms in a mouse model of PD.

physiology↗

bioRxiv2024

An in vivo genetic screen identifies Crq as a potent glial regulator of synapse elimination in development and aging

Neurons and glia work together to dynamically regulate neural circuit assembly and maintenance. In this study, we show Drosophila exhibit large-scale synapse formation and elimination as part of normal CNS circuit maturation, and that glia use conserved molecules to regulate these processes. Using a high throughput ELISA-based in vivo screening assay, we identify new glial genes that regulate synapse numbers in Drosophila in vivo, including the scavenger receptor ortholog Croquemort (Crq). Crq acts as an essential regulator of glial-dependent synapse elimination during development, with glial Crq loss leading to excess CNS synapses and progressive seizure susceptibility in adults. Loss of Crq in glia also prevents age-related synaptic, but not neuronal loss, in the adult brain. This work provides new insights into the cellular and molecular mechanisms that underlie synapse development and maintenance across the lifespan, and identifies glial Crq as a key regulator of these processes.

neuroscience↗

bioRxiv2023

Astrocyte-dependent local neurite pruning and Hox gene-mediated cell death in Beat-Va neurons

Neuronal remodeling is extensive and mechanistically diverse across the nervous systems of complex metazoans. To explore circuit refinement mechanisms, we screened for new neuronal subtypes in the Drosophila nervous system that undergo remodeling early in metamorphosis. We find Beat-VaM neurons elaborate a highly branched neurite network during larval stages that undergoes local neurite pruning during early metamorphosis. Surprisingly, Beat-VaM neurons remodel their branches despite blockade of steroid hormone signaling and instead depend on signaling from astrocytes to activate pruning. We show Beat-VaL neurons undergo steroid hormone-dependent cell death in posterior but not anterior abdominal segments. Correct activation of apoptotic cell death relies on segment-specific expression of the hox gene Abd-B, which is capable of activating cell death in any Beat-VaL neuron. Our work provides new model cells in which to study neuronal remodeling, highlights an important role for astrocytes in activating local pruning in Drosophila independent of steroid signaling, and defines a Hox gene-mediated mechanism for segment-specific cell elimination. SummaryLehmann et al. characterize two new populations of neurons that undergo remodeling during Drosophila metamorphosis. Beat-VaM neurons undergo drastic neurite pruning that is largely independent of ecdysone signaling and instead is driven by astrocytes. Beat-VaL neurons undergo Abd-B mediated, caspase driven cell death in a segmentally restricted manner.

neuroscience↗

bioRxiv2023

Tweek-dependent formation of ER-PM contact sites enables astrocyte phagocytic function and remodeling of neurons

Neuronal remodeling generates an enormous amount of cellular debris, which is cleared from the nervous system by glia. At the larva-to-adult transition, Drosophila astrocytes transform into phagocytes and engulf degenerating larval synapses, axonal and dendritic debris. Here we show Tweek, a member of the bridge-like lipid transfer protein family, is upregulated in astrocytes as they ramp up their phagocytic function early in metamorphosis, and is essential for internalization and degradation of neuronal debris. Tweek forms a bridge between the endoplasmic reticulum (ER) and plasma membrane (PM), and loss of Tweek disrupts ER-PM contact formation and membrane lipid distribution. Patient-identified mutations in the human homolog associated with Alkuraya-Kucinskas syndrome resulted in similar defects in neuronal remodeling, indicating these are loss of function mutations. We propose Tweek helps establish and maintain ER-PM contacts during astrocyte phagocytic function and drives bulk lipid transfer to the plasma membrane for continued efficient internalization and degradation of neuronal debris.

neuroscience↗

bioRxiv2023

Eta-secretase-like processing of the amyloid precursor protein (APP) by RHBDL4

The amyloid precursor protein (APP) has been extensively studied with regards to its contribution to the pathology of Alzheimers disease. APP is an ubiquitously expressed type I transmembrane protein synthesized in the endoplasmic reticulum (ER) and translocated to the plasma membrane where it undergoes proteolytic cleavages by several identified proteases. Conversely to other known proteases, we previously elucidated human rhomboid protease RHBDL4 as a novel APP processing enzyme where several cleavages likely occur already in the ER. Interestingly, the pattern of RHBDL4-derived large APP C-terminal fragments resemble those generated by the {eta}-secretase or MT5-MMP, which was described to generate so called A{eta} fragments. The similarity in large APP C-terminal fragments between both proteases raised the question whether RHBDL4 may contribute to {eta}-secretase activity and A{eta}-like fragments. Here, we identified two cleavage sites of RHBDL4 in APP by mass spectrometry, which, intriguingly, lie in close proximity to the cleavage sites of MT5-MMP. Indeed, we observed that RHBDL4 generates A{eta}-like fragments in vitro without contributions of -, {beta}-, or {gamma}-secretases. Such A{eta}-like fragments are likely generated in the ER since RHBDL4-derived APP-C-terminal fragments do not reach the cell surface. Inherited, familial APP mutations appear to not affect this processing pathway. In RHBDL4 knockout mice, we observed increased cerebral full length APP levels in comparison to WT brains in support of RHBDL4 being a physiologically relevant protease for APP. Furthermore, we found secreted A{eta} fragments in dissociated mixed cortical cultures from wild type mice, however significantly less A{eta} fragments in cultures from RHBDL4 knockout mice. Our data underscores that RHBDL4 contributes to {eta}-secretease-like processing of APP and that RHBDL4 is a physiologically relevant protease for APP.

biochemistry↗

bioRxiv2022

Cleavage of the pseudoprotease iRhom2 by the signal peptidase complex reveals an ER-to-nucleus signalling pathway

iRhoms are pseudoprotease members of the rhomboid-like superfamily and are cardinal regulators of inflammatory and growth factor signalling; they function primarily by recognising transmembrane domains of their clients. Here we report an unexpected, and mechanistically distinct, nuclear function of iRhoms. iRhom2 is a non-canonical substrate of the signal peptidase complex (SPC), the protease that removes signal peptides from secreted proteins. Cleavage of iRhom2 generates an N-terminal fragment that enters the nucleus and modifies the cellular transcriptome. We observed elevated nuclear iRhom2 in skin biopsies of patients with psoriasis and tylosis with oesophageal cancer (TOC); increased SPC-mediated iRhom2 cleavage in a psoriasis model; and overlapping transcriptional signatures between psoriasis and expression of the iRhom2 N-terminus. This work highlights the pathophysiological significance of this SPC-dependent ER-to-nucleus signalling pathway, and is the first example of a rhomboid-like protein that mediates protease-regulated nuclear signalling.

cell biology↗

bioRxiv2022

The Tre1/S1pr1 phospholipid-binding G protein-coupled receptor signaling pathway is required for astrocyte morphogenesis

Astrocytes play crucial roles in regulating neural circuit function by forming a dense network of synapse-associated membrane specializations, but signaling pathways regulating astrocyte morphogenesis remain poorly defined. Here we show the Drosophila lipid-binding G protein-coupled receptor (GPCR) Tre1, likely acting through Rac1, is required for astrocytes to elaborate their complex morphology in vivo. The lipid phosphate phosphatases Wunen/Wunen2, which process phospholipid ligands, also regulate astrocyte morphology, and, via Tre1, mediate astrocyte-astrocyte competition for growth promoting lipids. Loss of s1pr1, the functional analog of Tre1 in zebrafish disrupts astrocyte process elaboration. Live-imaging and pharmacology demonstrate that S1pr1 balances proper astrocyte process extension/retraction dynamics during morphogenesis, and that S1pr1 signaling is required throughout astrocyte development. Tre1 and S1pr1 are thus potent evolutionarily conserved regulators of astrocyte growth and elaboration of morphological complexity. O_LIThe GPCR Tre1 and LPPs Wun/Wun2 promote astrocyte process outgrowth in Drosophila C_LIO_LIAstrocytes compete for a growth{-}promoting phospholipid in the CNS C_LIO_LIWun/Wun2 act locally to regulate process outgrowth through Tre1 C_LIO_LIVertebrate S1pr1 regulates astrocyte growth early, through modulation of process dynamics C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/508188v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1e86db5org.highwire.dtl.DTLVardef@7abd6borg.highwire.dtl.DTLVardef@29f163org.highwire.dtl.DTLVardef@1a6c4b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

Functional unknomics: closing the knowledge gap to accelerate biomedical research

The human genome encodes [~]20,000 proteins, many still uncharacterised. Scientific and social factors have resulted in a focus on well-studied proteins, leading to a concern that poorly understood genes are unjustifiably neglected. To address this, we have developed an "Unknome database" that ranks proteins based on how little is known about them. We applied RNAi in Drosophila to 260 unknown genes that are conserved between flies and humans. About a quarter are required for viability, and functional screening of the rest revealed hits for fertility, development, locomotion, protein quality control and resilience to stress. CRISPR/Cas9 gene disruption validated a component of Notch signalling and two genes contributing to male fertility. Our work demonstrates the importance of poorly understood genes, provides a resource for future research acceleration, and highlights a need for our awareness of ignorance to be protected from erosion by automated database annotation.

molecular biology↗

bioRxiv2022

The HERV-K(HML-2) Transcriptome in Non-diseased Tissue

Human endogenous retrovirus (HERV) transcripts are known to be highly expressed in cancers, yet their activity in non-diseased tissue is largely unknown. Using the GTEx RNA-seq dataset from normal tissue sampled at autopsy, we characterized individual expression of the recent HERV-K(HML-2) provirus group across 13,000 different samples of 54 different tissues from 948 individuals. HML-2 transcripts could be identified in every tissue sampled, and were elevated in the cerebellum, pituitary, testis, and thyroid. 37 different individual proviruses were expressed in one or more tissues, representing all three LTR5 subgroups. 9 proviruses were identified as having LTR-driven transcription, 7 of which belonged to the most recent LTR5HS subgroup. Proviruses of different subgroups displayed a bias in tissue expression which may be associated with differences in transcription factor binding sites in their LTRs. Provirus expression was greater in evolutionarily older proviruses with an earliest shared ancestor of gorilla or older. HML-2 expression was significantly affected by biological sex in one tissue, while age and timing of death (Hardy score) had little effect. Proviruses containing intact gag, pro and env ORFs were expressed in the dataset, with almost every tissue measured potentially expressing at least one intact ORF (gag).

microbiology↗

bioRxiv2021

A serotonergic axon-cilium synapse drives nuclear signaling to maintain chromatin accessibility

Chemical synapses between axons and dendrites mediate much of the brains intercellular communication. Here we describe a new kind of synapse - the axo-ciliary synapse - between axons and primary cilia. By employing enhanced focused ion beam - scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between the serotonergic axons arising from the brainstem, and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, 5-hydroxytryptamine receptor 6 (HTR6), whose mutation is associated with learning and memory defects. Using a newly developed cilia-targeted serotonin sensor, we show that optogenetic stimulation of serotonergic axons results in serotonin release onto cilia. Ciliary HTR6 stimulation activates a non-canonical Gq/11-RhoA pathway. Ablation of this pathway results in nuclear actin and chromatin accessibility changes in CA1 pyramidal neurons. Axo-ciliary synapses serve as a distinct mechanism for neuromodulators to program neuron transcription through privileged access to the nuclear compartment.

cell biology↗

bioRxiv2021

Glial TGFβ activity promotes axon survival in peripheral nerves

Axons can represent the majority of the volume of a neuron and are energetically very demanding. Specialized glia ensheathe axons and are believed to support axon function and maintenance throughout life, but molecular details of glia-neuron support mechanisms remain poorly defined. Here we identify a collection of secreted and transmembrane genes that are required in glia for long-term axon survival in vivo. We show that key components of the TGF{beta} superfamily are required cell-autonomously in glia for peripheral nerve maintenance, although their loss does not disrupt glial morphology. We observe age-dependent neurodegeneration in the absence of glial TGF{beta} signaling that can be rescued by genetic blockade of Wallerian degeneration. Our data argue that glial TGF{beta} signaling normally acts to promote axon survival and suppress neurodegeneration. Significance StatementAxon maintenance is critical to preserving the functional integrity of the nervous system across animal lifespan. Glia contribute to axon maintenance, but their precise roles remain to be fully characterized. We identify glial genes that regulate axon support and provide new molecular insight into the means by which glia promote axon survival, which may help explain why neurodegeneration occurs when glia are lost in disease. We show that TGF{beta} signaling in mature glia is essential for long-term maintenance of axons, and that loss of TGF{beta} signaling activates an axon death signaling pathway.

neuroscience↗