Biology subjects

Chu, S.

Publications and source records attributed to Chu, S..

At least 19 recordsLinked to original sources

bioRxiv2026

A polysynaptic hypothalamuspelvic-ganglion circuit regulates prostate tumor growth via WNT3A

Brain-tumor communication is increasingly recognized. Peripheral autonomic nerves regulate prostate cancer progression, yet how the brain influences local nerve-tumor interactions remains unclear. We identify a polysynaptic circuit linking the hypothalamic paraventricular nucleus (PVN), dorsal motor nucleus of the vagus (DMV), major pelvic ganglion (MPG) and prostate tumor in mice. Although both nuclei were activated during tumor growth, PVN activity restrained, whereas DMV activity promoted tumor growth. MPG ablation abolished DMV-induced tumor promotion. Combining LMD with Smart-seq2 revealed transcriptional remodeling and increased Wnt3a expression in tumor-innervating, activated MPG neurons under upstream neural control. MPG explants released WNT3A, whose neutralization suppressed explant-induced cancer-cell proliferation. Together, our findings identify a polysynaptic PVN-DMV-MPG circuit that regulates prostate tumor growth and implicate WNT3A as a mediator of the pro-tumor effects of the MPG, highlighting the therapeutic potential of targeting the neural circuit and downstream molecular pathway.

cancer biology↗

bioRxiv2026

Functional orthogonality of WRN inhibitor resistance enables alternating therapy and mutation-tolerant inhibitor design in MSI-H cancers

Werner syndrome helicase (WRN) is a synthetic-lethal vulnerability in microsatellite instability-high (MSI-H)/mismatch repair-deficient (dMMR) cancers1-5, and non-covalent and covalent WRN inhibitors are now entering clinical development6-9. A central unresolved question is whether resistance to one WRN inhibitor class inevitably compromises the target, or instead creates actionable vulnerabilities to an alternate modality. Here we generated ten stable acquired-resistance models across three MSI-H cell lines using the non-covalent inhibitor HRO761 and the covalent inhibitor VVD-214. Whole-exome sequencing, biochemical reconstitution and isogenic knock-in models identified recurrent on-target WRN missense mutations as dominant resistance drivers, but with sharply modality-specific spectra: HRO761 resistance clustered at G729/F730/I852, whereas VVD-214 resistance concentrated at E846. These mutations impaired inhibitor engagement and abolished the canonical WRN inhibitor (WRNi)-induced pharmacodynamic cascade, including WRN reduction, DNA damage response (DDR) activation and G2/M arrest. Crucially, most resistance mutations retained biochemical, cellular and in vivo sensitivity to the alternate inhibitor modality, revealing a functional orthogonality that enabled a 7-day cyclic alternating regimen to delay tumour regrowth in xenografts. We further identified F730L as an engineered cross-resistant bottleneck model and used structure-guided, artificial intelligence (AI)-enabled optimization to generate GBA-007, a proof-of-concept mutation-tolerant WRN inhibitor candidate with promising activity against F730L in vitro and in vivo. Thus, clinically relevant WRN inhibitor classes impose distinct on-target resistance trajectories that can be exploited through schedule design, while cross-resistant bottlenecks can be addressed by rapid mutation-aware inhibitor engineering.

Cancer Biology↗

bioRxiv2026

A multi-agent system for spine MRI report generation from multi-sequence imaging

Spinal pathology is a leading cause of pain and disability worldwide. Spine magnetic resonance imaging (MRI) is central to clinical evaluation, yet its interpretation remains complex and time-consuming, requiring integration of information across multiple imaging sequences and anatomical regions. Despite recent advances in automated MRI analysis, effectively combining multi-sequence data while preserving sequence-specific diagnostic information remains an open challenge. Here we present SpineAgent, a multi-agent framework for spine MRI report generation built upon a multi-sequence foundation model trained on routine clinical data from 32,047 patients and 453,683 MRI series, comprising a total of 13,441,191 MRI slices. To accommodate diverse modalities of sequences, we first pre-train two DINOv3-based encoders separately on T1- and T2-weighted sequences. We then introduce a continual training strategy that learns a synthesizer to embed images of other sequences using the T1 and T2 encoders, producing patient-level embedding that integrates various signals across MRI sequences. Using these embeddings, SpineAgent achieves state-of-the-art performance, with mean 10.8% AUROC improvement across 17 spinal condition-prediction tasks compared to the best competing method, and demonstrates strong generalizability under cross-manufacturer and cross-cohort evaluation. Beyond classification, SpineAgent enables pathology localization by identifying findings-relevant slices and segmenting pathological regions. It also supports multimodal image-report retrieval, providing a solid foundation for scalable and explainable MRI report generation. We further integrate these validated capabilities of SpineAgent into 37 specialized agents for condition diagnosis, pathological-region localization, and clinically-similar-cases retrieval. Finally, we incorporate their outputs as structured tokens within a Medical Report Agent trained end-to-end for report generation. Through both automated metrics and expert evaluation by five radiologists, SpineAgent achieves leading performance in spine MRI report generation. Together, SpineAgent introduces a continual training approach for multi-sequence spine MRI understanding. By decomposing report generation into clinically grounded subtasks addressed by specialized agents, the SpineAgent framework enables accurate, interpretable and generalizable spine MRI reporting across diverse imaging sequences and anatomical regions.

bioinformatics↗

bioRxiv2026

Human hindbrain organoids with segment-specific 5-HT neurons for motor modulation

The serotonergic system regulates human mood, reward, and motor control, and its impairment contributes to debilitating psychiatric disorders. Serotonergic neurons (SNs) arise predominantly in the hindbrain and acquire segment-specific identities and functions; however, the lack of human models remains a challenge to understanding the human serotonergic system. Here, we generate human serotonergic-enriched rostral and caudal hindbrain organoids (hsrHOs and hscHOs) that produce segment-specific SNs with regional identities corresponding to rhombomeres r2-r3 and r5-r8, respectively. hsrHOs and hscHOs produce SNs that recapitulate their in vivo counterparts in transcriptomic profiles, function, and ascending or descending projection preferences. Assembly with human neuromusculoskeletal organoids enables modeling of serotonergic modulation of motor control. This system further reveals that deficiency of the schizophrenia risk gene DISC1 disrupts human serotonergic development and impairs motor modulation. Together, our study establishes a human organoid platform for modeling development, circuit function and disease-associated dysfunction of the serotonergic modulatory system.

neuroscience↗

bioRxiv2026

Characterising retinal function with optomotor visual performance in P23H rodent models of retinitis pigmentosa

Rhodopsin (RHO) P23H is one of the most common mutations causing autosomal dominant retinitis pigmentosa (adRP), yet the relationship between retinal electrophysiology, structure and visually guided behaviour in rodent models remains unclear. We characterised changes in heterozygous P23H (Sakami line) mice and P23H line 3 (P23H-3) rats using full-field electroretinography (ERG), optomotor response (OMR) assays and, in rats, optical coherence tomography (OCT). ERG assessed rod- and cone-mediated responses relative to wild-type controls, whereas OMR under scotopic and photopic conditions quantified contrast sensitivity and visual acuity. In P23H mice, scotopic ERG responses were significantly reduced from postnatal day 16 and declined further from 4 months. Scotopic OMR contrast sensitivity remained largely preserved until 2 months, and photopic acuity was comparable to wild-type up to 6 months. In 13-week-old P23H-3 rats, ERG amplitudes were significantly reduced, and OCT revealed retinal thinning. OMR showed a decline in contrast sensitivity at 7 and 15 weeks, whereas photopic acuity was maintained. Thus, in both models, electrophysiological and structural abnormalities precede detectable OMR deficits, with implications for the selection of outcome measures in preclinical studies. Summary StatementThis study compares electrical and behavioural measures of vision in rodent models of inherited blindness, revealing that retinal dysfunction appears well before measurable vision loss.

animal behavior and cognition↗

bioRxiv2026

Optimization of AAV tools to target M&uumlller glial cells for retinal gene therapy

Reprogramming of Muller glial (MG) cells into retinal neurons has the potential to treat vision loss by regenerating the retina. Development of efficient gene delivery systems to target the MG cells is critical. Adeno-associated virus (AAV) serotypes and promoter specificity are important factors that influence AAV transduction profile in the retina. However, studies that optimize these parameters to specifically target MG cells are limited, in particular in rats which are commonly used for eye research. Here we tested 4 AAV serotypes and 14 promoters to optimize gene delivery to human MG cells in vitro and/or rat MG cells in vivo. We showed that the combinatorial use of MG-specific serotypes and promoters achieved high specificity for MG cell targeting, with ShH10Y serotype and the GFAP (gfaABC1D) promoter as the best performing tool to target rat MG cells in vivo. We developed new AAV vectors using known and novel MG-specific promoters and engineered short promoter variants to improve the cargo capacity of AAV delivery. Our results highlighted a number of promoters that can target MG cells in vitro or in vivo. This study further expands the AAV toolbox to target MG cells, which has important implications for retinal gene therapy development.

bioengineering↗

bioRxiv2026

Optimization and Characterization of SHIP1 Ligands for Cellular Target Engagement and Activity in Alzheimer's Disease Models

Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1), encoded by the gene INPP5D, is a lipid phosphatase that negatively regulates immune receptor signaling in hematopoietic cells and microglia. Here, we describe a pyridyl-pyrazole-piperidine scaffold and the lead compound 3-((2-chlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine (32), which demonstrates SHIP1 target engagement, brain exposure, and evidence of a central pharmacodynamic response in vivo. Structure-activity relationship studies, guided by biochemical and cellular assays using multiple human and murine protein constructs and cells, identified SHIP1-active ligands. A thermal shift assay using full-length SHIP1 was used to assess compounds for cellular target engagement, while studies in IL-4 conditioned THP-1 cells was used to demonstrate changes in downstream AKT signaling. Targeted lipidomics revealed changes in the overall phosphoinositide pool consistent with SHIP1 target engagement and reduction of phospho-AKT levels. In a protein-lipid overlay assay, compound 32 induced changes in the relative association of SHIP1 with multiple phosphatidylinositols on a membrane surface. In high-content cellular imaging assays, compound 32 enhanced the uptake of myelin/membrane debris and fibrillar amyloid by primary murine microglia, phenocopying a genetic model with reduced SHIP1 expression. Finally, oral administration of compound 32 resulted in brain exposure sufficient to alter gene expression and reduce IL-1{beta} levels as pharmacodynamic markers of microglial activation and neuroinflammation in an amyloidosis mouse model of Alzheimers disease. Collectively, these results define a scaffold with SHIP1 target engagement, CNS exposure, and in vivo activity, providing a foundation for the optimization of brain-penetrant SHIP1 ligands suitable for further mechanistic studies and therapeutic development for the treatment of Alzheimers disease.

pharmacology and toxicology↗

bioRxiv2025

Optimization of AAV tools to target Muller glial cells for retinal gene therapy

molecular biology↗

bioRxiv2025

Intranigral injection of Alpha-Synuclein pre-formed fibrils leads to BBB compromise and Bilateral Dopaminergic Neurodegeneration in A53T Alpha-Synuclein transgenic mice.

Parkinsons disease (PD) is a progressive neurodegenerative disorder characterized by alpha-()-Synuclein neuronal aggregation and loss of dopaminergic (DA) neurons. Developing animal models that replicate PDs neuropathological phenotypes is critical for understanding its pathophysiology and evaluating potential therapeutic targets. In this study, we show that direct unilateral injection of human -Synuclein PFFs into the Substantia Nigra (SN) of mutant A53T -synuclein overexpressing mice induce bilateral phosphorylated -Synuclein (pS129) pathology in the SN. This pathology spreads to the striatum, cerebral cortex, and midbrain within 60 days and is accompanied by neuroinflammation in the midbrain and cerebral cortex. Additionally, we observed synuclein-dependent neurodegeneration, with a 50% reduction in Tyrosine Hydroxylase (TH) intensity in the SN and a 40% reduction in Striatum, both bilaterally. The model also revealed a compromised blood-brain barrier (BBB) and T-cell infiltration in the PFF injected animals, correlating with pS129 pathology and neuroinflammation. Taken together, we developed a mouse model that recapitulates multiple PD phenotypes, providing a valuable platform for testing therapeutic strategies targeting human -Synuclein pathology and for exploring CNS-peripheral immune interactions in PD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/690780v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@19c6234org.highwire.dtl.DTLVardef@1f36bbaorg.highwire.dtl.DTLVardef@25ac6borg.highwire.dtl.DTLVardef@15ad61d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Development and validation of an ultra-low-cost, open source normothermic ex vivo organ perfusion platform

BackgroundNormothermic ex vivo organ perfusion (NEVOP) promises to catalyze organ preservation, therapeutic discovery, and organ-specific disease modeling. Existing technology platforms remain inaccessible for research due to restricted access to commercial organ perfusion devices, high costs of both devices and proprietary consumables, and steep technical learning curves. Additionally, the available technology is not optimized to perfuse smaller organs such as the kidney. MethodsTo overcome these barriers, a custom NEVOP circuit was developed using recycled, repurposed, and low-cost components. Porcine kidneys and autologous blood were used to iteratively optimize circuit design. A porcine kidney autotransplantation protocol was adapted to evaluate in vivo kidney function after ex vivo perfusion. To pilot the flexibility of this system as a multi-organ platform for ex vivo human biology, non-transplantable human donor kidney, spleen, and pancreas specimens were stably perfused using human blood products and analyzed. ResultsAn ultra low-cost NEVOP system engineered to perfuse porcine kidneys and diverse human organs (kidney, pancreas, and spleen) sustained viable organs for up to 24 hours with evidence of both function and viability. Key innovations included a parallel flow resistor to facilitate low-flow perfusion in non-heparinized organs and a containment bag with adjustable magnets to provide vascular stability and recycling of venous overflow. The circuit costs less than 1,500USD to construct, and porcine kidneys perfused for 24 hours on this platform demonstrated healthy in vivo function upon autotransplantation. ConclusionsCustom NEVOP platforms constitute novel and potentially transformative research platforms which use low-cost and readily available materials. Paired with access to non-transplantable research organs from altruistic donors, this model provides a road map for investigators to advance biomedical discovery and human ex vivo biology.

bioengineering↗

bioRxiv2025

Ligand-dependent G protein dynamics underlying opioid signaling efficacy

Activation of heterotrimeric G proteins by G protein-coupled receptors (GPCRs) requires large-scale opening of the G -helical domain (AHD) to expose the nucleotide-binding site and facilitate GDP-GTP exchange. While orthosteric ligands are known to modulate GPCR conformation and signaling efficacy, how these effects propagate to the G protein itself remains unclear. Using single-molecule fluorescence resonance energy transfer (smFRET) imaging, we monitored AHD motions in Gi proteins coupled to the -opioid receptor (OR) across a spectrum of ligand- and nucleotide-bound states. We find that receptor ligands differentially modulate these dynamics from over 70 [A] away, with higher-efficacy agonists more effectively promoting transitions to an open, low-nucleotide-affinity conformation. These data also capture transient OR-Gi intermediates during nucleotide binding and suggest that -opioid ligand efficacy arises in part from allosteric control over G protein conformational equilibria that kinetically gate activation.

biophysics↗

bioRxiv2024

Protein degradation by small tag artificial bacterial E3 ligase

Targeting of proteins for degradation in a reversible manner is a powerful approach to decipher gene function and mimic drug effects, with great potential for drug target discovery and validation. A generalized approach is to tag a protein of interest and then use this tag to recruit an endogenously or exogenously expressed E3 ligase for its polyubiquitination and subsequent degradation via 26S proteasome. However, the often bulky size of the tag and the great variability of substrate-dependent degradation efficiency of mammalian E3 ligases pose great challenges in practice. Here we show that small tags (10-15 amino acids) can be used to efficiently tag endogenous proteins for degradation when coupled with an exogenously expressed artificial bacterial E3 ligase (ABEL) consisting of a tag-interacting moiety and the catalytic domain of the bacterial E3 ligase IpaH9.8. We name this versatile and efficient platform degradation by small tag ABEL (DESTABEL). Furthermore, we show that an ABEL containing a nanobody against human -synuclein mediates efficient degradation in primary neurons as well as in the adult mouse brain. Taken together, our data show that tag-dependent and independent ABELs are powerful yet flexible tools for studies of protein function and drug target validation.

molecular biology↗

bioRxiv2024

Yersinia pseudotuberculosis growth arrest during type-III secretion system expression is associated with altered ribosomal protein expression and decreased gentamicin susceptibility

It has been long appreciated that expression of the Yersinia type-III secretion system (T3SS) in culture is associated with growth arrest. Here we sought to understand whether this impacts expression of ribosomal protein genes, which were among the most highly abundant transcripts in exponential phase Yersinia pseudotuberculosis based on RNA-seq analysis. To visualize changes in ribosomal protein expression, we generated a fluorescent transcriptional reporter with the promoter upstream of rpsJ/S10 fused to a destabilized gfp variant. We confirmed reporter expression significantly increases in exponential phase and decreases as cells transition to stationary phase. We then utilized a mouse model of systemic Y. pseudotuberculosis infection to compare T3SS and S10 reporter expression during clustered bacterial growth in the spleen, and found that cells expressing high levels of the T3SS had decreased S10 levels, while cells with lower T3SS expression retained higher S10 expression. In bacteriological media, growth inhibition with T3SS induction and a reduction in S10 expression were observed in subsets of cells, while cells with high expression of both T3SS and S10 were also observed. Loss of T3SS genes resulted in rescued growth and heightened S10 expression. To understand if clustered growth impacted bacterial gene expression, we utilized droplet-based microfluidics to encapsulate bacteria in spherical agarose droplets, and also observed growth inhibition with high expression of T3SS and reduced S10 levels that better mirrored phenotypes observed in the mouse spleen. Finally, we show that T3SS expression is sufficient to promote tolerance to the ribosome-targeting antibiotic, gentamicin. Collectively, these data indicate that the growth arrest associated with T3SS induction leads to decreased expression of ribosomal protein genes, and this results in reduced antibiotic susceptibility. Author SummarySlow-growing bacterial cells have reduced antibiotic susceptibility, rendering them very difficult to eliminate during antibiotic treatment. However, for many key virulence factors (bacterial factors required to promote infection), it remains unclear whether expression is sufficient to slow bacterial growth and impact antibiotic susceptibility. Using Yersinia pseudotuberculosis, we found ribosomal protein expression fluctuated based on growth rate, and we generated a fluorescent reporter construct to detect altered ribosomal protein expression within individual bacterial cells. We then asked if expression of a key virulence factor in Yersinia, the type-III secretion system (T3SS), is sufficient to lower ribosomal protein expression, since it has been well established that T3SS induction results in growth arrest. We found high levels of T3SS expression promotes slowed growth and antibiotic tolerance, and bacterial cells that survive treatment with a ribosome-targeting antibiotic, gentamicin, have heightened levels of T3SS and lower levels of S10 expression.

microbiology↗

bioRxiv2024

Effects of short-term isolation on vocal and non-vocal social behaviors in prairie voles

Social isolation affects the brain and behavior in a variety of animals, including humans. Studies in traditional laboratory rodents, including mice and rats, have supported the idea that short-term social isolation promotes affiliative social behaviors, while long-term isolation promotes anti-social behaviors, including increased aggression. Whether the effects of isolation on the social behaviors of mice and rats generalize to other rodents remains understudied. In the current study, we characterized the effects of short-term (3-days) social isolation on the vocal and non-vocal social behaviors of adult prairie voles (Microtus ochrogaster) during same-sex and opposite-sex social interactions. Our experiments revealed that short-term isolation did not affect rates of ultrasonic vocalizations or time spent in non-aggressive social behaviors and huddling. In contrast, short-term isolation affected aggression in a sex- and context-specific manner during male-male interactions. Our findings highlight the importance of comparative work across species and the consideration of social context to understand the diverse ways in which social isolation can impact social behavior.

animal behavior and cognition↗

bioRxiv2024

Alleviation of salt stress in strawberries by hydrogen-rich water: physiological, transcriptomic and metabolomic responses

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/605184v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1aa6488org.highwire.dtl.DTLVardef@1ae580borg.highwire.dtl.DTLVardef@11656bcorg.highwire.dtl.DTLVardef@1985c7b_HPS_FORMAT_FIGEXP M_FIG C_FIG With the continuous advancement of climate change and human activities, cultivable land is becoming increasingly susceptible to the influence of salt ions. Therefore, it is crucial to mitigate the salt stress on plants through various approaches. In this study, strawberry seedlings were selected as the experimental plants, and a comprehensive transcriptomic and metabolomic analysis was conducted to elucidate the effects of hydrogen-rich water (HRW) on strawberries under salt stress. The results indicated that HRW significantly promoted plant growth, particularly increasing root biomass by 49.50%. Additionally, HRW regulated the levels of soluble sugars, malondialdehyde (MDA), and antioxidant enzymes, enhancing the cellular uptake of potassium ions and the expulsion of sodium ions. The levels of Ca2+ and Mg2+ in organelles increased by 2.06 and 2.45-fold, respectively. Transcriptomic analysis revealed that HRW substantially altered gene expression in strawberry roots; under salt stress, HRW up-regulated beneficial biological processes. Furthermore, genes related to ion absorption and transport, antioxidant enzymes, and cell wall biosynthesis were screened. Meanwhile, key common pathways were identified in differentially expressed metabolites (DEMs) and differentially expressed genes (DEGs) related to phenylpropane biosynthesis, alanine, aspartate, and glutamate metabolism, amino and nucleotide sugar metabolism, and galactose metabolism. A molecular mechanism for mitigating salt stress in strawberry seedlings by HRW was provided by the integrated approaches in this research, reflecting the potential applications of hydrogen gas in agriculture.

physiology↗

bioRxiv2023

INTEGRATED GLOBAL CHICKEN REFERENCE PANEL FROM 13,187 CHICKEN GENOMES

Chickens are a crucial source of protein for humans and a popular model animal for bird research. Despite the emergence of imputation as a reliable genotyping strategy for large populations, the lack of a high-quality chicken reference panel has hindered progress in chicken genome research. To address this issue, here we introduce the first phase of the 100 K Global Chicken Reference Panel Project (100 K GCRPP). The project includes 13,187 samples and provides services for varied applications on its website (http://farmrefpanel.com/GCRP/). Currently, two panels are available: a Comprehensive Mix Panel (CMP) for domestication diversity research and a Commercial Breed Panel (CBP) for breeding broilers specifically. Evaluation of genotype imputation quality showed that CMP had the highest imputation accuracy compared to imputation using existing chicken panel in animal SNPAtlas, whereas CBP performed stably in the imputation of commercial populations. Additionally, we found that genome-wide association studies using GCRP-imputed data, whether on simulated or real phenotypes, exhibited greater statistical power. In conclusion, our study indicates that the GCRP effectively fills the gap in high-quality reference panels for chickens, providing an effective imputation platform for future genetic and breeding research.

bioinformatics↗

bioRxiv2023

Multiplicative frequency and angular speckle reduction in ultrasound imaging

Speckle is the major artifact in ultrasound imaging, and it is well-known that speckle can be reduced by compounding (averaging) images taken either at different frequencies or from different angles. By averaging images of a phantom taken over a frequency range [Formula] and a 90{degrees} span of angles, the combined speckle reduction is demonstrated to be ~ 9x compared to non-compounded images, while the reduction with frequency or angle averaging resulted in reductions of ~ 3x individually. The rf input to the transducer is altered to vary the sound frequency and the phantom is rotated with respect to the transducer to obtain different imaging angles. Numerical simulations of sound scattered by randomly distributed point scatterers showed quantitative agreement with the experiment. Using a commercial system, a 6x reduction in speckle is demonstrated imaging a human wrist. A robot arm is used to move the transducer along a circular path to acquire images at 9 angles separated by 10{degrees}. The commercial system does not allow direct control of the input to the transducer, so the broadband signal detected is Fourier filtered to obtain images at different frequencies with ~ 2x reduced frequency range. Images taken at different angles contain distortions from speed of sound variations and pressure induced by the probe. Two forms of non-rigid image registration are applied to correct for the distortions and create a higher resolution composite image. A design for achieving ~10x speckle reduction with essentially no loss in imaging speed is described.

bioengineering↗

bioRxiv2023

SHIP1 modulation and proteome characterization of microglia

Microglia, the resident macrophage in brain, has gained significant attention due to their involvement in neurodegenerative diseases. Disease associated microglia (DAM) have been identified at sites of amyloid-beta plaques and neurodegeneration. Understanding microglial states in the aging brain has become crucial, especially with the discovery of numerous Alzheimers disease (AD) risk and protective variants in genes such as TREM2, CD33, APOE, ABCA7, PLCG2, and INPP5D, which are essential to microglia function1. Here we present a thorough examination of microglia-like cell lines and primary mouse microglia at the proteomic and transcriptomic levels to help illuminate the roles these genes and the proteins they encode play in various cell states. This analysis serves as a guide to the exploration of potential therapeutic targets in the context of neurodegeneration. INPP5D, which encodes the SHIP1 protein, is essential for microglia function. SHIP1 has emerged as a target of interest having been nominated as a therapeutic target by three teams within the Accelerating Medicines Partnership for Alzheimers Disease (AMP-AD)2. In this study, we compared the proteomic profiles of wildtype, SHIP1 heterozygous knockout, and homozygous knockout primary microglia. Our findings revealed significant proteomic alterations only in the homozygous knockout of the SHIP1 gene, revealing its impact on the microglial proteome. Additionally, we compared the proteomic and transcriptomic profiles of BV2 and HMC3 cells with primary mouse microglia because these cell lines are often used as microglial cellular models. Our results demonstrated a substantial similarity between the proteome of BV2 cells and mouse primary cells, while notable differences were observed between BV2 and human HMC3 cells, with some shared characteristics. Since SHIP1 functions as a lipid phosphatase that modulates phosphatidylinositol (PI) species, we conducted lipidomic analysis to quantify different phosphatidylinositols (PIs), phosphatidylinositol monophosphate (PIPs), and polyphosphoinositides (PPIs) in the HMC3 and BV2 cells. Under basal conditions, PI(3,4,5)P3 and PI(3,4)P2 species were detected at extremely low levels, making confident quantification challenging; however, PIP species within the overall pool were significantly changed upon SHIP1 overexpression in HMC3. This in-depth proteomic analysis of both mouse and human microglia, complemented by targeted lipidomic studies, enhances our understanding of these cellular models. The similarities between primary mouse microglia and the BV2 cell line is especially encouraging, supporting the use of this model for further investigations into the role that SHIP1 and other potential drug targets may play in the regulation of microglial states.

neuroscience↗