Biology subjects

Lin, S.

Publications and source records attributed to Lin, S..

At least 145 records · Page 8Linked to original sources

bioRxiv2023

The CDK12 inhibitor SR-4835 functions as a molecular glue that promotes cyclin K degradation in melanoma

CDK12 is a transcriptional cyclin-dependent kinase (CDK) that interacts with cyclin K to regulate different aspects of gene expression. The CDK12-cyclin K complex phosphorylates several substrates, including RNA polymerase II (Pol II), and thereby regulates transcription elongation, RNA splicing, as well as cleavage and polyadenylation. Because of its implication in cancer, including breast cancer and melanoma, multiple pharmacological inhibitors of CDK12 have been identified to date, including THZ531 and SR-4835. While both CDK12 inhibitors affect Poll II phosphorylation, we found that SR-4835 uniquely promotes cyclin K degradation via the proteasome. Using loss-of-function genetic screening, we found that SR-4835 cytotoxicity depends on a functional CUL4-RBX1-DDB1 ubiquitin ligase complex. Consistent with this, we show that DDB1 is required for cyclin K degradation, and that SR-4835 promotes DDB1 interaction with the CDK12-cyclin K complex. Docking studies and structure-activity relationship analyses of SR-4835 revealed the importance of the benzimidazole side-chain in molecular glue activity. Together, our results indicate that SR-4835 acts as a molecular glue that recruits the CDK12-cyclin K complex to the CUL4-RBX1-DDB1 ubiquitin ligase complex to target cyclin K for degradation.

molecular biology↗

bioRxiv2023

Low-dose carbon monoxide therapy is neuroprotective in rodent models of Parkinson's disease

Paradoxically, cigarette smoking is associated with a reduced risk of Parkinsons disease (PD). This led us to hypothesize that carbon monoxide (CO) levels, which are constitutively but modestly elevated in smokers, might contribute to neuroprotection. Using rodent models of PD based on -synuclein (Syn) accumulation and oxidative stress, we show that low-dose CO mitigates neurodegeneration and reduces Syn pathology. Oral CO administration activated signaling cascades mediated by heme oxygenase-1 (HO-1), which have been implicated in limiting oxidative stress, and in promoting Syn degradation, thereby conferring neuroprotection. Consistent with a neuroprotective effect of smoking, HO-1 levels in cerebrospinal fluid were higher in human smokers compared to nonsmokers. Moreover, in PD brain samples, HO-1 levels were higher in neurons without Syn pathology. Thus, CO in rodent PD models reduces pathology and increases oxidative stress responses, phenocopying possible protective effects of smoking evident in PD patients. These data highlight the potential for low-dose CO modulated pathways to slow symptom onset and limit pathology in PD patients.

neuroscience↗

bioRxiv2023

Frequency-dependent Distribution of Different Subtypes of Spiral Ganglion Neurons in the Cochlea and the Changes During Aging

Sound information is transmitted from the cochlea to the brain mainly by type I spiral ganglion neurons (SGNs), which consist of different subtypes with distinct physiological properties and selective expression of molecular markers. It remains unclear how these SGN subtypes distribute along the tonotopic axis, and how the distribution pattern changes during aging that might underlie age-related hearing loss (ARHL). We investigated these questions using immunohistochemistry in three age groups of CBA/CaJ mice of either sex, including 2-5 months (young), 17-19 months (middle-age), and 28-32 months (old). Mouse cochleae were cryo-sectioned and triple-stained using antibodies against Tuj1, calretinin (CR) and calbindin (CB), which are reportedly expressed in all type I, subtype Ia, and subtype Ib SGNs, respectively. Labeled SGNs were classified into four groups based on the expression pattern of stained markers, including CR+ (subtype Ia), CB+ (subtype Ib), CR+CB+ (dual-labeled Ia/Ib), and CR-CB- (subtype Ic) neurons. The distribution of these SGN groups was analyzed in the apex, middle, and base regions of the cochleae. It showed that the prevalence of subtype Ia, Ib and dual-labeled Ia/Ib SGNs are high in the apex and low in the base. In contrast, the distribution pattern is reversed in Ic SGNs. Such frequency-dependent distribution is largely maintained during aging except for a preferential reduction of Ic SGNs, especially in the base. These findings suggest that sound processing of different frequencies involves distinct combinations of SGN subtypes, and the age-dependent loss of Ic SGNs in the base may especially impact high-frequency hearing during ARHL.

neuroscience↗

bioRxiv2023

Short- and long-term high-fat diet exposure differentially alters phasic and tonic GABAergic signalling onto lateral orbitofrontal pyramidal neurons

The chronic consumption of caloric dense high-fat foods is a major contributor to increased body weight, obesity, and other chronic health conditions. The orbitofrontal cortex (OFC) is critical in guiding decisions about food intake and is altered with diet-induced obesity. Obese rodents have altered morphological and synaptic electrophysiological properties in the lateral orbitofrontal cortex (lOFC). Yet the time course by which exposure to a high-fat diet (HFD) induces these changes is poorly understood. Here male mice are exposed to either short- (7 day) or long-term (90 day) HFD. Long-term HFD exposure increases body weight, and glucose signaling compared to short-term HFD or a standard chow control diet (SCD). Both short and long-term HFD exposure increased the excitability of lOFC pyramidal neurons. However, phasic and tonic GABAergic signalling was differentially altered depending on HFD exposure length, such that tonic GABAergic signaling was decreased with early exposure to the HFD and phasic signaling was changed with long-term diet exposure. Furthermore, alterations in the short-term diet exposure were transient, as removal of the diet restored electrophysiological characteristics similar to mice fed SCD whereas long-term HFD electrophysiological changes were persistent and remained after HFD removal. Finally, we demonstrate that changes in reward devaluation occur early with diet exposure. Together, these results suggest that the duration of HFD exposure differentially alters lOFC function and provides mechanistic insights into the susceptibility of the OFC to impairments in outcome devaluation. Significant statementThis study provides mechanistic insight on the impact of short- and long-term high fat diet (HFD) exposure on GABAergic function in the lateral orbitofrontal cortex (lOFC), a region known to guide decision making. We find short-term HFD exposure induces transient changes in firing and tonic GABA action on lOFC pyramidal neurons, whereas long-term HFD induces obesity and has lasting changes on firing, tonic GABA and inhibitory synaptic transmission onto lOFC neurons. Given that GABAergic signaling in the lOFC can influence decision making around food, these results have important implications in present society as palatable energy dense foods are abundantly available.

neuroscience↗

bioRxiv2023

The LRRK2 kinase substrates Rab8a and Rab10 contribute complementary but distinct disease-relevant phenotypes in human neurons

Mutations in the LRRK2 gene cause familial Parkinsons disease presenting with pleomorphic neuropathology that can involve -synuclein or tau accumulation. LRRK2 mutations are thought to converge toward a pathogenic increase in LRRK2 kinase activity. A subset of small Rab GTPases have been identified as LRRK2 substrates, with LRRK2-dependent phosphorylation resulting in Rab inactivation. We used CRISPR/Cas9 genome editing to generate a novel series of isogenic iPSC lines deficient in the two most well validated LRRK2 substrates, Rab8a and Rab10, from two independent, deeply phenotyped healthy control lines. Thorough characterization of NGN2-induced neurons revealed divergent effects of Rab8a and Rab10 deficiency on lysosomal pH, LAMP1 association with Golgi, -synuclein insolubility and tau phosphorylation, while parallel effects on lysosomal numbers and Golgi clustering were observed. Our data demonstrate largely antagonistic effects of genetic Rab8a or Rab10 inactivation which provide discrete insight into the pathologic features of their biochemical inactivation by pathogenic LRRK2 mutation. HighlightsO_LIRab8a and Rab10 deficiency induce lysosomal and Golgi defects C_LIO_LIRab8a and Rab10 deficiency induce opposing effects on lysosomal pH C_LIO_LIRab8a KO and Rab10 KO neurons show divergent effects on synuclein and tau proteostasis C_LIO_LIInactivation of different Rab GTPases can induce distinct disease-relevant phenotypes C_LI

neuroscience↗

bioRxiv2023

Stricturing Crohn's disease single-cell RNA sequencing reveals fibroblast heterogeneity and intercellular interactions

BackgroundFibroblasts play a key role in stricture formation in Crohns disease (CD) but understanding its pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. MethodsWe performed scRNAseq of 13 fresh full thickness CD resections containing non-involved, inflamed non-strictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next generation sequencing, proteomics and animal models. ResultsOur integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and upregulated, and its pro-fibrotic function was validated by NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and two animal models of experimental colitis. ConclusionA full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and opens potential therapeutic developments.

immunology↗

bioRxiv2023

Keypoint-MoSeq: parsing behavior by linking point tracking to pose dynamics

Keypoint tracking algorithms have revolutionized the analysis of animal behavior, enabling investigators to flexibly quantify behavioral dynamics from conventional video recordings obtained in a wide variety of settings. However, it remains unclear how to parse continuous keypoint data into the modules out of which behavior is organized. This challenge is particularly acute because keypoint data is susceptible to high frequency jitter that clustering algorithms can mistake for transitions between behavioral modules. Here we present keypoint-MoSeq, a machine learning-based platform for identifying behavioral modules ("syllables") from keypoint data without human supervision. Keypoint-MoSeq uses a generative model to distinguish keypoint noise from behavior, enabling it to effectively identify syllables whose boundaries correspond to natural sub-second discontinuities inherent to mouse behavior. Keypoint-MoSeq outperforms commonly used alternative clustering methods at identifying these transitions, at capturing correlations between neural activity and behavior, and at classifying either solitary or social behaviors in accordance with human annotations. Keypoint-MoSeq therefore renders behavioral syllables and grammar accessible to the many researchers who use standard video to capture animal behavior.

neuroscience↗

bioRxiv2023

Ultra-high throughput mapping of genetic design space

Massively parallel genetic screens have been used to map sequence-to-function relationships for a variety of genetic elements. However, because these approaches only interrogate short sequences, it remains challenging to perform high throughput (HT) assays on constructs containing combinations of multiple sequence elements arranged across multi-kb length scales. Overcoming this barrier could accelerate synthetic biology; by screening diverse gene circuit designs and learning "composition-to-function" mappings that reveal genetic part composability rules and enable rapid identification of behavior-optimized variants. Here, we introduce CLASSIC, a genetic screening platform that combines long- and short-read next-generation sequencing (NGS) modalities to quantitatively assess pools of constructs of arbitrary length containing diverse part compositions. We show that CLAS-SIC can measure expression profiles of >105 gene circuit designs (from 5-20 kb) in a single experiment in human cells. The resulting datasets can be used to train ML models that accurately predict circuit behavior across expansive circuit design landscapes, revealing part composability rules that govern circuit performance. Our work shows that by expanding the throughput of each design-build-test-learn (DBTL) cycle, CLASSIC enhances the pace and scale of synthetic biology and establishes an experimental basis for data-driven design of complex genetic systems.

synthetic biology↗

bioRxiv2023

Destabilization of neuromuscular junctions and deregulation of activity-dependent signalling pathways in Myotonic Dystrophy type I

Myotonic Dystrophy type I (DM1) is the most common muscular dystrophy in adults. Previous reports have highlighted that neuromuscular junctions (NMJs) deteriorate in skeletal muscle from DM1 patients and mouse models thereof. However, the underlying pathomechanisms and their contribution to muscle dysfunction remain unknown. We compared changes in NMJs and activity-dependent signalling pathways in HSALR and Mbnl1{Delta}E3/{Delta}E3 mice, two established mouse models for DM1. DM1 muscle showed major deregulation of calcium/calmodulin-dependent protein kinases II (CaMKIIs), which are key activity sensors regulating synaptic gene expression and acetylcholine receptor (AChR) recycling at the NMJ. Both mouse models displayed increased fragmentation of the endplate, which preceded muscle degeneration. Endplate fragmentation was not accompanied by changes in AChR turnover at the NMJ. However, expression of synaptic genes was up-regulated in DM1 muscle, which may be linked to the abnormally high activity of histone deacetylase 4 (HDAC4), a known target of CaMKII. Consistently, expression of myosin heavy chains was deregulated as well, leading to a major switch to type IIA fibres in Mbnl1{Delta}E3/{Delta}E3 muscle, and to a lesser extent in HSALR muscle. Interestingly, although HDAC4 was efficiently induced upon nerve injury, synaptic gene up-regulation was abrogated in DM1 muscle, together with a reduced increase in AChR turnover. This suggested that HDAC4-independent mechanisms lead to the defective response to denervation in DM1 muscle. Our study shows that activity-dependent signalling pathways are disturbed in DM1 muscle, which may contribute to NMJ destabilization and muscle dysfunction in DM1 patients.

physiology↗

bioRxiv2023

Design-Rules for Stapled Alpha-Helical Peptides with On-Target In Vivo Activity: Application toMdm2/X dual antagonists

Stapled -helical peptides can bind to and modulate historically intractable targets while addressing the traditional liabilities associated with peptide therapeutics. However, their pipeline advancement has been impeded by the challenges of identifying peptides with sufficient cellular uptake to engage the target protein while lacking off-target toxicities. Here, we advance the field to arrive at a workflow for identifying advanced stapled peptide lead molecules with on-target in vivo activity with no off-target cell proliferation effects. Specifically, we generated a >350-member library based on ATSP-7041, a stapled peptide Mdm2(X) antagonist with validated on-target cellular effects but with significant off-target activity. Key insights from library analysis include 1) a clear correlation between lipophilicity and permeability, 2) removal of positive charge to avoid off-target toxicities, 3) judicious placement of anionic residues to enhance peptide solubility/behavior, 4) optimization of C-terminal length and helicity to enhance cell activity, 5) optimization of staple type/number to avoid polypharmacology. Incorporation of one or more of these attributes led to molecules with improved in vitro and in vivo activities (up to a >292x improved cell proliferation EC50). A subset of peptides were devoid of off-target cell proliferation effects in cell lines lacking wild-type p53 protein (up to a >3800x on-target index). This latter improvement contrasted with clinical Mdm2 antagonistic molecules. Application of these design rules to a distinct Mdm2(X) peptide series resulted in rapid improvement in cellular activity (>150x) and removal of off-target toxicities. Overall, the detailed workflow outlined here should help researchers identify stapled -helical peptides for therapeutic impact.

biochemistry↗

bioRxiv2023

Fast, multiplexable and highly efficient somatic gene deletions in adult mouse skeletal muscle fibers using AAV-CRISPR/Cas9

Molecular screens comparing different disease states to identify candidate genes rely on the availability of fast, reliable and multiplexable systems to interrogate genes of interest. CRISPR/Cas9- based reverse genetics is a promising method to eventually achieve this. However, such methods are sorely lacking for multi-nucleated muscle fibers, since highly efficient nuclei editing is a requisite to robustly inactive candidate genes. Here, we couple Cre-mediated skeletal muscle fiber-specific Cas9 expression with myotropic adeno-associated virus-mediated sgRNA delivery to establish a system for highly effective somatic gene mutation in mice. Using well-characterized genes, we show that local or systemic inactivation of these genes copy the phenotype of traditional gene-knockout mouse models. Thus, this proof-of-principle study establishes a method to unravel the function of individual genes or entire signaling pathways in adult skeletal muscle fibers without the cumbersome requirement of generating knockout mice.

molecular biology↗

bioRxiv2023

Integrated Cell Landscape and Dynamics in the Progression of Bone Repair

Bone homeostasis and repair is a systematic progress with spatiotemporal interaction of multiple cell types involved in skeletal and immune system. Precise spatiotemporal regulation of cell type-specific functions in bone repair contributes to further development of tissue engineering and regenerative medicine. Here, we utilized single-cell RNA sequencing to illustrate a map of cell landscape and dynamics in the progression of rodent bone self-healing and a perturbation by lymphoid cell-deficiency. We identified different functions of myeloid cell and lymphoid cell to osteogenesis and angiogenesis during bone repair and their mutual complementation under lymphoid cell-deficient condition. Additionally, we used CD34+ humanized reconstituted mice to reveal further insights into the mechanism of human bone homeostasis and repair. Our integrated cellular analysis of bone repair explores the functional diversity and complementation between myeloid cells and lymphoid cells during bone healing process and provides further therapeutic implications for the treatment of bone disease and degeneration following ageing.

cell biology↗

bioRxiv2023

Unraveling codon usage of Escherichia coli using machine learning

Machine learning is used to investigate the codon usage of protein-encoding genes, which is one of the fundamental questions of molecular biology. The presentation, parameter learning, and decoding of the conditional random field (CRF) model are implemented and then utilized to analyze the codon usage of the genes of Escherichia coli and its phages. Most genes of E. coli use codons conforming to the weights of the model determined by all E. coli genes. Phages use the codons like their host E. coli. Finally, the study evaluates the codon usage of several example genes in the context of the model. These results help to understand the codon usage in E. coli.

systems biology↗

bioRxiv2023

CAM5, WRKY53, and TGA5 regulate defense gene expression mediated by the volatile organic compound ethyl vinyl ketone

Plants produce ethyl vinyl ketone (evk) in response to biotic stress, but the evks identification and downstream defense response remain unclear. In this paper, it is predicted by docking for the first time that evk can be recognized by RBOH protein and assist the electron transfer of RBOHD/RBOHF by binding to its FAD or NADPH binding site. Here, we show that evk treatment increased H2O2 and intracellular calcium concentrations in Arabidopsis thaliana mesophyll cells, as observed by confocal laser scanning microscopy and non-invasive micro-test technology, and that H2O2 signaling functioned upstream of Ca2+ signaling. Yeast two-hybrid, firefly luciferase complementation imaging, and in vitro pull-down assays demonstrated that the ACA8 (AUTOINHIBITED Ca2+-ATPASE, ISOFORM 8)-CML8 (CALMODULIN-LIKE 8) interaction promoted Ca2+ efflux to return Ca2+ levels to the resting state. Evk treatment led to the antagonism of salicylic acid (SA) and jasmonic acid (JA). CALMODULIN 5 (CAM5) positively regulates WRKY53 expression, and CAM5 and WRKY53 positively regulate SA-related gene expression. These proteins physically interact and form a complex that is unlocked by Ca2+ to release WRKY53. An electrophoretic mobility shift assay and dual-luciferase reporter assay demonstrated that WRKY53 and TGA5 cooperate to enhance the expression of the defense gene PATHOGENESIS-REALTED 1 (PR1) and that WRKY53 enhances the binding of TGA5 to the PR1 promoter. This paper proposes a framework that evk, as a RES substance, can achieve plants REScue through complete defense response.

plant biology↗

bioRxiv2022

Enterotypes of the human gut mycobiome

The fungal component of the human gut microbiome, also known as the mycobiome, plays a vital role in intestinal ecology and human health. Here, we identify and characterize four mycobiome enterotypes using ITS profiling of 3,363 samples from 16 cohorts across three continents, including 572 newly profiled samples from China. These enterotypes exhibit stability across populations and geographical locations and significant correlation with bacterial enterotypes. Particularly, we notice that fungal enterotypes have a strong age preference, where the enterotype dominated by Candida (i.e., fun_C_E enterotype) is enriched in the elderly population and confers an increased risk of multiple diseases associated with compromised intestinal barrier. In addition, bidirectional mediation analysis reveals that the fungi-contributed aerobic respiration pathway associated with fun_C_E enterotype might mediate the association between the compromised intestinal barrier and aging. TeaserAs an integral part of the human gut microbiome, the fungi, which co-habit with prokaryotic microbiome in the gut, play important role in the intestinal ecology and human health. Yet, the overall structure of the human gut mycobiome and the inter-individual variation worldwide remain largely unclear. Lai et al. analyzed the fungal profiles of 3,363 samples from 16 cohorts across three continents, and identified four fungal enterotypes that exhibit stability across populations. They found that fungal enterotypes showed age preference, where a Candida dominated enterotype was enriched in the elderly population and confers an increased risk of multiple diseases and more severe compromised intestinal barrier. Furthermore, they determined one fungi-contributed aerobic respiration pathway could mediate the association between the compromised intestinal barrier and aging.

bioinformatics↗

bioRxiv2022

Species-dependent effects of seawater acidification on alkaline phosphatase activity in dinoflagellates

Global climate change is widely shown to cause ocean acidification (OA) and projected to intensify nutrient limitation. Phosphorus (P) is an essential nutrient for phytoplankton to grow. While dissolved inorganic phosphate (DIP) is the preferred form of P, phytoplankton have evolved alkaline phosphatase (AP) to utilize dissolved organic phosphorus (DOP) when DIP is deficient. Although AP is known to require pH>7, how OA may affect AP activity and hence the capacity of phytoplankton to utilize DOP is poorly understood. Here, we examine the effects of pH conditions (5.5 to 11) on AP activity from six species of dinoflagellates, an important group of marine phytoplankton. We observed a general pattern that AP activity declined sharply at pH 5.5, peaked between pH 7 and 8, and dropped at pH>8. However, our data revealed remarkable interspecific variations in optimal pH and niche breadth of pH. Among the species examined, Fugacium kawagutii and Prorocentrum cordatum had an optimal pH at 8, Alexandrium pacificum, Amphidinium carterae, Effrenium voratum, and Karenia mikimotoi showed an optimal pH of 7. However, whereas A. pacificum, F. kawagutii and K. mikimotoi had a broad pH range for AP (7-11), A. carterae, E. voratum, and P. cordatum exhibited a narrow pH range. The response of AP activity of A. carterae to pH changes was verified using purified AP heterologously expressed in Escherichia coli. These findings suggest that OA will likely differentially impact the capacity of different phytoplankton species to utilize DOP in the projected acidified and nutrient-limited future ocean.

ecology↗

bioRxiv2022

Brain region-specific changes in neurons and glia and dysregulation of dopamine signaling in Grin2a mutant mice

Schizophrenia disease mechanisms remain poorly understood, in large part due to a lack of valid animal models. Rare heterozygous loss-of-function mutations in GRIN2A, encoding a subunit of the NMDA (N-methyl-d-aspartate) receptor, greatly increase the risk of schizophrenia. By transcriptomic, proteomic, electroencephalogram (EEG) recording and behavioral analysis, we report that heterozygous Grin2a mutant mice show: (i) large-scale gene expression changes across multiple brain regions and in neuronal (excitatory and inhibitory) and non-neuronal cells (astrocytes, oligodendrocytes); (ii) evidence of reduced activity in prefrontal cortex and increased activity in hippocampus and striatum; (iii) elevated dopamine signaling in striatum; (iv) altered cholesterol biosynthesis in astrocytes; (v) reduction of glutamatergic receptor signalin g proteins in the synapse; (iv) heightened gamma oscillation power in EEG; (vi) aberrant locomotor behavioral pattern opposite of that induced by antipsychotic drugs. These findings reveal potential pathophysiologic mechanisms, provide support for both the "hypo-glutamate" and "hyper-dopamine" hypotheses of schizophrenia, and underscore the utility of Grin2a-deficient mice as a new genetic model of schizophrenia.

neuroscience↗

bioRxiv2022

Dendritic degeneration and altered synaptic innervation of a central auditory neuron during age-related hearing loss

Cellular morphology and synaptic configuration are key determinants of neuronal function and are often modified under pathological conditions. In the first nucleus of the central auditory system, the cochlear nucleus (CN), principal bushy neurons specialize in processing temporal information of sound critical for hearing. These neurons alter their physiological properties during aging that contribute to age-related hearing loss (ARHL). The structural basis of such changes remains unclear, especially age-related modifications in their dendritic morphology and the innervating auditory nerve (AN) synapses. Using young (2-5 months) and aged (28-33 months) CBA/CaJ mice of either sex, we filled individual bushy neurons with fluorescent dye in acute brain slices to characterize their dendritic morphology, followed by immunostaining against vesicular glutamate transporter 1 (VGluT1) and calretinin (CR) to identify innervating AN synapses. We found that dendritic morphology of aged bushy neurons had significantly reduced complexity, suggesting age-dependent dendritic degeneration, especially in neurons with predominantly non-CR-expressing synapses on the soma. These dendrites were innervated by AN bouton synapses, which were predominantly non-CR-expressing in young mice but had increased proportion of CR-expressing synapses in old mice. While somatic AN synapses degenerated substantially with age, as quantified by VGluT1-labeled puncta volume, no significant difference was observed in the total volume of dendritic synapses between young and old mice. Consequently, synaptic density on dendrites was significantly higher in old mice. The findings suggest that dendritic degeneration and altered synaptic innervation in bushy neurons during aging may underlie their changed physiological activity and contribute to the development of ARHL. HIGHLIGHTSO_LIDendrites of bushy neurons degenerate during aging with reduced complexity C_LIO_LIBushy dendrites receive AN input primarily from SGNs with medium/high threshold C_LIO_LIDendritic synapses show similar total volume but increased density during aging C_LIO_LIDendritic degeneration in bushy neurons and altered AN input may underlie ARHL C_LI

neuroscience↗