Biology subjects

Milani, M.

Publications and source records attributed to Milani, M..

11 recordsLinked to original sources

bioRxiv2026

Calcium-Driven Activation of Gelsolin: Mechanism and the Active-State Ensemble

Gelsolin is a master regulator of the actin cytoskeleton whose activity is controlled by large Ca2+ dependent conformational transitions of its 6 domains (G1-G6). Despite decades of biochemical and cellular studies, the structural basis of activation of the full-length protein remains poorly understood because active gelsolin is highly dynamic and has largely escaped structural characterization. Here, we combine small-angle X-ray scattering, DEER-EPR spectroscopy, and structure-based molecular dynamics simulations to investigate the transition from the compact inactive state to the actin-binding active form. This integrative approach reveals that activation proceeds through a sequential and domain-dependent mechanism, where different regions of the protein respond to Ca2+ with distinct kinetics and conformational trajectories. Rather than converging toward a single active structure, activated gelsolin populates a heterogeneous ensemble of plastic conformations characterized by asymmetric domain arrangements and extensive structural variability. The two major domain triplets (G1-G3 and G4-G6) display markedly different dynamic behaviors, while actin-binding surfaces are exposed according to a defined hierarchy and remain differentially accessible within the active ensemble, suggesting specialized functional roles for different domains during filament engagement. Together, these findings provide a time- and domain-resolved view of gelsolin activation, establish a structural framework for interpreting its diverse biological functions, and offer new opportunities for targeting gelsolin-associated diseases.

biophysics↗

bioRxiv2026

The lysosomal channel RECS1 regulates extracellular vesicle biogenesis

Lysosomal ion channels play key roles in regulating membrane trafficking, autophagy, and cell death. RECS1 is a pH-sensitive lysosomal calcium channel previously implicated in lysosome-mediated apoptosis. Here, we identify a novel role for RECS1 in exosome biology. Using immunoprecipitation followed by mass spectrometry, we mapped RECS1 interactors under apoptotic and lysosomal stress conditions. Notably, Syntenin-1, a key scaffolding protein in ESCRT-independent exosome biogenesis, emerged as the top hit. We validated the physical interaction between RECS1-Syntenin-1 using various approaches. RECS1 localized to secreted exosomes, and its overexpression increased exosome production, as measured by nanoparticle tracking analysis. Intriguingly, a channel-dead RECS1 retained both Syntenin-1 interaction and the ability to promote exosome release, suggesting a channel-independent mechanism. Our findings identify RECS1 as a structural component of the exosomal trafficking machinery and a modulator of extracellular vesicle biogenesis. This work connects lysosomal signalling with intercellular communication and suggests a broader role for RECS1 in stress-responsive secretion.

cell biology↗

bioRxiv2024

Cytoplasmic accumulation of a splice variant of hnRNP A2/B1 contributes to FUS-associated toxicity in a mouse model of ALS

Genetic and experimental findings point to a crucial role of RNA dysfunction in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). Evidence suggests that mutations in RBPs such as FUS, a gene associated with ALS, affect the regulation of alternative splicing. We have previously shown that the overexpression of wild-type FUS in mice, a condition that induces ALS-like phenotypes, impacts the splicing of hnRNP A2/B1, a protein with key roles in RNA metabolism, suggesting that a pathological connection between FUS and hnRNP A2/B1 might promote FUS-associated toxicity. Here we report that the expression and distribution of different hnRNP A2/B1 splice variants are modified in the affected tissues of mice overexpressing wild-type FUS. Notably, degenerating motor neurons are characterized by the cytoplasmic accumulation of splice variants of hnRNP A2/B1 lacking exon 9 (hnRNP A2b/B1b). In vitro studies show that exon 9 skipping affects the nucleocytoplasmic distribution of hnRNP A2/B1, promoting its localization into stress granules (SGs), and demonstrate that cytoplasmic localization is the primary driver of hnRNP A2b recruitment into SGs and cell toxicity. Finally, boosting exon 9 skipping using splicing switching oligonucleotides exacerbates disease phenotypes in wild-type FUS mice. Altogether, these findings reveal that alterations of the nucleocytoplasmic distribution of hnRNP A2/B1, driven by FUS-induced splicing changes, likely contribute to motor neuron degeneration in ALS.

neuroscience↗

bioRxiv2024

A Mutation in Tmem135 Causes Progressive Sensorineural Hearing Loss

Transmembrane protein 135 (TMEM135) is a 52 kDa protein with five predicted transmembrane domains that is highly conserved across species. Previous studies have shown that TMEM135 is involved in mitochondrial dynamics, thermogenesis, and lipid metabolism in multiple tissues; however, its role in the inner ear or the auditory system is unknown. We investigated the function of TMEM135 in hearing using wild-type (WT) and Tmem135FUN025/FUN025 (FUN025) mutant mice on a CBA/CaJ background, a normal-hearing mouse strain. Although FUN025 mice displayed normal auditory brainstem response (ABR) at 1 month, we observed significantly elevated ABR thresholds at 8, 16, and 64 kHz by 3 months, which progressed to profound hearing loss by 12 months. Consistent with our auditory testing, 13-month-old FUN025 mice exhibited a severe loss of outer hair cells and spiral ganglion neurons in the cochlea. Our results using BaseScope in situ hybridization indicate that TMEM135 is expressed in the inner hair cells, outer hair cells, and supporting cells. Together, these results demonstrate that the FUN025 mutation in Tmem135 causes progressive sensorineural hearing loss, and suggest that TMEM135 is crucial for maintaining key cochlear cell types and normal sensory function in the aging cochlea.

neuroscience↗

bioRxiv2024

BAX and BAK expression triggers multiple IRE1 signaling outputs under ER stress

Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the unfolded protein response (UPR) stress sensor inositol-requiring enzyme 1 alpha (IRE1). IRE1 is a central ER stress sensors, that signals through the activation of its RNase domain to catalyze the splicing the mRNA encoding the transcription factor X-box binding protein 1 (XBP1), resulting on the expression of a stable and active transcription factor termed XBP1s. The kinetics and amplitude of IRE1 signaling are regulated by different posttranslational modifications and the physical interaction of different factors. Early studies demonstrated that the expression of the proapoptotic proteins BAX and BAX enhance UPR signaling. However, the possible effects on the RNase activity were not defined. Here we provide preliminary evidence indicating that BAX and BAK deficiency increases the in 10 folds the threshold of ER stress to induce XBP1 mRNA splicing, and the upregulation of its target genes. In addition, the degradation of RIDD substrates was strongly reduced in BAX and BAK null cells. BAX and BAK double deficiency also attenuated the levels of IRE1 phosphorylation under mild ER stress. These results reinforce previous findings indicating that proapoptotic BAX and BAK have alternative functions at the ER regulating the UPR.

cell biology↗

bioRxiv2024

Loss of Esr1 Does Not Affect Hearing and Balance

Although estrogen affects the structure and function of the nervous system and brain and has a number of effects on cognition, its roles in the auditory and vestibular systems remain unclear. The actions of estrogen are mediated predominately through two classical nuclear estrogen receptors, estrogen receptor 1 (ESR1) and estrogen receptor 2 (ESR2). In the current study, we investigated the roles of ESR1 in normal auditory function and balance performance using 3-month-old wild-type (WT) and Esr1 knockout (KO) mice on a CBA/CaJ background, a normal-hearing strain. As expected, body weight of Esr1 KO females was lower than that of Esr1 KO males. Body weight of Esr1 KO females was higher than that of WT females, while there was no difference in body weight between WT and Esr1 KO males. Similarly, head diameter was higher in Esr1 KO vs. WT females. Contrary to our expectations, there were no differences in auditory brainstem response (ABR) thresholds, ABR waves I-V amplitudes and ABR waves I-V latencies at 8, 16, 32, and 48 kHz, distortion product otoacoustic emission (DPOAE) thresholds and amplitudes at 8, 16, and 32 kHz, and rotarod balance performance (latency to fall) between WT and Esr1 KO mice. Furthermore, there were no sex differences in ABRs, DPOAEs, and rotarod balance performance in Esr1 KO mice. Taken together, our findings show that Esr1 deficiency does not affect auditory function or balance performance in normal hearing mice, and suggest that loss of Esr1 is likely compensated by ESR2 or other estrogen receptors to maintain the structure and function of the auditory and vestibular systems under normal physiological conditions. HighlightsO_LIHead diameter of female Esr1 KO mice was higher than that of female WT mice. C_LIO_LIABRs and DPOAEs were not different in WT and Esr1 KO mice. C_LIO_LIThere were no sex differences in ABRs and DPOAEs in Esr1 KO mice. C_LIO_LIRotarod balance performance was not different in WT and Esr1 KO mice. C_LIO_LIThere were no sex differences in rotarod balance performance in Esr1 KO mice. C_LIO_LILoss of Esr1 does not affect auditory function or balance performance under normal physiological conditions. C_LI

neuroscience↗

bioRxiv2023

Frataxin Deficiency Drives a Shift from Mitochondrial Metabolism to Glucose Catabolism, Triggering an Inflammatory Phenotype in Microglia

Immunometabolism investigates the complex interplay between the immune system and cellular metabolism. This study highlights the effects of mitochondrial frataxin (FXN) depletion, which causes Friedreichs ataxia (FRDA), a neurodegenerative condition characterized by coordination and muscle control deficiencies. Using single-cell RNA sequencing, we identified specific cell groups in the cerebellum of a FRDA mouse model, emphasizing a notable inflammatory microglial response. These FXN-deficient microglia cells exhibited enhanced inflammatory reactions. Furthermore, our metabolomic analyses revealed increased glycolysis and itaconate production in these cells, possibly driving the inflammation. Remarkably, butyrate treatment counteracted these immunometabolic changes, triggered an antioxidant response via the itaconate-Nrf2-GSH pathways, and dampened inflammation. The study also pinpointed Hcar2 (GPR109A) as a potential agent for butyrate anti-inflammatory impact on microglia. Tests on FRDA mice highlighted the neuroprotective attributes of butyrate intake, bolstering neuromotor performance. In essence, our findings shed light on how cerebellar microglia activation contributes to FRDA and highlight butyrate potential to alleviate neuroinflammation, rectify metabolic imbalances, and boost neuromotor capabilities in FRDA and similar conditions.

physiology↗

bioRxiv2023

The endoplasmic reticulum stress sensor IRE1 regulates collagen secretion through the enforcement of the proteostasis factor P4HB/PDIA1 contributing to liver damage and fibrosis

Collagen is one the most abundant proteins and the main cargo of the secretory pathway, contributing to hepatic fibrosis and cirrhosis due to excessive deposition of extracellular matrix. Here we investigated the possible contribution of the unfolded protein response, the main adaptive pathway that monitors and adjusts the protein production capacity at the endoplasmic reticulum, to collagen biogenesis and liver disease. Genetic ablation of the ER stress sensor IRE1 reduced liver damage and diminished collagen deposition in models of liver fibrosis triggered by carbon tetrachloride (CCl4) administration or by high fat diet. Proteomic and transcriptomic profiling identified the prolyl 4-hydroxylase (P4HB, also known as PDIA1), which is known to be critical for collagen maturation, as a major IRE1-induced gene. Cell culture studies demonstrated that IRE1 deficiency results in collagen retention at the ER and altered secretion, a phenotype rescued by P4HB overexpression. Taken together, our results collectively establish a role of the IRE1/P4HB axis in the regulation of collagen production and its significance in the pathogenesis of various disease states.

cell biology↗

bioRxiv2020

Combined in silico docking and in vitro antiviral testing for drug repurposing identified lurasidone and elbasvir as SARS-CoV-2 and HCoV-OC43 inhibitors

The current emergency of the novel coronavirus SARS-CoV-2 urged the need for broad-spectrum antiviral drugs as the first line of treatment. Coronaviruses are a large family of viruses that already challenged humanity in at least two other previous outbreaks and are likely to be a constant threat for the future. In this work we developed a pipeline based on in silico docking of known drugs on SARS-CoV RNA-dependent RNA polymerase combined with in vitro antiviral assays on both SARS-CoV-2 and the common cold human coronavirus HCoV-OC43. Results showed that certain drugs displayed activity for both viruses at a similar inhibitory concentration, while others were specific. In particular, the antipsychotic drug lurasidone and the antiviral drug elbasvir showed promising activity in the low micromolar range against both viruses with good selective index.

pharmacology and toxicology↗

bioRxiv2020

SPARC regulation of PMN clearance protects from pristane induced lupus and rheumatoid arthritis

One step along the pathogenesis of Systemic lupus erythematosus (SLE) is associated with polymorphonuclear leukocyte (PMN) death and their ineffective removal by M2-macrophages. The secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein with unexpected immunosuppressive function in M2-macrophages and myeloid cells. To investigate the role of SPARC in autoimmunity, we adopted a pristane-induced model of lupus in mice, which recapitulates clinical manifestations of human SLE. Sparc-/- mice developed earlier and more severe renal disease, lung and liver parenchymal damage than the WT counterpart. Most prominently, Sparc-/- mice had anticipated and severe occurrence of arthritis. An intermediate phenotype was obtained in Sparc+/- hemizygous mice, a result that suggests Sparc gene-dosage as relevant in autoimmune-related events. Mechanistically, a defective Sparc expression in PMN blocks their clearance by macrophages, through a defective delivery of eat-me and dont eat-me signals. Sparc-/- PMN that escape macrophage scavenging becomes a source of autoantigens for dendritic cell (DC) presentation and a direct stimulus for IL-17 expression in {gamma}{delta}-T-cells. Gene profile analysis of synovial biopsies of knees affected by SLE-associated arthritis showed an inverse correlation between SPARC and key autoimmune genes. These results point to SPARC down-regulation as a key event characterizing SLE and associated rheumatoid arthritis pathogenesis.

immunology↗

bioRxiv2018

ER shaping proteins regulate mitochondrial fission, outer membrane permeabilization and apoptosis

The mitochondrial fission machinery, comprising a dynamin-related GTPase, DRP-1, is crucial for the regulation of mitochondrial membrane dynamics. Recent reports suggest that the tubular architecture of the endoplasmic reticulum (ER) marks the constriction sites on the mitochondria to facilitate DRP-1-mediated mitochondrial fission. However, the role of several ER shaping proteins that maintain the elaborate network of tubes and sheets in mitochondrial constriction and fission is not yet known. In this report, we demonstrate that modulation of the expression levels of key ER shaping proteins, namely Reticulon1 (RTN-1), Reticulon 4 (RTN-4), Lunapark-1 (LNP-1) and CLIMP-63, markedly decreased the extent of mitochondrial fission mediated by BH3 mimetics, despite no detectable changes in DRP-1 recruitment to the mitochondria. Furthermore, modulation of ER shaping proteins significantly decreased other hallmarks of apoptosis, such as mitochondrial outer membrane permeabilization, caspase activation and phosphatidylserine externalization, and functioned independently of mitochondrial cristae remodeling, thus demonstrating a requirement of ER shaping proteins and ER structural integrity for the efficient execution of the instrinsic apoptotic pathway.

cell biology↗