Biology subjects

Milosevic, I.

Publications and source records attributed to Milosevic, I..

13 recordsLinked to original sources

bioRxiv2026

β-Adrenergic inhibition of exocytotic surface deposition of MHCII molecules in reactive astrocytes is mediated by amisyn

Degeneration of the locus coeruleus, a noradrenergic nucleus, reduces noradrenaline bioavailability in the central nervous system and promotes neuroinflammation via reactive astrocytes, although the underlying mechanisms remain unclear. We investigated whether interferon-{gamma}-induced expression of major histocompatibility complex class II (MHCII), a marker of pro-inflammatory reactive astrocytes, is regulated by adrenergic receptors and amisyn. {beta}-Adrenergic, but not -adrenergic, stimulation increased cyclic adenosine monophosphate (cAMP) and reduced MHCII expression, as detected immunocytochemically, in human and rat astrocytes. {beta}-Adrenergic treatment altered transient exocytosis of lysosome-like vesicles, increasing event frequency and reducing fusion-pore conductance and dwell time, thereby limiting MHCII surface expression. Overexpression of wild-type amisyn inhibited surface expression of MHCII and the lysosomal marker CD63 and reduced fusion-pore conductance and dwell time. Conversely, amisyn knockdown enhanced full fusion exocytosis of larger vesicles and abolished {beta}-adrenergic effects, indicating that amisyn mediates {beta}-adrenergic inhibition of exocytosis and MHCII surface deposition.

neuroscience↗

bioRxiv2026

Dopamine release from Parkinson's patient-derived neurons is disrupted due to impaired synaptic vesicle loading

Striatal dopamine release defects are an early pathological feature observed in diverse models of Parkinsons disease. However, the underlying molecular mechanisms responsible, and potential links to disease aetiology in humans, have been elusive. Here, we tested the hypothesis that dopamine release deficits are a characteristic feature of disease-relevant human neurons, using human Parkinsons patient iPSC-derived dopamine neurons carrying the SNCA-triplication mutation. We reveal deficits in dopamine release from SNCA-triplication patient-derived neurons, and identify that this is due to reduced dopamine content arising from a lower capacity to store dopamine through reduced expression and function of vesicular monoamine transporter 2 (VMAT2) compared to healthy controls. In turn, by imaging VMAT substrate FFN206, and reporters for synaptic vesicular dynamics, SynaptopHluorin and CypHer5E, we reveal corresponding deficits in the size of either VMAT-containing, presynaptic releasing or recycling vesicle pools. Consistent with diminished synaptic vesicle loading and recycling, the cytosolic turnover of dopamine indicated by the ratio of concentrations of dopamine metabolite DOPAC to dopamine was elevated. By contrast, glutamate release events and VGLUT2 levels in neurons in the same preparations were not disturbed, demonstrating that vesicular dysfunction is limited to vesicles for dopamine. These findings therefore reveal dopamine loading into vesicles as a locus of dysfunction in human Parkinsons-derived neurons. These disturbances will not only drive deficits in dopamine release but could potentially also be detrimental to dopamine neuron viability through an increased burden of oxidative stress associated with elevated cytosolic dopamine, thus contributing to both symptoms and aetiology of Parkinsons pathology and offering a strategic target for improved therapies.

neuroscience↗

bioRxiv2025

Nucleophagy removes cytotoxic trapped PARP1

Poly (ADP-Ribose) Polymerase inhibitors (PARPi) induce cytotoxicity in homologous recombination repair (HRR)-deficient cancers by causing PARP1 to become trapped on chromatin, resulting in irreparable replication-associated DNA damage. Although increased clearance of trapped PARP1 from chromatin reduces the sensitivity of cancer cells to PARPi, details surrounding this process remain unclear. PARPi exposure is known to cause increased autophagy flux, whilst autophagy inhibition can hypersensitise cells to PARPi. Using various biochemical, cell biological and live imaging-based assays, we found that trapped PARP1 is cleared by nucleophagy, the selective autophagy of nuclear substrates. Specifically, the nucleophagy of trapped PARP1 was orchestrated by the selective autophagy receptor TEX264 and its partner segregase p97/VCP. TEX264 mediates this process by directly interacting with trapped PARP1, thus bridging PARP1 to the autophagosomal resident protein LC3 for processing via autophagy. Impeding this process, either chemically or genetically, heightened PARP1 trapping, leading to accumulation of protein aggregates, replication-associated DNA damage and cell lethality, re-sensitising PARPi-resistant cells to various PARPi. In conclusion, we show that nucleophagy acts in a cytoprotective manner to directly target PARPi-induced trapped PARP1 for degradation.

cell biology↗

bioRxiv2025

Micronuclear collapse under oxidative stress drives amphisome-mediated export of DNA in Parkinson disease

The diversity of extracellular vesicle (EV) subpopulations and their impact on intercellular communication are increasingly recognized, but how organelle dysfunction shapes EV content in neurodegenerative diseases remains unclear. Mitochondrial and lysosomal functional defects are hallmarks of Parkinsons disease (PD). Here we uncover a novel pathway linking this dysfunctional axis to EV remodeling and immune activation. We show that mitochondrial reactive oxygen species (ROS) induce genomic instability and micronuclei formation in PD fibroblasts, with ruptured micronuclei being sequestered into amphisomes and exported through small EVs. These EVs are enriched in oxidized mitochondrial and nuclear DNA, which potently stimulate microglial inflammatory responses. Mechanistically, this work identifies micronuclei not as passive byproducts of genome instability but as active intermediates in EV cargo loading. Importantly, treatment with the mitochondria-targeted antioxidant AntiOxCIN4 elicited a mitohormetic response, enhancing ATM-mediated DNA damage repair, restoring mitochondrial dynamics, and improving lysosomal function. This reduced the incorporation of oxidized DNA into EVs and blunted their pro-inflammatory activity. Together, our findings reveal a previously unrecognized mechanism by which mitochondrial-lysosomal dysfunction drives the release of DNA-enriched EVs that fuel neuroinflammation in a neurodegenerative context. Targeting mitochondrial quality control to limit oxidized cargo in EVs emerges as a potential strategy to mitigate early inflammatory events in PD.

neuroscience↗

bioRxiv2024

Loss of the lysosomal protein CLN3 modifies the lipid content of the nuclear envelope leading to DNA damage and activation of YAP1 pro-apoptotic signaling

Batten disease is characterized by early-onset blindness, juvenile dementia and death during the second decade of life. The most common genetic causes are mutations in the CLN3 gene encoding a lysosomal protein. There are currently no therapies targeting the progression of the disease, mostly due to the lack of knowledge about the disease mechanisms. To gain insight into the impact of CLN3 loss on cellular signaling and organelle function, we generated CLN3 knock-out cells in a human cell line (CLN3-KO), and performed RNA sequencing to obtain the cellular transcriptome. Following a multi-dimensional transcriptome analysis, we identified the transcriptional regulator YAP1 as a major driver of the transcriptional changes observed in CLN3-KO cells. We further observed that YAP1 pro-apoptotic signaling is hyperactive as a consequence of CLN3 functional loss in retinal pigment epithelia cells, and in the hippocampus and thalamus of CLN3ex{Delta}7/8 mice, an established model of Batten disease. Loss of CLN3 activates YAP1 by a cascade of events that starts with the inability of releasing glycerophosphodiesthers from CLN3-KO lysosomes, which leads to perturbations in the lipid content of the nuclear envelope and nuclear dysmorphism. This results in increased number of DNA lesions, activating the kinase c-Abl, which phosphorylates YAP1, stimulating its pro-apoptotic signaling. Altogether, our results highlight a novel organelle crosstalk paradigm in which lysosomal metabolites regulate nuclear envelope content, nuclear shape and DNA homeostasis. This novel molecular mechanism underlying the loss of CLN3 in mammalian cells and tissues may open new c-Abl-centric therapeutic strategies to target Batten disease.

cell biology↗

bioRxiv2024

Up-regulation of cholesterol synthesis by lysosomal defects requires a functional mitochondrial respiratory chain

Mitochondria and lysosomes are two organelles that carry out both signaling and metabolic roles in the cells. Recent evidence has shown that mitochondria and lysosomes are dependent on one another, as primary defects in one cause secondary defects in the other. Nevertheless, the signaling consequences of primary mitochondrial malfunction and of primary lysosomal defects are not similar, despite in both cases there are impairments of mitochondria and of lysosomes. Here, we used RNA sequencing to obtain transcriptomes from cells with primary mitochondrial or lysosomal defects, to identify what are the global cellular consequences that are associated with malfunction of mitochondria or lysosomes. We used these data to determine what are the pathways that are affected by defects in both organelles, which revealed a prominent role for the cholesterol synthesis pathway. This pathway is transcriptionally up-regulated in cellular and mouse models of lysosomal defects and is transcriptionally down-regulated in cellular and mouse models of mitochondrial defects. We identified a role for post-transcriptional regulation of the transcription factor SREBF1, a master regulator of cholesterol and lipid biosynthesis, in models of mitochondrial respiratory chain deficiency. Furthermore, the retention of Ca2+ in the lysosomes of cells with mitochondrial respiratory chain defects contributes to the differential regulation of the cholesterol synthesis pathway in the mitochondrial and lysosomal defects tested. Finally, we verified in vivo, using models of mitochondria-associated diseases in C. elegans, that normalization of lysosomal Ca2+ levels results in partial rescue of the developmental arrest induced by the respiratory chain deficiency.

biochemistry↗

bioRxiv2023

Intersectin and Endophilin condensates prime synaptic vesicles for release site replenishment

Neurotransmitter is released from dedicated sites of synaptic vesicle fusion within a synapse. Following fusion, the vacated sites are replenished immediately by new vesicles for subsequent neurotransmission. These replacement vesicles are assumed to be located near release sites and used by chance. Here, we find that replacement vesicles are clustered around this region by Intersectin-1. Specifically, Intersectin-1 forms dynamic molecular condensates with Endophilin A1 near release sites and sequesters vesicles around this region. In the absence of Intersectin-1, vesicles within 20 nm of the plasma membrane are reduced, and consequently, vacated sites cannot be replenished rapidly, leading to depression of synaptic transmission. Similarly, mutations in Intersectin-1 that disrupt Endophilin A1 binding result in similar phenotypes. However, in the absence of Endophilin, this replacement pool of vesicles is available but cannot be accessed, suggesting that Endophilin A1 is needed to mobilize these vesicles. Thus, our work describes a distinct physical region within a synapse where replacement vesicles are harbored for release site replenishment.

neuroscience↗

bioRxiv2021

Functional peroxisomes are required for heat shock-induced hormesis in Caenorhabditis elegans

Exact mechanisms of heat shock induced lifespan extension, while documented across species, are still not well understood. Here we put forth evidence that fully functional peroxisomes are required for the activation of the canonical heat shock response and heat-induced hormesis in C. elegans. While during heat shock the HSP-70 chaperone is strongly upregulated in the wild-type (WT) as well as in the absence of peroxisomal catalase ({Delta}ctl-2 mutant), the small heat shock proteins display modestly increased expression in the mutant. Nuclear localization of HSF-1 is reduced in the{Delta} ctl-2 mutant. In addition, heat-induced lifespan extension, observed in the WT, is absent in the{Delta} ctl-2 mutant. Activation of the antioxidant response, the pentose phosphate pathway and increased triglyceride content are the most prominent changes observed during heat shock in the WT worm, but not in the{Delta} ctl-2 mutant. Involvement of peroxisomes in the cell-wide response to transient heat shock reported here gives new insight into the role of organelle communication in the organisms stress response.

cell biology↗

bioRxiv2021

Co-localization of different Neurotransmitter Transporters on the same Synaptic Vesicle is Bona-fide yet Sparse

Vesicular transporters (VTs) define the type of neurotransmitter that synaptic vesicles (SVs) store and release. While certain neurons in mammalian brain release multiple transmitters, the prevalence, physiology of such pluralism and if the release occurs from same or distinct vesicle pools is not clear. Using quantitative single vesicle imaging, we show that a small population of neuronal SVs indeed contain different VTs to accomplish corelease. Surprisingly, this population is highly diverse (27 types), expressing distinct dual transporters suggesting corelease of various combinations of neurotransmitters. Using glutamatergic vesicles as an example, we demonstrate that transporter colocalization not only determine the transmitter type but also influences the SV content and synaptic quantal size. Thus, presence of diverse transporters on the same vesicle is bona-fide and, depending on the VT types, this may act as one mechanism to regulate neurotransmitter type, content and release in space and time.

neuroscience↗

bioRxiv2020

Cooperativity of membrane-protein and protein-protein interactions control membrane remodeling by epsin 1 and regulate clathrin-mediated endocytosis

Membrane remodeling is a critical process for many membrane trafficking events, including clathrin-mediated endocytosis. Several molecular mechanisms for protein induced membrane curvature have been described in some detail. Contrary, the effect that the physico-chemical properties of the membrane has on these processes is far less well understood. Here, we show that the membrane binding and curvature-inducing ENTH domain of epsin1 is regulated by phosphatidylserine (PS). ENTH binds to membranes in a PI(4,5)P2-dependent manner but only induces curvature in the presence of PS. On PS-containing membranes, the ENTH domain forms rigid homo-oligomers and assembles into clusters. Membrane binding and membrane remodeling can be separated by structure-to-function mutants. Such oligomerization mutants bind to membranes but do not show membrane remodeling activity. In vivo they are not able to rescue defects in epidermal growth factor receptor (EGFR) endocytosis in epsin knock-down cells. Together, these data show that the membrane lipid composition is important for the regulation of protein-dependent membrane deformation during clathrin-mediated endocytosis.

biophysics↗

bioRxiv2019

Impaired lysosomal acidification triggers iron deficiency, necrotic cell death and inflammation in vivo

Lysosomal acidification is a key feature of healthy cells. Inability to maintain lysosomal acidic pH is associated with aging and neurodegenerative diseases. However, the mechanisms elicited by impaired lysosomal acidification remain unknown. We show here that inhibition of lysosomal acidification triggers cellular iron deficiency, which results in impaired mitochondrial function and necrotic cell death. These effects are recovered by supplying iron via a lysosome-independent pathway. Notably, iron deficiency is sufficient to trigger inflammatory signaling in cultured primary neurons. Using a mouse model of impaired lysosomal acidification, we observed a robust iron deficiency response in the brain, verified by in vivo magnetic resonance imaging. Furthermore, the brains of these mice present a pervasive inflammatory signature associated with instability of mitochondrial DNA (mtDNA), both corrected by supplementation of the mice diet with iron. Our results highlight a novel mechanism linking lysosomal dysfunction, mitochondrial malfunction and inflammation in vivo.

cell biology↗

bioRxiv2019

Endophilin-A controls recruitment, priming and fusion of neurosecretory vesicles

Endophilins-A are conserved endocytic adaptors with membrane curvature-sensing and - inducing properties. We show here that, independently of their role in endocytosis, endophilin-A1 and endophilin-A2 regulate exocytosis of neurosecretory vesicles. The number of neurosecretory vesicles was not altered in chromaffin cells without endophilin, yet fast capacitance and amperometry measurements revealed reduced exocytosis, smaller vesicle pools and changed fusion kinetics. Both endophilin-A1 (brain-enriched) and A2 (ubiquitous) rescued exocytic defects, but endophilin-A2 was more efficient. Distribution of neurosecretory vesicles was altered in the plasma membrane proximity, but levels and distributions of main exocytic and endocytic factors were unchanged, and slow compensatory endocytosis was not robustly affected. Endophilins role in exocytosis is mediated through its SH3-domain and, at least in part, interaction with intersectin, a coordinator of exocytic and endocytic traffic. Altogether, we report that endophilins-A, key endocytic proteins linked to neurodegeneration, directly regulate exocytosis by controlling vesicle recruitment, priming and fusion. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/540864v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@b5cabeorg.highwire.dtl.DTLVardef@1119afborg.highwire.dtl.DTLVardef@d5da6aorg.highwire.dtl.DTLVardef@1f67e32_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIRecruitment, priming and fusion of secretory vesicles is controlled by endophilin C_LIO_LILack of endophilins alters the distribution of secretory vesicles near the PM C_LIO_LIEndophilins role in exocytosis is mediated through its SH3-domain C_LIO_LIEndophilin regulates intersectin localization by keeping it away from the PM C_LI

neuroscience↗

bioRxiv2018

Mitochondrial biogenesis is transcriptionally repressed in lysosomal lipid storage diseases

Perturbations in mitochondrial function and homeostasis are pervasive in lysosomal storage diseases, but the underlying mechanisms remain unknown. Here, we report a transcriptional program that represses mitochondrial biogenesis and function in lysosomal storage diseases Niemann-Pick type C (NPC) and acid sphingomyelinase deficiency (ASM), in patient cells and mouse tissues. This mechanism is mediated by the transcription factors KLF2 and ETV1, which are both induced in NPC and ASM patient cells. Mitochondrial biogenesis and function defects in these cells are rescued by the silencing of KLF2 or ETV1. Increased ETV1 expression is regulated by KLF2, while the increase of KLF2 protein levels in NPC and ASM stems from impaired signaling downstream sphingosine-1-phosphate receptor 1 (S1PR1), which normally represses KLF2. In patient cells, S1PR1 is undetectable at the plasma membrane and thus unable to repress KLF2. This manuscript provides a mechanistic pathway for the prevalent mitochondrial defects in lysosomal storage diseases.

cell biology↗