Biology subjects

Savas, J. N.

Publications and source records attributed to Savas, J. N..

At least 19 recordsLinked to original sources

bioRxiv2026

Single-cell transcriptional landscape of muscle-derived stem/progenitor cells reveals hallmarks of aging and rejuvenation

Muscle-derived stem/progenitor cells (MDSPCs) are an adult stem cell population with demonstrated regenerative and rejuvenative potential distinct from other muscle progenitor cells. However, their molecular identity and developmental status remain poorly defined. Using single-cell transcriptomics and proteomics, we comprehensively profiled murine MDSPCs across age groups. We show that MDSPCs exist along a transcriptional continuum of maturation--ranging from metabolically active, proliferative early-stage cells to late-stage, lineage-committed myogenic populations. While lacking canonical pluripotency markers, early-stage MDSPCs express gene programs associated with embryonic progenitor identity, suggesting a non-canonical, multipotent-like state. These features distinguish them from both satellite cells and committed myoblasts. Aging reshapes this continuum by reducing stemness-associated signatures while enhancing differentiation programs and oxidative stress. Our identification of distinct MDSPC states provide critical insights into mechanisms that underly tissue regeneration and aging. These findings offer a blueprint for development of future regenerative therapies to combat age-related functional decline.

cell biology↗

bioRxiv2026

Extracellular neuroligin-ICAM5 coupling drives dendritic growth via actin remodeling

Neuroligins (NLGNs) organize neuronal connectivity by engaging a diverse set of interaction partners, yet how extracellular recognition couples to intracellular growth programs remains unclear. Using affinity proteomics, we identify intercellular adhesion molecule-5 (ICAM5), a cell-surface protein localized to dendritic filopodia, as a novel neuroligin interactor. Surface plasmon resonance and cell-based assays demonstrate direct binding between the ICAM5 and NLGN3 extracellular domains and reveal that ICAM5 engages all neuroligin isoforms. ICAM5 is required for NLGN-induced dendritic outgrowth, but the NLGN3-ICAM5 complex does not contribute to synaptogenesis. Mechanistically, ICAM5 sustains PAK-Cofilin signaling and F-actin organization in growth cones, positioning it as a downstream effector that links neuroligin engagement to actin remodeling. Together, these findings define a neuroligin-ICAM5 axis that couples extracellular recognition to intracellular actin remodeling to control neuronal structural development.

neuroscience↗

bioRxiv2025

Global Analysis of Aggregation Determinants in Small Protein Domains

Protein aggregation is an obstacle for engineering effective recombinant proteins for biotechnology and therapeutic applications. Predicting protein aggregation propensity remains challenging due to the complex interplay of sequence, structure, environmental factors, and external stress conditions, particularly for globular proteins. To understand the determinants of aggregation and improve its prediction, we quantified insoluble aggregation following high temperature and acidic stress in custom libraries of small protein domains (40-72 amino acids) using a high-throughput, in vitro, mass spectrometry-based method. In total, we quantified aggregation for 18,987 small protein domains, revealing diverse stress-dependent aggregation phenotypes that were consistent in different library contexts. We also found that aggregation measurements on individually purified proteins strongly correlated with high-throughput mixed-pool data (Pearsons r = 0.65-0.79), supporting the use of multiplexed approaches to study aggregation. Using machine learning, we identified sequence and structural features that correlate with aggregation and fine-tuned the protein language model SaProt, which explained 43-55% of the observed variation in a held-out test set of unrelated protein domains. Our model shows promising utility for engineering aggregation-resistant proteins, and our dataset serves as an important resource for developing improved models of protein aggregation.

biophysics↗

bioRxiv2025

Response dynamics of discrete subiculum->retrosplenial cortex projections underlying trace fear conditioning

Associating events separated in time depends on the CA1, subiculum (SUB), and retrosplenial cortex (RSP). The degree to which their connectivity and underlying circuit mechanisms contribute to the association of such temporally discontiguous events is not known. Here we showed, using trace fear conditioning (TFC), wherein mice learn to associate tone and shock separated by a temporal trace, that molecularly distinct excitatory VGluT1+ and VGluT2+ SUB[->]RSP projections subserve the associative and temporal components of TFC. During trace memory formation, VGluT2+ SUB[->]RSP projections showed increased and decreased bulk calcium activity at tone and trace onset, respectively, an activity pattern that was reestablished during memory recall. Such pattern was not observed in CA subfields, suggesting that associative and temporal components of TFC are integrated at the SUB or SUB[->]RSP synapses before being presented to the RSP. Our findings establish a circuit mechanism for representing complex temporal information in episodic memory.

neuroscience↗

bioRxiv2024

The ALS-associated co-chaperone DNAJC7 mediates neuroprotection against proteotoxic stress by modulating HSF1 activity

The degeneration of neurons in patients with amyotrophic lateral sclerosis (ALS) is commonly associated with accumulation of misfolded, insoluble proteins. Heat shock proteins (HSPs) are central regulators of protein homeostasis as they fold newly synthesized proteins and refold damaged proteins. Heterozygous loss-of- function mutations in the DNAJC7 gene that encodes an HSP co-chaperone were recently identified as a cause for rare forms of ALS, yet the mechanisms underlying pathogenesis remain unclear. Using mass spectrometry, we found that the DNAJC7 interactome in human motor neurons (MNs) is enriched for RNA binding proteins (RBPs) and stress response chaperones. MNs generated from iPSCs with the ALS-associated mutation R156X in DNAJC7 exhibit increased insolubility of its client RBP HNRNPU and associated RNA metabolism alterations. Additionally, DNAJC7 haploinsufficiency renders MNs increasingly susceptible to proteotoxic stress and cell death as a result of an ablated HSF1 stress response pathway. Critically, expression of HSF1 in mutant DNAJC7 MNs is sufficient to rescue their sensitivity to proteotoxic stress, while postmortem ALS patient cortical neurons exhibit a reduction in the expression of HSF1 pathway genes. Taken together, our work identifies DNAJC7 as a crucial mediator of HNRNPU function and stress response pathways in human MNs and highlights HSF1 as a therapeutic target in ALS.

neuroscience↗

bioRxiv2024

Levetiracetam prevents Aβ42 production through SV2a-dependent modulation of App processing in Alzheimer's disease models

In Alzheimers disease (AD), amyloid-beta (A{beta}) peptides are produced by proteolytic cleavage of the amyloid precursor protein (APP), which can occur during synaptic vesicle (SV) cycling at presynapses. Precisely how amyloidogenic APP processing may impair presynaptic proteostasis and how to therapeutically target this process remains poorly understood. Using App knock-in mouse models of early A{beta} pathology, we found proteins with hampered degradation accumulate at presynaptic sites. At this mild pathological stage, amyloidogenic processing leads to accumulation of A{beta}42 inside SVs. To explore if targeting SVs modulates A{beta} accumulation, we investigated levetiracetam (Lev), a SV-binding small molecule drug that has shown promise in mitigating AD-related pathologies despite its mechanism of action being unclear. We discovered Lev reduces A{beta}42 levels by decreasing amyloidogenic processing of APP in a SV2a-dependent manner. Lev corrects SV protein levels and cycling, which results in increased surface localization of APP, where it favors processing via the non-amyloidogenic pathway. Using metabolic stable isotopes and mass spectrometry we confirmed that Lev prevents the production of A{beta}42 in vivo. In transgenic mice with aggressive pathology, electrophysiological and immunofluorescent microscopy analyses revealed that Lev treatment reduces SV cycling and minimizes synapse loss. Finally, we found that human Down syndrome brains with early A{beta} pathology, have elevated levels of presynaptic proteins, confirming a comparable presynaptic deficit in human brains. Taken together, we report a mechanism that highlights the therapeutic potential of Lev to modify the early stages of AD and represent a promising strategy to prevent A{beta}42 pathology before irreversible damage occurs. One Sentence SummaryWe discovered that the SV-binding drug levetiracetam prevents A{beta}42 production by modulating SV cycling which alters APP localization and thus proteolytic processing, highlighting its therapeutic potential for AD.

neuroscience↗

bioRxiv2024

Isotope Encoded Chemical Imaging Identifies Amyloid Plaque Age Dependent Structural Maturation, Synaptic Loss, and Increased Toxicity

It is of critical importance to our understanding of Alzheimers disease (AD) pathology to determine how key pathological factors are interconnected and implicated in nerve cell death, clinical symptoms, and disease progression. The formation of extracellular beta-amyloid (A{beta}) plaques is the major pathological hallmark of AD and A{beta} has been suggested to be a critical inducer of AD, driving disease pathogenesis. Exactly how A{beta} plaque formation begins and how ongoing plaque deposition proceeds and initiates subsequent neurotoxic mechanisms is not well understood. The primary aim of our research is to elucidate the biochemical processes underlying early A{beta} plaque formation in brain tissue. We recently introduced a chemical imaging paradigm based on mass spectrometry imaging (MSI) and metabolic isotope labelling to follow stable isotope labelling kinetics (iSILK) in vivo to track the in vivo build-up and deposition of A{beta}. Herein, knock-in A{beta} mouse models (AppNL-F) that develop A{beta} pathology gradually are metabolically labeled with stable isotopes. This chemical imaging approach timestamps amyloid plaques during the period of initial deposition allowing the fate of aggregating A{beta} species from before and during the earliest events of plaque pathology through plaque maturation to be tracked. To identify the molecular and cellular response to plaque maturation, we integrated iSILK with single plaque transcriptomics performed on adjacent tissue sections. This enabled changes in gene expression to be tracked as a function of plaque age (as encoded in the A{beta} peptide isotopologue pattern) distinct from changes due to the chronological age or pathological severity. This approach identified that plaque age correlates negatively with gene expression patterns associated with synaptic function as early as in 10-month-old animals but persists into 18 months. Finally, we integrated hyperspectral confocal microscopy into our multiomic approach to image amyloid structural isomers, revealing a positive correlation between plaque age and amyloid structural maturity. This analysis identified three categories of plaques, each with a distinct impact on the surrounding microenvironment. Here, we identified that older, more compact plaques were associated with the most significant synapse loss and toxicity. These data show how isotope-encoded MS imaging can be used to delineate A{beta} toxicity dynamics in vivo. Moreover, we show for the first time a functional integration of dynamic MSI, structural plaque imaging and whole genome-wide spatial transcriptomics at the single plaque level. This multiomic approach offers an unprecedented combination of temporal and spatial resolution enabling a description of the earliest events of precipitating amyloid pathology and how A{beta} modulates synaptotoxic mechanisms.

neuroscience↗

bioRxiv2024

Keratinocyte-derived exosomes in painful diabetic neuropathy

Painful diabetic neuropathy (PDN) is a challenging complication of diabetes with patients experiencing a painful and burning sensation in their extremities. Existing treatments provide limited relief without addressing the underlying mechanisms of the disease. PDN involves the gradual degeneration of nerve fibers in the skin. Keratinocytes, the most abundant epidermal cell type, are closely positioned to cutaneous nerve terminals, suggesting the possibility of bi-directional communication. Exosomes are small extracellular vesicles released from many cell types that mediate cell to cell communication. The role of keratinocyte-derived exosomes (KDEs) in influencing signaling between the skin and cutaneous nerve terminals and their contribution to the genesis of PDN has not been explored. In this study, we characterized KDEs in a well-established high-fat diet (HFD) mouse model of PDN using primary adult mouse keratinocyte cultures. We obtained highly enriched KDEs through size exclusion chromatography and then analyzed their molecular cargo using proteomic analysis and small RNA sequencing. We found significant differences in the protein and microRNA content of HFD KDEs compared to KDEs obtained from control mice on a regular diet (RD), including pathways involved in axon guidance and synaptic transmission. Additionally, using an in vivo conditional extracellular vesicle (EV) reporter mouse model, we demonstrated that epidermal-originating GFP-tagged KDEs are retrogradely trafficked into the DRG neuron cell body. Overall, our study presents a potential novel mode of communication between keratinocytes and DRG neurons in the skin, revealing a possible role for KDEs in contributing to the axonal degeneration that underlies neuropathic pain in PDN. Moreover, this study presents potential therapeutic targets in the skin for developing more effective, disease-modifying, and better-tolerated topical interventions for patients suffering from PDN, one of the most common and untreatable peripheral neuropathies.

neuroscience↗

bioRxiv2023

Exceptional longevity of mammalian ovarian and oocyte macromolecules throughout the reproductive lifespan

The mechanisms contributing to age-related deterioration of the female reproductive system are complex, however aberrant protein homeostasis is a major contributor. We elucidated exceptionally stable proteins, structures, and macromolecules that persist in mammalian ovaries and gametes across the reproductive lifespan. Ovaries exhibit localized structural and cell-type specific enrichment of stable macromolecules in both the follicular and extrafollicular environments. Moreover, ovaries and oocytes both harbor a panel of exceptionally long-lived proteins, including cytoskeletal, mitochondrial, and oocyte-derived proteins. The exceptional persistence of these long-lived molecules suggest a critical role in lifelong maintenance and age-dependent deterioration of reproductive tissues. One sentence summaryExceptionally long-lived macromolecules in mammalian ovaries and oocytes as pillars for lifelong reproductive health span.

physiology↗

bioRxiv2023

A pathogenic missense mutation in kainate receptors elevates dendritic excitability and synaptic integration through dysregulation of SK channels

Numerous rare de novo variants that cause neurodevelopmental disorders (NDDs) occur within genes encoding synaptic proteins, including ionotropic glutamate receptors (iGluRs). However, in many cases it remains unclear how damaging missense variants affect brain function. Here we determined the physiological consequences of an NDD causing missense mutation in the GRIK2 kainate receptor (KAR) gene, that results in a single amino acid change p.Ala657Thr in the GluK2 receptor subunit. We engineered the equivalent mutation in the mouse Grik2 gene, yielding a GluK2(A657T) mouse, to better understand the human disorder and determine how hippocampal neuronal function is disrupted. Synaptic KAR currents in hippocampal CA3 pyramidal neurons from heterozygous A657T mice exhibited slow decay kinetics, consistent with incorporation of the mutant subunit into functional receptors. Unexpectedly, CA3 neurons demonstrated elevated action potential spiking due to down-regulation of the small conductance Ca2+ activated K+ channel (SK), which mediates the post-spike afterhyperpolarization (AHP). The reduction in SK activity resulted in increased CA3 dendritic excitability, increased EPSP-spike coupling and lowered the threshold for the induction of LTP of the associational commissural (AC) synapses in the distal dendrites of CA3 neurons. Pharmacological inhibition of SK channels in wild-type (WT) mice increased dendritic excitability and EPSP-spike coupling, mimicking the phenotype in A657T mice and suggesting a causative role for attenuated SK activity in aberrant excitability observed in the mutant mice. These findings demonstrate that a disease-associated missense mutation in GRIK2 leads to altered signaling through neuronal KARs, pleiotropic effects on neuronal and dendritic excitability, and implicate these processes in neuropathology in patients with genetic NDDs.

neuroscience↗

bioRxiv2023

Do perineuronal nets stabilize the engram of a synaptic circuit?

Perineuronal nets (PNN), a specialized form of ECM (?), surround numerous neurons in the CNS and allow synaptic connectivity through holes in its structure. We hypothesis that PNNs serve as gatekeepers that guard and protect synaptic territory, and thus may stabilize an engram circuit. We present high-resolution, and 3D EM images of PNN- engulfed neurons showing that synapses occupy the PNN holes, and that invasion of other cellular components are rare. PNN constituents are long-lived and can be eroded faster in an enriched environment, while synaptic proteins have high turnover rate. Preventing PNN erosion by using pharmacological inhibition of PNN-modifying proteases or MMP9 knockout mice allowed normal fear memory acquisition but diminished remote-memory stabilization, supporting the above hypothesis. SignificanceIn this multidisciplinary work, we challenge the hypothesis that the pattern of holes in the perineuronal nets (PNN) hold the code for very-long-term memories. The scope of this work might lead us closer to the understanding of how we can vividly remember events from childhood to death bed. We postulate that the PNN holes hold the code for the engram. To test this hypothesis, we used three independent experimental strategies; high-resolution 3D electron microscopy, Stable Isotop Labeling in Mammals (SILAM) for proteins longevity, and pharmacologically and genetically interruption of memory consolidation in fear conditioning experiments. All of these experimental results did not dispute the PNN hypothesis.

neuroscience↗

bioRxiv2023

Neuron-specific protein expression at striatal excitatory synapses

Combinatorial expression of postsynaptic proteins underlies synapse diversity within and between neuron types 1-5. Thus, characterization of neuron-type-specific postsynaptic proteomes is key to obtaining a deeper understanding of discrete synaptic properties and how selective dysfunction manifests in synaptopathies 6-9. To overcome the limitations associated with bulk measures of synaptic protein abundance 6,10, we developed a biotin proximity protein tagging probe to characterize neuron-type-specific postsynaptic proteomes in vivo. We found Shank3 protein isoforms are differentially expressed by direct and indirect pathway spiny projection neurons (dSPNs and iSPNs). Studies in mice lacking Shank3 gene exons 13-16 revealed a robust postsynaptic proteome alteration in iSPNs. We report unexpected cell-type specific synaptic protein isoform expression which could play a key causal role in specifying synapse diversity and selective synapse dysfunction in synaptopathies.

neuroscience↗

bioRxiv2023

Amyloid fibril proteomics of AD brains reveals modifiers of aggregation and toxicity

BackgroundThe accumulation of amyloid beta (A{beta}) peptides in fibrils is prerequisite for Alzheimers disease (AD). Our understanding of the proteins that promote A{beta} fibril formation and mediate neurotoxicity has been limited due to technical challenges in isolating pure amyloid fibrils from brain extracts. MethodsTo investigate how amyloid fibrils form and cause neurotoxicity in AD brain, we developed a robust biochemical strategy. We benchmarked the success of our purifications using electron microscopy, amyloid dyes, and a large panel of A{beta} immunoassays. Tandem mass-spectrometry based proteomic analysis workflows provided quantitative measures of the amyloid fibril proteome. These methods allowed us to compare amyloid fibril composition from human AD brains, three amyloid mouse models, transgenic A{beta}42 flies, and A{beta}42 seeded cultured neurons. ResultsAmyloid fibrils are primarily composed by A{beta}42 and unexpectedly harbor A{beta}38 but generally lack A{beta}40 peptides. Multidimensional quantitative proteomics allowed us to redefine the fibril proteome by identifying 17 new amyloid-associated proteins. Notably, we confirmed 126 previously reported plaque-associated proteins. We validated a panel of these proteins as bona fide amyloid-interacting proteins using antibodies and orthogonal proteomic analysis. One metal-binding chaperone metallothionein-3 is tightly associated with amyloid fibrils and modulates fibril formation in vitro. Lastly, we used a transgenic A{beta}42 fly model to test if knock down or over-expression of fibril-interacting gene homologues modifies neurotoxicity. Eight RNAi lines suppressed and 11 enhanced A{beta}42 toxicity. ConclusionsThese discoveries and subsequent confirmation indicate that fibril-associated proteins play a key role in amyloid formation and AD pathology.

neuroscience↗

bioRxiv2021

Sex-Dependent Effects of Chronic Microdrive Implantation on Acquisition of Trace Eyeblink Conditioning

Neuroscience techniques, including in vivo recording, have allowed for a great expansion in knowledge; however, this technology may also affect the very phenomena researchers set out to investigate. Including both female and male mice in our associative learning experiments shed light on sex differences on the impact of chronic implantation of tetrodes on learning. While previous research showed intact female mice acquired trace eyeblink conditioning faster than male and ovariectomized females, implantation of chronic microdrive arrays showed sexually dimorphic effects on learning. Microdrive implanted male mice acquired the associative learning paradigm faster than both intact and ovariectomized females. These effects were not due to the weight of the drive alone, as there were no significant sex-differences in learning of animals that received "dummy drive" implants without tetrodes lowered into the brain. Tandem mass tag mass spectrometry and western blot analysis suggest that significant alterations in the MAPK pathway, acute inflammation, and brain derived neurotrophic factor may underlie these observed sex- and surgery-dependent effects on learning.

neuroscience↗

bioRxiv2020

A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

Membrane fusion, an essential process in all eukaryotes, is driven by SNARE proteins. Ykt6 is an essential SNARE that plays critical roles throughout the secretory, endocytic, and autophagy pathways. Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition is unknown. Through genetic, pharmacologic, and structural modeling approaches in mammalian cells, we found that phosphorylation regulates Ykt6 conversion from a closed to an open state. The phosphorylation site we identified is highly conserved in evolution and is regulated by the Ca2+-dependent phosphatase, calcineurin. We found that phosphorylation is a key determinant for intracellular localization of Ykt6 and its function in macroautophagy. Our studies reveal a novel mechanism by which Ykt6 conformation and activity is regulated by Ca2+ signaling with implications in Parkinsons Disease in which Ykt6 has been shown to play a role.

cell biology↗

bioRxiv2019

Mapping the cell-surface proteome underlying hippocampal mossy fiber synapse identity

Synaptic diversity is a key feature of neural circuits. The structural and functional diversity of closely spaced inputs converging on the same neuron suggests that cell-surface interactions are essential in organizing input properties. Here, we analyzed the cell-surface protein (CSP) composition of hippocampal mossy fiber (MF) inputs on CA3 pyramidal neurons to identify regulators of MF-CA3 synapse properties. We uncover a rich cell-surface repertoire that includes adhesion proteins, guidance cue receptors, extracellular matrix (ECM) proteins, and uncharacterized CSPs. Interactome screening reveals multiple ligand-receptor modules and identifies ECM protein Tenascin-R (TenR) as a ligand of the uncharacterized neuronal receptor IgSF8. Presynaptic Igsf8 deletion impairs MF-CA3 synaptic architecture and robustly decreases the density of bouton filopodia that provide feedforward inhibition of CA3 neurons. Consequently, loss of IgSF8 increases CA3 neuron excitability. Our findings identify IgSF8 as a regulator of CA3 microcircuit development and suggest that combinations of CSP modules define input identity.

neuroscience↗

bioRxiv2019

Nucleocytoplasmic Proteomic Analysis Uncovers ETF1 and Nonsense Mediated Decay as Modifiers of ALS C9orf72 Toxicity

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide repeat expansion in C9orf72 (C9-HRE). While RNA and dipeptide repeats produced by the C9-HRE disrupt nucleocytoplasmic transport, the proteins that become redistributed remain unknown. Here, we utilized subcellular fractionation coupled with tandem mass spectrometry and identified 126 proteins, enriched for protein translation and RNA metabolism pathways, which collectively drive a shift towards a more cytosolic proteome in C9-HRE cells. Amongst these was eRF1, which regulates translation termination and nonsense-mediated decay (NMD). eRF1 accumulates within elaborate nuclear envelope invaginations in patient iPSC-neurons and postmortem tissue and mediates a protective shift from protein translation to NMD-dependent mRNA degradation. Overexpression of eRF1 and the NMD-driver UPF1 ameliorate C9-HRE toxicity in vivo. Our findings provide a resource for proteome-wide nucleocytoplasmic alterations across neurodegeneration-associated repeat expansion mutations and highlight eRF1 and NMD as therapeutic targets in C9orf72-associated ALS/FTD.

neuroscience↗

bioRxiv2019

CNTNAP2 ectodomain, detected in neuronal and CSF sheddomes, modulates Ca2+ dynamics and network synchrony

SUMMARYWhile many neuronal membrane-anchored proteins undergo proteolytic cleavage, little is known about the biological significance of neuronal ectodomain shedding. Using mass spectrometry (MS)-based proteomics, we showed that the neuronal sheddome mirrors human cerebrospinal fluid (hCSF). Among shed synaptic proteins in hCSF was the ectodomain of CNTNAP2 (CNTNAP2-ecto), a risk factor for neurodevelopmental disorders (NDD). Using structured-illumination microscopy (SIM), we mapped the spatial organization of neuronal CNTNAP2-ecto shedding. Using affinity chromatography followed by MS, we identified the ATP2B/PMCA Ca2+ extrusion pumps as novel CNTNAP2-ecto binding partners. CNTNAP2-ecto coimmunoprecipitates with PMCA2, a known autism risk factor, and enhances its activity, thereby modulating neuronal Ca2+ levels. Finally, we showed that CNTNAP2-ecto regulates neuronal network synchrony in primary cultures and brain slices. These data provide new insights into the biology of synaptic ectodomain shedding and reveal a novel mechanism of regulation of Ca2+ homeostasis and neuronal network synchrony.

neuroscience↗