Biology subjects

Stamenkovic, V.

Publications and source records attributed to Stamenkovic, V..

5 recordsLinked to original sources

bioRxiv2025

Genetic rescue of disrupted synaptic protein interaction network dynamics following SYNGAP1 reactivation

Synaptic protein interaction networks (PINs) dynamically translate neural activity into biochemical signals that regulate synaptic structure and plasticity. Disruption of these coordinated networks is a common feature of autism spectrum disorder (ASD) risk genes, yet it remains unclear whether the molecular organization of a perturbed network can be restored after development. Here, we examined how post-developmental re-expression of the synaptic Ras GTPase-activating protein SynGAP1 affects network structure and signaling dynamics in a conditional SynGAP1 haploinsufficient mouse. Quantitative multiplex co-immunoprecipitation (QMI) across development revealed that SynGAP haploinsufficiency selectively reduced SynGAP-containing complexes without broadly disrupting NMDA-dependent network responses. Tamoxifen-inducible re-expression of SynGAP at postnatal day 21 fully restored both steady-state and activity-dependent interactions within the SynGAP module in hippocampus, and additionally normalized secondary alterations in Shank-Homer scaffolding complexes in somatosensory cortex. These data demonstrate that biochemical restoration of a disrupted synaptic network is achievable, even after early developmental windows have closed. Our findings suggest that while critical periods may constrain functional recovery, molecular network normalization remains possible through genetic reactivation of haploinsufficient synaptic regulators.

neuroscience↗

bioRxiv2025

Targeting dendritic cells with RNA-loaded nanoparticles grafted with short peptides

Nanoparticles encapsulating therapeutic RNA have emerged as a transformative strategy in precision medicine, capable of mobilizing the immune system to induce specific responses, ranging from immune tolerance to fighting tumors. However, most current preclinical and clinical efforts rely on non-targeted delivery systems, limiting their safety, therapeutic efficacy, and selectivity. To enhance the therapeutic index of RNA-based therapeutic systems for immunomodulatory purposes, we report on the design of a novel Clec9A-targeted polymeric nanoparticle, aimed at selectively engaging dendritic cells responsible for antigen presentation. We began by evaluating in silico the binding potential of the previously reported 12-amino-acid WH peptide, known for its high affinity to mouse Clec9A, the human ortholog. Using computational tools, we designed and screened truncated variants of the peptide and identified promising candidates with retained or enhanced binding capacity to human Clec9A. These optimized short peptides were synthesized and covalently conjugated to our proprietary poly(beta amino ester) (pBAE) polymers. We evaluated the impact of conjugation site, comparing terminal versus lateral chain attachment on receptor targeting and confirmed in vitro that peptide orientation significantly influences binding efficiency. Additionally, we computationally generated and validated shorter mutant peptide variants with improved Clec9A affinity over the original sequences. Our findings demonstrate that rationally engineered short peptides, when site-specifically conjugated to pBAE polymers, can provide high-affinity, selective targeting of dendritic cells via Clec9A. This strategy lays the groundwork for the next generation of targeted RNA-based immunotherapeutics, offering improved selectivity, immune activation, and therapeutic potential. Graphical abstractSchematic representation of the workflow used in this work. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/686747v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@17f2d4eorg.highwire.dtl.DTLVardef@779825org.highwire.dtl.DTLVardef@1a5ded2org.highwire.dtl.DTLVardef@c7cb96_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

The Alzheimer's disease risk gene SORL1 is a regulator of excitatory neuronal function

BackgroundSynaptic dysfunction is an early feature of Alzheimers disease (AD) and a significant contributor to cognitive decline and neurodegeneration. Proper localization of proteins involved in pre-and post-synaptic composition is dependent on endosomal recycling and trafficking. Alterations in trafficking complexes, such as retromer, have been shown to impair neuronal synaptic function. The SORL1 gene has been strongly implicated in AD pathogenesis and its protein product, SORLA, is an endosomal receptor that works in conjunction with retromer to regulate endosomal recycling. MethodsWe utilized our established human induced pluripotent stem cell (hiPSC) derived excitatory cortical neuron model to examine SORL1s role in synaptic protein composition and neuronal function. We used Quantitative Multiplex co-Immunoprecipitation (QMI), a mesoscale proteomics assay to measure synaptic protein interactions, immunocytochemistry to assay synapses and AMPA receptor subunits, and multi-electrode arrays (MEAs) to measure neuronal function of SORL1 KO and isogenic control hiPSC derived neurons. ResultsWe show that loss of SORL1 expression significantly changes many synaptic protein-protein interactions and patterns of expression. We demonstrate that SORL1 deficient neurons are hyperactive and that the increased activity is driven by glutamatergic neurotransmission. Hyperexcitability has been seen in other models of AD with familial AD variants in amyloid precursor protein and presenilin genes, due to the increases in amyloid beta (A{beta}) peptides. In the case of SORL1 deficiency, the hyperexcitability we observe is primarily due to mis-trafficking of synaptic proteins, rather than an overall increase in A{beta}. Finally, we find that SORL1 deficient neurons have impaired synaptic plasticity. ConclusionsThese findings further support a growing body of literature implicating early endosomal recycling defects as drivers of AD pathogenesis. Furthermore, our work supports further emphasis on exploring the SORL1-retromer pathway for therapeutic development in AD.

neuroscience↗

bioRxiv2025

Dissociation of the mTOR protein interaction network following neuronal activation is altered by Shank3 mutation

The mechanistic target of Rapamycin (mTOR) kinase pathway plays critical roles in neuronal function and synaptic plasticity, and its dysfunction is implicated in numerous neurological and psychiatric disorders. Traditional linear models depict mTOR signaling as a sequential phosphorylation cascade, but accumulating evidence supports a model that includes signaling through dynamic protein-protein interaction networks. To examine how neuronal mTOR signaling discriminates between distinct stimuli, we quantified phosphorylation events and protein co-association networks in primary mouse cortical neurons. Unexpectedly, neuronal mTOR activation by IGF or glutamate triggered dissociation--rather than the anticipated assembly--of protein complexes involving mTOR complex1 (TORC1), mTOR complex 2 (TORC2), and translational machinery, distinguishing neurons from proliferative cells. Applying in vitro homeostatic scaling paradigms revealed distinct combinatorial encoding of synaptic scaling direction: both up- and down-scaling induced dissociation of translational complexes, but downscaling uniquely included dissociation of upstream pathway regulators. Cortical neurons from Shank3B knockout mice, modeling autism-associated Phelan-McDermid Syndrome, displayed baseline hyperactivation of the mTOR network, which reduced the dynamic range of network responses to homeostatic scaling and pharmacological inhibition. These findings reveal that neuronal mTOR signaling employs stimulus-specific combinations of dissociative protein interaction modules to encode opposing forms of synaptic plasticity.

biochemistry↗

bioRxiv2023

SRC family kinase inhibition rescues molecular and behavioral phenotypes, but not protein interaction network dynamics, in a mouse model of Fragile X syndrome

Glutamatergic synapses encode information from extracellular inputs using dynamic protein interaction networks (PINs) that undergo widespread reorganization following synaptic activity, allowing cells to distinguish between signaling inputs and generate coordinated cellular responses. Here, we investigated how Fragile X Messenger Ribonucleoprotein (FMRP) deficiency disrupts signal transduction through a glutamatergic synapse PIN. In cultured cortical neurons or acute cortical slices from P7, P17 and P60 FMR1-/y mice, the unstimulated protein interaction networks state resembled that of wildtype littermates stimulated with neurotransmitter agonists, demonstrating resting state pre-activation of signaling networks. We identified the Src family kinase (SFK) Fyn as a network hub, because many interactions involving Fyn were pre-activated. We tested whether targeting Fyn in FMR1-/y mice could modify disease phenotypes, and found that Saracatinib (AZD-0530), an SFK inhibitor, normalized elevated basal protein synthesis, novel object recognition memory and social behavior in FMR1-/y mice. However, SCB treatment did not normalize the PIN to a wild-type-like state in vitro or in vivo, but rather induced extensive changes to protein complexes containing Shank3, NMDARs and Fyn. We conclude that targeting abnormal nodes of a PIN can identify potential disease-modifying drugs, but behavioral rescue does not correlate with PIN normalization.

neuroscience↗