ACS Chemical Neuroscience

Serotonin 5-HT4 receptors (5-HT4Rs) have emerged as potential therapeutic targets in neuropsychiatric and neurodegenerative disorders by modulating circuits that shape mood, cognition, and motor function. Ligands for 5-HT4Rs can modify dopamine (DA) and acetylcholine (ACh) transmission but mechanisms and circuits have not been fully resolved. Some 5-HT4R agonists have been suggested to have effects that include inhibition of acetylcholinesterase (AChE), raising speculation that 5-HT4R ligands might modulate ACh and/or DA through this action. Here, we investigated the impact of RS67333, a partial 5-HT4R agonist, on DA and ACh release dynamics in the striatum detected ex vivo in mouse brain slices using fast-scan cyclic voltammetry and genetically encoded ACh sensor GRABACh3.0 respectively. We found that RS67333 significantly modulated electrically evoked DA release in dorsolateral striatum and nucleus accumbens core, effects that were abolished by a nicotinic receptor (nAChR) antagonist. In parallel, RS67333 altered evoked ACh signals by extending extracellular ACh lifetime, and correspondingly, RS67333 was found to inhibit striatal AChE enzymatic activity. By contrast, BIMU8, an alternative 5-HT4R ligand that did not inhibit striatal AChE, had no effect on evoked striatal ACh or DA release. These findings indicate that RS67333 modulates striatal ACh transmission, which shapes downstream regulation of DA release by nAChRs, not through 5-HT4Rs but through AChE inhibition. These findings emphasize the caution due in attributing functions to 5-HT4Rs, but also highlight an alternative pharmacological profile of some purported 5-HT4R ligands as AChE inhibitors of potential utility for treating ACh/DA disorders.

Decreases in brain pH are associated with numerous neurological and neuropsychiatric conditions yet the molecular mechanisms linking decreased brain pH with these disorders are incomplete. The ionotropic glutamate receptors (iGluRs) mediate the majority of excitatory neurotransmission in the brain and are inhibited by extracellular protons; however, the proton sensitivity of the delta-glutamate receptor subclass of iGluRs is unknown. Using whole-cell patch-clamp recordings of serotonin neurons in mouse brain slices and activating alpha 1-adrenergic receptors to induce delta 1 glutamate receptor (GluD1R) current, we demonstrated that GluD1R current is inhibited by physiological drops in extracellular pH. Unlike other iGluRs, protons inhibited GluD1R current via a voltage-independent decrease in unitary current. Moreover, mice lacking GluD1R showed impaired behavioral responses to inhalation of CO2. Taken together, this study continues to expand on the growing body of evidence positing GluD1R as functional ion channels and suggests that GluD1R facilitate pH sensing in vivo.

Tonic glutamate signaling by ambient levels of extracellular glutamate has been implicated in development, brain injury, pathologies, and physiological activity. However, it has been difficult to assay extracellular glutamate changes with spatial and temporal resolution. Here, we utilize the rarely used ratiometric excitations properties of the fluorescence glutamate sensor iGluSnFr to enable the characterization of ambient glutamate levels in acute brain slices. This ratiometric imaging enables a spatial, temporal and calibratable assay of ambient glutamate and demonstrates regional differences in ambient glutamate and sensitivity to glutamate transporters and system Xc inhibition.

The hippocampus is essential for memory consolidation, a process mediated by high-frequency oscillations known as ripples during non-rapid eye movement (NREM) sleep. Ramelteon, a selective MT1/MT2 receptor agonist, has been reported to possess cognitive-enhancing properties; however, its impact on the fine-scale dynamics of hippocampal ripples remains unclear. We performed chronic local field potential recordings from the dorsal hippocampus and prefrontal cortex in mice. Following the intraperitoneal administration of either vehicle or ramelteon, we evaluated sleep architecture and characterized ripple properties, including occurrence rate, amplitude, instantaneous frequency, and duration during NREM sleep. Ramelteon administration significantly increased NREM sleep occupancy. Notably, we found that ramelteon significantly enhanced both the occurrence rate and amplitude of hippocampal ripples compared to the control group. While a slight increase in intra-ripple frequency was observed, other structural features, such as ripple duration and asymmetry index, remained unaffected. Our findings demonstrate that ramelteon facilitates hippocampal ripple dynamics by increasing their occurrence and synchrony during NREM sleep. Given the critical role of ripples in memory consolidation, these neurophysiological changes may underlie the procognitive effects of ramelteon. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=55 SRC="FIGDIR/small/723673v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@c798c7org.highwire.dtl.DTLVardef@1ff616eorg.highwire.dtl.DTLVardef@1557dc8org.highwire.dtl.DTLVardef@1b4e89e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Amyloid {beta} peptides (A{beta}) trigger Alzheimers disease (AD) but how has remained elusive. A{beta} stimulates the {beta}2 adrenergic receptor ({beta}2AR), which forms a unique signaling complex with the L-type Ca2+ channel (LTCC) CaV1.2. LTCCs have been implicated in the etiology of dementia and AD. We show that A{beta} acutely potentiates CaV1.2 via the {beta}2AR, which triggers postsynaptic recruitment of Ca2+ permeable (CP) AMPARs in hippocampal cultures and impairs LTP in hippocampal slices within minutes. The long-term consequence is a loss of postsynaptic structure of glutamatergic synapses and neurotoxicity. Disrupting this signaling cascade with highly specific tools prevented all of these effects, unifying a number of currently divergent findings on A{beta} synaptotoxicity including dysregulation of AMPARs and synaptic plasticity. TEASERAmyloid {beta} peptide is the primary pathological agent in Alzheimers disease. It affects the nanoscale structure and function of glutamatergic synapses. The molecular mechanisms are largely unknown except for identification of several binding proteins including the {beta}2 adrenergic receptor. We show that this binding potently (EC50<100 nM) augments Ca2+ influx through the L-type Ca channel CaV1.2. This effect leads to improper recruitment of Ca2+-permeable glutamate receptors to postsynaptic sites (EC50<100 nM), synaptic dysfunction and ultimately neuronal death. This work identifies an essential mechanism in amyloid {beta} neurotoxicity and explains many of the observed postsynaptic alterations. HighlightsImmediate effects of A{beta}-induced stimulation of {beta}2AR on Cav1.2: O_LIA{beta} induces phosphorylation of Cav1.2 on S1928 by PKA C_LIO_LIA{beta} augments Cav1.2 activity via {beta}2AR-induced S1928 phosphorylation within seconds C_LI A{beta}-induced {beta}2AR - Cav1.2 signaling has the following synaptotoxic effects. O_LIA{beta} induces postsynaptic accumulation of Ca-permeable AMPARs via {beta}2AR - Cav1.2 signaling within 20 min C_LIO_LIA{beta} impairs long-term potentiation (LTP) via {beta}2AR - Cav1.2 signaling C_LIO_LIA{beta} impairs postsynaptic structure and neuronal viability over 24 h C_LIO_LIPotency of A{beta} in all the above effects is very high (100 nM A{beta} is saturating!) C_LIO_LIAll effects are prevented in S1928A KI mice and acute displaces {beta}2AR from Cav1.2 with tat-Pep1923 C_LI

Colony stimulating factor 1 receptor (CSF1R) is a tyrosine kinase receptor that is expressed exclusively in microglia within the CNS. Its endogenous ligands, colony stimulating factor-1 (CSF1) and interleukin-34 (IL-34), are released from neurons, positioning CSF1R as a key mediator receptor of neuron-glia communication. CSF1R is considered not only a potential drug target, but also a biomarker of neuroinflammation. From that perspective, selective radioligands for neuroimaging are of great interest for imaging neuroinflammation and determining drug occupancy. In this study, we have validated the binding characteristics of a CSF1R inhibitor, 4-((5-MethOxy-6-((5-methoxypyridin-2-yl)methoxy)pyridin-3-yl)methyl)-2-(1-methyl-1H-pyrazol-4-yl)pyrimidine (5-MOP) as a novel CSF1R radioligand, by performing in vitro saturation binding experiments in human and murine tissues. 5-MOP was found to be selective for CSF1R among a broad range of kinases. Autoradiography revealed that [3H]5-MOP binds with high affinity (KD = 9.8 nM) to a single saturable binding site in human meningioma tissues, and this binding was displaced with known CSF1R inhibitors, including CPPC, sCSF1inh and GW-2580. In contrast, CPPC, which has been extensively used as a CSF1R radioligand showed substantial cross-reactivity to other brain kinases, including Trk A/B/C, and [3H]CPPC could only be displaced with CPPC itself, not by other ligands, including 5-MOP. These results identify [3H]5-MOP as the most selective radioligand currently available, enabling accurate detection of drug occupancy and activated microglia. Significance of the studyThis study identifies and validates a novel selective radioligand that binds CSF1R with high selectivity and low nanomolar affinity. Because CSF1R is selectively expressed in activated microglia, this radioligand could be useful for detecting neuroinflammatory activity.

Elucidating the structure and dynamics of insulin and its analogs has been of broad interest, while presenting challenges due to the unique structural dynamics of insulin (composed itself of two multiply cross-linked peptides A and B) and its ability to assemble in a variety of oligomeric structures under physiological conditions. Here, we present two distinct X-ray crystallographic structures of the long-acting human insulin analog detemir (INSD) resolved in hexameric and dodecameric (or dihexameric) states at 2.85 [A] and 2.70 [A] resolution, respectively, using diffraction data collected under ambient temperature conditions. Characterization of the collective dynamics of these oligomers using the Gaussian Network Model (GNM) reveals several key features: (i) Oligomerization imparts high cooperativity in structural dynamics evidenced by dissection of the cross-correlations at various hierarchical levels; (ii) detemir monomers conformational flexibility is highly suppressed within oligomeric constructs, the effect being particularly strong in the dihexamer due to the asymmetric packing of the hexamers and the presence of myristoyl groups at B peptides termini whose interactions imparts further heterogeneities; and (iii) a number of key residues retain, however, their intrinsic dynamics, to be deployed upon release from the oligomers. We distinguish in particular residues serving as hinge sites that mediates the conformational dynamics of the asymmetric units (dimers) and monomers (I2A-V3A and Y19 A -C20A, and L11B-L15B and Y26B of the respective peptides A and B), or as anchors supporting structural stability (disulfide-bridge forming cysteines, plus selected residues such as L16A, G8B and R22B-F24B. Overall, this study provides a structural-dynamic framework for gaining new insights into the dynamics of long-acting analog INSD and helps identify actionable sites for modulating insulin (analogs) dynamics toward designing more effective therapeutics.

Positron emission tomography (PET) tracers targeting amyloid-{beta} (A{beta}) are central to the diagnosis and staging of Alzheimers disease (AD). However, growing evidence indicates that these tracers can engage off-target molecules, complicating signal interpretation. Sulfotransferases (SULTs) have been experimentally identified as binding partners of 11C-Pittsburgh Compound-B (PiB). However, whether the clinically used fluorinated PiB derivatives flutemetamol and flutafuranol interact with brain-expressed SULTs is yet unexplored. Here, we combined multi-omic transcriptomic profiling with molecular docking and molecular dynamics (MD) simulations to assess the structural interactions of SULT-tracer complexes. Analysis of the Genotype-Tissue Expression project and the Human Protein Atlas identified SULT1A1, SULT1A3, and SULT4A1 as the SULT isoforms predominantly expressed in the human brain. Docking and MD simulations showed that all three tracers form energetically comparable complexes within the catalytic pockets of these isoforms, yet their dynamic stability varied in an enzyme- and tracer-specific manner. PiB and flutemetamol were stably accommodated in SULT1A1, but PiB lost its initial pose in SULT4A1. Flutafuranol showed weaker binding in SULT1A1, yet formed stable complexes in SULT1A3 and SULT4A1. Notably, SULT1A1, SULT1A3, and SULT4A1 are all expressed in the cerebellum, the brain region used as a reference for A{beta} PET signal normalization. These findings provide a structural framework for off-target tracer interaction with brain SULTs and suggest that the intracellular enzymatic environment may contribute to variability in A{beta} PET signals beyond fibrillar A{beta} deposition.

CAPON (NOS1AP) is an adaptor protein involved in neuronal nitric oxide synthase (nNOS) signaling and has been implicated in Alzheimers disease (AD), excitotoxicity, and tau-associated neurodegeneration. Here, we report the identification of cyclic peptide ligands targeting CAPON using phage display screening of a disulfide-constrained peptide library. Phage enrichment, ELISA validation, microscale thermophoresis (MST), and biolayer interferometry (BLI) identified CAP1 as the lead peptide, exhibiting low micromolar binding affinity toward CAPON. Computational studies further supported stable CAPON-CAP1 interactions through complementary hydrophobic and electrostatic contacts. Functionally, CAP1 attenuated A{beta}42-induced neuronal toxicity, suppressed NMDA-driven nitric oxide production, and reduced pathological tau phosphorylation in neuronal models under AD-relevant stress conditions. In addition, CAP1 demonstrated favorable preliminary pharmacokinetic properties, including good aqueous solubility, plasma stability, and measurable membrane permeability. Collectively, these findings establish the first cyclic peptide ligands targeting CAPON and identify CAP1 as a promising scaffold for modulation of CAPON-dependent neurodegenerative signaling.

Activity of the mesolimbic dopamine system has long been implicated in encoding primary rewards and contributing to the addictive properties of drugs of abuse. Dopamine neurons in the ventral tegmental area (VTADA) of the midbrain typically show patterns of spontaneous burst activity that align with the onset of salient events or rewarding stimuli, resulting in phasic dopamine release in the nucleus accumbens (NAc). Fiber photometry is increasingly being used as an accessible technique to quantify neural activity with high temporal resolution at sensors offering signal specificity in stable recordings over extended periods of time. It has been well established by multiple techniques that opioids increase mesolimbic dopamine activity, likely through disinhibition of VTADA neurons. Here we used fiber photometry to compare sub-second transient events from VTADA neurons with GCaMP6f and dopamine release in the lateral shell of the NAc with dLight1.3b and GRABDA2h in response to morphine treatment. In weekly sessions, one dose of morphine was administered in escalating order (2.5, 5,7.5, and 10 mg/kg, intraperitoneal). Consistent with prior literature, both GCaMP6f in VTADA neurons and dLight1.3b in NAc showed patterns of increased signal following morphine treatment. In contrast, morphine suppressed transient activity at GRABDA2h sensors. Further analyses of whole signal streams from each sensor showed a generalized increase, but reduction in variability of the GRABDA2h signal, consistent with the interpretation of sensor saturation. Such results emphasize the importance of the inclusion of appropriate controls to contextualize the interpretation of biosensor responses, particularly in response to pharmacological treatment. HIGHLIGHTSO_LIMorphine elicited increased signaling in VTADA GCaMP6f and NAc dLight1.3b, consistent with prior literature C_LIO_LIMorphine suppressed NAc GRABDA2h signaling of transient events, suggesting saturation of GRABDA2h sensor C_LIO_LISensor validation with pharmacological challenges is critical for interpretation of data C_LI

Tau aggregation at synapses is a key process driving Alzheimers disease but the mechanism(s) that cause this have not been established. We used a model system of forward-programming induced glutamatergic neurons (iNeurons) with three independent cell lines treated with TNF[a]. Using aggregate-specific SIMOA, STED microscopy, and SynPull to detect nanoscopic tau aggregates in bulk samples and at individual synapses, we found that TNF[a]-driven tau aggregation occurs preferentially at the pre-synapse, forming predominantly non-fibrillar aggregates that are larger than ones in the extra- and post-synaptic regions. Using mathematical models of aggregate formation, we fitted the frequency of AT8-positive tau aggregates in synaptosomes, which showed that aggregate replication is the dominant process and is much faster than de-novo aggregate formation, leading to rapid local amplification once one aggregate is formed. Our results provide direct evidence for tau aggregate replication at the pre-synapse, linking inflammation induced tau aggregation with synaptic pathology. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/728997v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@169dacorg.highwire.dtl.DTLVardef@c9894corg.highwire.dtl.DTLVardef@1551253org.highwire.dtl.DTLVardef@260ae7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Apolipoprotein E (APOE) and Triggering Receptor Expressed on Myeloid cells 2 (TREM2) are the two strongest genetic risk factors of late-onset Alzheimers disease. ApoE binds to the low-density lipoprotein receptor (LDLR) to facilitate the uptake of ApoE-lipoprotein particles. TREM2 is a cell surface receptor expressed on microglia in the brain. The activation of TREM2 is essential for microglia to carry out protective functions against AD pathology. Several studies have shown that TREM2 signaling is activated through direct interaction between TREM2 and ApoE. In addition to its important role in AD pathogenesis, the ApoE/TREM2 interaction has been shown to induce immunosuppression of neutrophils within the tumor microenvironment. Despite its clinical importance, a high-resolution molecular understanding of the complex remains elusive. Here, we carried out chemical cross-linking mass spectrometry (XL-MS) analysis of the ApoE3/TREM2ECD complex to identify intra- and inter-protein cross-links, which were used as restraints to guide integrative protein-protein docking. Our data support a binding model in which a helix-loop-helix motif within the ApoE3 hinge and C-terminal region forms a transient hydrophobic pocket that wraps around the hydrophobic tip of the TREM2 ectodomain. This model is further supported by de novo-designed mini-protein binders, which show the same binding mode as identified by our XL-MS experiment. These results establish a robust framework for developing mini-protein-based TREM2 agonists.

Pigment epithelial-derived factor (PEDF) is a multifunctional protein produced predominantly by the retinal pigment epithelium and expressed in many tissues, including the brain, highlighting its participation in crucial processes, such as neuroprotection and angiogenesis. Some neurodevelopmental disorders, such as ASD, are characterized by neurodevelopmental abnormalities, including altered neurite formation, spine formation, and neuronal activities. Many efforts have been made to resolve NDDs, but until now, some symptoms remain untargeted. PEDF is involved in many steps of neurodevelopment. The treatment of PEDF peptide might improve the outcome of NDD symptoms by altering neuronal morphologies. We used PEDF peptides that contain different functional domains to study the effect of administering PEDF peptides on neuronal morphology in a prenatal valproic acid (VPA)-exposed mouse model. We identified that the treatment with PEDF peptides rectified the abnormalities in neurite formation and spine formation in VPA-exposed cortical neurons. In vitro calcium imaging showed abnormalities in the spontaneous activity in VPA-exposed cortical neurons. Treatment of a short PEDF peptide normalized intracellular calcium response to the control level. Accordingly, PEDF peptides have the prospect of serving as potential treatments for patients with neurodevelopmental disorders, such as ASD.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a key regulator of microglial function and a promising therapeutic target in Alzheimers disease. While current strategies have largely focused on antibody-based agonists, alternative modalities capable of modulating TREM2 signaling remain underexplored. Here, we report the discovery of TREM2-binding cyclic peptides using a disulfide-constrained phage display library. Screening and biophysical validation identified multiple binders, with TREM2-6 and TREM2-12 exhibiting micromolar affinity. Both peptides modulated microglial responses in human iPSC-derived model of amyloid stress and in neuron-microglia co-cultures. Molecular dynamics simulations supported stable peptide-TREM2 interactions, with TREM2-12 displaying a more constrained binding mode. In vitro pharmacokinetic profiling revealed favorable plasma and intestinal stability but limited permeability, consistent with cyclic peptide scaffolds. Together, these findings establish cyclic peptides as a viable modality for targeting TREM2 and provide a foundation for the development of tunable neuroimmune therapeutics. Insert Table of Contents artwork here O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/720287v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1977a2aorg.highwire.dtl.DTLVardef@1d571c1org.highwire.dtl.DTLVardef@1f6a22org.highwire.dtl.DTLVardef@70eea7_HPS_FORMAT_FIGEXP M_FIG C_FIG

The p21-activated kinases (PAKs) are a group of serine-threonine kinases central to multiple signaling pathways that govern cell survival and proliferation. Aberrant activity of PAK1, the most well characterized member of the PAK family, drives progression of several malignancies and brain disorders, including Alzheimers disease and neurodevelopmental disorders. Despite growing interest in PAK1 as a drug target for these diseases, there is no assay to evaluate the intracellular target engagement of PAK1 inhibitors. To address this need, we developed first-in-class NanoBRET assays for wild-type PAK1 and a neurodevelopmental disorder-causing gain-of-function PAK1 mutant. Furthermore, we executed our novel PAK1 NanoBRET assay to evaluate target engagement of PAK1 inhibitors in primary hippocampal neurons. To the best of our knowledge, this is the first demonstration of a NanoBRET cellular target engagement assay in primary neurons, thereby increasing the relevance of our work by confirming PAK1 inhibitor binding to the aberrant form of the protein in primary neurons.

Apolipoprotein E (APOE) associates with amyloid plaques (A{beta}) in Alzheimer disease (AD). The {varepsilon}4 allele of apolipoprotein E (APOE{varepsilon}4) is the strongest genetic risk factor for sporadic AD and exacerbates A{beta} plaque burden relative to APOE{varepsilon}3 and APOE{varepsilon}2. The majority of ApoE associates with multiple lipid classes to form lipoproteins both in the brain and the periphery. However, the lipidation status of A{beta} plaque-associated ApoE is not yet fully defined. Here, we use fluorescence lifetime imaging microscopy coupled with Forster resonance energy transfer (FLIM-FRET) to determine the lipidation status of ApoE in plaques, as well as the nanoscale spatial proximity of ApoE and A{beta} to anionic lipids and cholesterol within human AD brain tissue. We demonstrate that lipids are in close nanoscale proximity to ApoE and A{beta} within A{beta} plaques. Our results reveal that lipidated ApoE complexes enriched in anionic lipids and cholesterol are core constituents of AD plaques in-situ. We propose a pathological mechanism in which the surface presentation of anionic lipids on ApoE lipoproteins facilitates initial interaction with and subsequent aggregation of A{beta}.

The development of small synthetic catalytic peptides, or "catalytides," offers a promising therapeutic strategy for the targeted degradation of amyloid-beta (A{beta}). Among these, the pentapeptide SKGQA mimics the proteolytic activity of serine proteases despite its minimal size. However, the molecular mechanism enabling such a short peptide to achieve effective cleavage at multiple sites remains unclear. In this study, we utilized HADDOCK docking and molecular dynamics (MD) simulations to investigate the interaction between SKGQA and the A{beta}(17-42) region. Our results demonstrate that SKGQA operates through a highly dynamic process, where the substrate serves as a scaffold to stabilize "serine protease-like" active geometries from a flexible conformational ensemble. We identified distinct "stable binding" and "stochastic attack" modes, explaining the peptides ability to facilitate both high-probability and multi-site cleavage. Given its minimal size, SKGQA may also benefit from enhanced accessibility to dense amyloid environments compared to larger proteases. These findings provide a fundamental understanding of minimal enzymatic function and offer a transformative platform for designing next-generation, cost-effective catalytides.

Migraine headaches affect over one billion people internationally and can be defined as episodes of acute severe pain wrapping around the head and are normally accompanied by nausea, blurry vision, and sensitivity to light and sound. While triggers that cause migraines may vary among patients, evidence shows they are involved with the trigeminovascular system (a network of blood vessels in the brain in conjunction with the trigeminal nerve). Activation of the trigeminal neurons triggers the release of vasoactive neuropeptides, such as calcitonin gene-related peptide (CGRP), leading to neurogenic inflammation and vasodilation of cranial blood vessels. The development of 5-HT1B serotonin agonist drugs, commonly known as triptans, have been an effective measure of migraine relief. The drugs created in this research were found to have improved docking scores within the 5-HT1B binding site compared to that of naratriptan. The two drugs proposed in this paper would need to undergo further investigation to determine the feasibility of laboratory synthesis and clinical trials.

Schizophrenia (SCZ) is a severe and debilitating neurodevelopmental disorder with lifelong impact on everyday life. Disruptions in synapse functions play a key role in its complex and poorly understood etiological and pathological mechanisms. Here, we investigated both the molecular composition and the spontaneous and stimulated functional properties of synapses in neural organoids from SCZ individuals. Air-liquid interface forebrain organoids (ALI-FOs) were generated from induced pluripotent stem cells (iPSCs) derived from three individuals with SCZ and three healthy controls. At day 170 synaptosomes were enriched and analyzed by data-independent acquisition mass spectrometry to profile the proteome, alongside with TMT-labeled phosphoproteomics both before and after acute KCl-induced depolarization. In parallel, we characterized the PTMome of the surrounding cellular environment, comprising phosphorylation, peptides with free and reversibly modified cysteines, and sialylated N-linked glycopeptides. Functional glutamatergic and GABAergic activity was assessed using calcium imaging to capture spontaneous neuronal signaling. Both conditions exhibited mature synaptic structures, while growth cones were observed only in SCZ-derived ALI-FOs, indicative of ongoing or delayed synaptogenesis. Proteomic analysis of synaptosome preparations revealed 358 differentially regulated proteins between SCZ and controls and 125 phophoproteins with altered phosphorylation, which clustered into three major categories: (1) synaptogenesis and synapse signaling; (2) cytoskeleton and cell junctions; (3) growth cone dynamics and neurite outgrowth. Analysis of the PTMs in the surrounding cellular environment revealed regulation of key regulatory mechanisms in 526 proteins, supporting the synaptic alterations observed. Notably, components of the Wnt signaling pathway were consistently dysregulated across both the synaptosome preparation and the PTMome in SCZ-derived ALIFOs as compared to controls. Depolarization-induced phospho-signaling revealed SCZ-specific response enriched in synaptic vesicle trafficking pathways. Together, these findings provide new insights into early synaptic alterations in SCZ, highlighting changes not only in protein composition, but more in protein regulatory mechanisms underlying synaptic signaling.

Alpha-Synuclein (-syn) aggregation is central to Parkinson's disease (PD), yet measurements in biofluids are confounded by the coexistence of monomeric and aggregated species. Using Syn-IMAGR, a single-molecule imaging platform with sub-femtomolar sensitivity, we show that purified -syn aggregates undergo dilution-induced disassembly, revealing a concentration-dependent equilibrium. Applied to postmortem brain lysates, Syn-IMAGR distinguishes physiological -syn multimers, which are dimmer and readily dissociate upon dilution, from PD-associated aggregates, which remain detectable and exhibit greater structural resistance to disruption. These results indicate that -syn assemblies occupy distinct stability regimes, with PD-associated aggregates representing a more persistent and less dilution-sensitive structural state. Syn-IMAGR thus provides a quantitative framework for resolving -syn species and for probing their concentration-dependent equilibrium.