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.

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.

BACKGROUND:Chimeric antigen receptor (CAR) T cells targeting CD19 achieve remarkable remissions in refractory B cell malignancies, yet deleterious side effects such as cytokine release syndrome (CRS) limit their broader application. Current preclinical assays on manufactured cell products do not predict human clinical function. We hypothesized that variability in the CAR proximal protein interaction networks that mediate CAR signal transduction may correlate with patient-to-patient differences in toxicity. METHODS:Using banked, preinfusion 41BB-CD3ζ CAR T cell products with known clinical outcomes, we applied quantitative multiplex co-immunoprecipitation (QMI) to profile ∼200 binary interactions among 21 key signaling proteins following CD19 stimulation. Bioinformatic analysis clustered interactions into functional modules, and correlated protein interaction patterns with clinical outcomes. RESULTS:Correlation network analysis, which clusters interactions into coregulated modules, identified a stimulation-responsive module with similar behavior in all products, and a second module that correlated with the presence of CRS. The CRS module was enriched for interactions among CD28, FYB, and the SRC family kinases LCK and FYN. In a head-to-head validation cohort, a similar CD28-FYB-kinase module again correlated with the presence of CRS. Using a combined dataset, a machine learning classifier trained on top QMI features retrospectively identified CRS samples with high accuracy. CONCLUSIONS:These data indicate that subtle, batch-to-batch differences in CAR signalosome assembly may correlate with CRS, and they support the further development of a preinfusion proteomic assay to forecast CRS risk in CAR T cell products.

Background:Synaptic dysfunction is an early feature of Alzheimer's 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. Methods:We utilized our established human induced pluripotent stem cell (hiPSC) derived excitatory cortical neuron model to examine SORL1's 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. Results:We 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 (Aβ) 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 Aβ. Finally, we find that SORL1 deficient neurons have impaired synaptic plasticity. Conclusions:These 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.

Cells rely on activity-dependent protein-protein interactions to convey biological signals. For chimeric antigen receptor (CAR) T cells containing a 4-1BB costimulatory domain, receptor engagement is thought to stimulate the formation of protein complexes similar to those stimulated by T cell receptor (TCR)-mediated signaling, but the number and type of protein interaction-mediating binding domains differ between CARs and TCRs. Here, we performed coimmunoprecipitation mass spectrometry analysis of a second-generation, CD19-directed 4-1BB:ζ CAR (referred to as bbζCAR) and identified 128 proteins that increased their coassociation after target engagement. We compared activity-induced TCR and CAR signalosomes by quantitative multiplex coimmunoprecipitation and showed that bbζCAR engagement led to the activation of two modules of protein interactions, one similar to TCR signaling that was more weakly engaged by bbζCAR as compared with the TCR and one composed of TRAF signaling complexes that was not engaged by the TCR. Batch-to-batch and interindividual variations in production of the cytokine IL-2 correlated with differences in the magnitude of protein network activation. Future CAR T cell manufacturing protocols could measure, and eventually control, biological variation by monitoring these signalosome activation markers.

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 investigate how Fragile X Messenger Ribonucleoprotein (FMRP) deficiency disrupts signal transduction through a glutamatergic synapse PIN downstream of NMDA receptor or metabotropic glutamate receptor (mGluR) stimulation. In cultured cortical neurons or acute cortical slices from P7, P17 and P60 FMR1-/y mice, the unstimulated protein interaction network state resembled that of wildtype littermates stimulated with mGluR agonists, demonstrating resting state pre-activation of mGluR signaling networks. In contrast, interactions downstream of NMDAR stimulation were similar to WT. We identified the Src family kinase (SFK) Fyn as a network hub, because many interactions involving Fyn were pre-activated in FMR1-/y animals. We tested whether targeting SFKs in FMR1-/y mice could modify disease phenotypes, and found that Saracatinib (SCB), 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.

The Pediatric Leukemia Adoptive Therapy (PLAT) trials at Seattle Children’s Hospital have pioneered the use of chimeric antigen receptor (CAR) T cell (CAR-T) therapies in pediatric patients with B cell Acute Lymphoblastic Leukemia. The landmark PLAT-02 trial (n=45), utilizing a second-generation CD19-directed CAR, observed a 93% remission rate following CAR-T infusion. However, approximately half of these subjects relapsed within one year and nearly a third of those tumors no longer expressed the CD19 target. The PLAT-04 trial (n=4) was subsequently conceived with a second-generation CAR (SCRI-C22v1) engineered to target CD22, an alternative antigen expressed on the B cell surface. Despite promising preclinical data, SCRI-C22v1 failed to generate anti-leukemic activity in all four subjects and the trial was halted. In response, a different second-generation CD22-directed CAR (SCRI-C22v2) was engineered, replacing the CD28 transmembrane domain and IgG4 hinge domain in SCRI-C22v1 with a continuous CD8 hinge and transmembrane domain while retaining identical 4-1BB and CD3ζ intracellular signaling domains. The modified SCRI-C22v2 displayed improved anti-tumor activity in preclinical models and was ultimately tested in patients in the PLAT-07 trial (n=4), where it exhibited strong in vivo expansion and persistence while inducing complete remission after 1 month in all four subjects. To investigate the molecular signaling responsible for clinical differences observed between SCRI-C22v1 and SCRI-C22v2, we applied an emerging proteomic technology, Quantitative Multiplex Immunoprecipitation (QMI), that captures medium-throughput quantitative data about fold changes in protein interaction networks downstream of the CAR. CAR-T products from healthy donors expressing either SCRI-C22v1 or SCRI-C22v2 were stimulated with fixed K562 cells expressing CD22 and protein interaction networks were profiled with QMI, revealing distinct differences in signal transduction downstream of the CAR. Contrary to expectation, it was the ineffective SCRI-C22v1 that exhibited significantly enhanced signalosome formation, engaging more strongly than SCRI-C22v2 with both the classical CD3ζ-ZAP70-LAT-SLP76 pathway as well as the non-classical TRAF pathway. Interestingly, the clinically successful SCRI-C22v2 demonstrated stronger baseline engagement with inhibitory molecules that attenuate canonical TCR signaling such as SHP2 and UBASH3A, suggesting regulatory control of CAR signaling is crucial for optimal clinical responses. Since the CAR is a synthetic receptor, it should be possible to bioengineer optimized signal transduction to some extent, and our group continues to work towards identifying an optimized QMI signature that can be used as a CAR-tune network for the rational design of more effective CAR-T therapies. Citation Format: Eric Bueter, Corinne Summers, Isabella Draper, Joshua Gustafson, Stephen EP Smith, Kamila Gwiazda, Julie Park, Colleen Annesley, Rebecca Gardner, Michael Jensen. Quantitative multiplex immunoprecipitation reveals distinct protein interaction networks responsible for discrepant in vivo activity exhibited by two second generation CD22-targeted chimeric antigen receptors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A042.

The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that acts as a central mediator of translation, and plays important roles in cell growth, synaptic plasticity, cancer, and a wide range of developmental disorders. The signaling cascade linking lipid kinases (PI3Ks), protein kinases (AKT) and translation initiation complexes (EIFs) to mTOR has been extensively modeled, but does not fully describe mTOR system behavior. Here, we use quantitative multiplex co-immunoprecipitation to monitor a protein interaction network (PIN) composed of 300+ binary interactions among mTOR-related proteins. Using a simple model system of serum deprived or fresh-media-fed mouse 3T3 fibroblasts, we observed extensive PIN remodeling involving 27+ individual protein interactions after one hour, despite phosphorylation changes observed after only five minutes. Using small molecule inhibitors of PI3K, AKT, mTOR, MEK and ERK, we define subsets of the PIN, termed 'modules', that respond differently to each inhibitor. Using primary fibroblasts from individuals with overgrowth disorders caused by pathogenic PIK3CA or MTOR variants, we find that hyperactivation of mTOR pathway components is reflected in a hyperactive PIN. Our data define a "modular" organization of the mTOR PIN in which coordinated groups of interactions respond to activation or inhibition of distinct nodes, and demonstrate that kinase inhibitors affect the modular network architecture in a complex manner, inconsistent with simple linear models of signal transduction.

(R,R′)-4′-Methoxy-1-naphthylfenoterol (MNF) promotes growth inhibition and apoptosis of human HepG2 hepatocarcinoma cells via cannabinoid receptor (CBR) activation. The synthetic CB1R inverse agonist, AM251, has been shown to block the anti-mitogenic effect of MNF in these cells; however, AM251 is also an agonist of the recently deorphanized, lipid-sensing receptor, GPR55, whose upregulation contributes to carcinogenesis. Here, we investigated the role of MNF in GPR55 signaling in human HepG2 and PANC-1 cancer cell lines in culture by focusing first on internalization of the fluorescent ligand Tocrifluor 1117 (T1117). Initial results indicated that cell pretreatment with GPR55 agonists, including the atypical cannabinoid O-1602 and l-α-lysophosphatidylinositol, dose-dependently reduced the rate of cellular T1117 uptake, a process that was sensitive to MNF inhibition. GPR55 internalization and signaling mediated by O-1602 was blocked by MNF in GPR55-expressing HEK293 cells. Pretreatment of HepG2 and PANC-1 cells with MNF significantly abrogated the induction of ERK1/2 phosphorylation in response to AM251 and O-1602. Moreover, MNF exerted a coordinated negative regulation of AM251 and O-1602 inducible processes, including changes in cellular morphology and cell migration using scratch wound healing assay. This study shows for the first time that MNF impairs GPR55-mediated signaling and, therefore, may have therapeutic potential in the management of cancer.

Neurons encode information by rapidly modifying synaptic protein complexes, which changes the strength of specific synaptic connections. Homer1 is abundantly expressed at glutamatergic synapses, and is known to alter its binding to metabotropic glutamate receptor 5 (mGlu5) in response to synaptic activity. However, Homer participates in many additional known interactions whose activity-dependence is unclear. Here, we used co-immunoprecipitation and label-free quantitative mass spectrometry to characterize activity-dependent interactions in the cerebral cortex of wildtype and Homer1 knockout mice. We identified a small, high-confidence protein network consisting of mGlu5, Shank2 and 3, and Homer1–3, of which only mGlu5 and Shank3 were significantly reduced following neuronal depolarization. We identified several other proteins that reduced their co-association in an activity-dependent manner, likely mediated by Shank proteins. We conclude that Homer1 dissociates from mGlu5 and Shank3 following depolarization, but our data suggest that direct Homer1 interactions in the cortex may be more limited than expected.

In late 2019, a novel coronavirus began circulating within humans in central China. It was designated SARS-CoV-2 because of its genetic similarities to the 2003 SARS coronavirus (SARS-CoV). Now that SARS-CoV-2 has spread worldwide, there is a risk of it establishing new animal reservoirs and recombination with native circulating coronaviruses. To screen local animal populations in the United States for exposure to SARS-like coronaviruses, we developed a serological assay using the receptor binding domain (RBD) from SARS-CoV-2. SARS-CoV-2's RBD is antigenically distinct from common human and animal coronaviruses, allowing us to identify animals previously infected with SARS-CoV or SARS-CoV-2. Using an indirect enzyme-linked immunosorbent assay (ELISA) for SARS-CoV-2's RBD, we screened serum from wild and domestic animals for the presence of antibodies against SARS-CoV-2's RBD. Surprisingly prepandemic feline serum samples submitted to the University of Tennessee Veterinary Hospital were ∼50% positive for anti-SARS RBD antibodies. Some of these samples were serologically negative for feline coronavirus (FCoV), raising the question of the etiological agent generating anti-SARS-CoV-2 RBD cross-reactivity. We also identified several white-tailed deer from South Carolina with anti-SARS-CoV-2 antibodies. These results are intriguing, as cross-reactive antibodies toward SARS-CoV-2 RBD have not been reported to date. The etiological agent responsible for seropositivity was not readily apparent, but finding seropositive cats prior to the current SARS-CoV-2 pandemic highlights our lack of information about circulating coronaviruses in other species. IMPORTANCE We report cross-reactive antibodies from prepandemic cats and postpandemic South Carolina white-tailed deer that are specific for that SARS-CoV RBD. There are several potential explanations for this cross-reactivity, each with important implications to coronavirus disease surveillance. Perhaps the most intriguing possibility is the existence and transmission of an etiological agent (such as another coronavirus) with similarity to SARS-CoV-2's RBD region. However, we lack conclusive evidence of prepandemic transmission of a SARS-like virus. Our findings provide impetus for the adoption of a One Health Initiative focusing on infectious disease surveillance of multiple animal species to predict the next zoonotic transmission to humans and future pandemics.

Cells rely on activity-dependent protein-protein interactions to convey biological signals, but the state-dependent interactome is notoriously cell-specific and undercharacterized[1][1]. In the case of chimeric antigen receptor (CAR) T cells containing a 4-1BB costimulatory domain, receptor engagement is thought to trigger the formation of protein complexes similar to those triggered by T cell receptor (TCR)-mediated signaling, but the number and type of protein-interaction-mediating binding domains differ between CARs and TCRs. Here, we performed co-immunoprecipitation mass spectrometry of a 2nd generation CD19-directed 4-1BB:zeta CAR (referred to as bbζCAR) and identified 67 proteins that increased their co-association after target engagement. We compared activity-induced TCR and CAR signalosomes using quantitative multiplex co-immunoprecipitation and showed that bbζCAR engagement leads to activation of two modules of protein interactions, one similar to TCR signaling that is more weakly engaged in bbζCAR vs. TCR, and one composed of TRAF signaling complexes that is not engaged by the TCR. Batch-to-batch and inter-individual variations in IL2 production correlated with differences in the magnitude of protein network activation. Future CAR T cell manufacturing protocols could measure, and eventually control, biological variation by monitoring these signalosome activation markers.One Sentence Summary We define a network of protein interactions engaged by chimeric antigen receptors following target binding, and show that the magnitude of network activation correlates with IL-2 secretion, a proxy measure for CAR T cell function.### Competing Interest StatementThe authors have declared no competing interest. [1]: #ref-1

Abstract The approval and commercial launch of multiple first-generation CD19- or BCMA-directed, autologous CAR-T cell products have laid the foundation and opened a path for the development of more advanced cellular therapeutics, including CAR-T cell products with next-generation capabilities. Among these newer designs, allogeneic cell therapies are positioned to unlock the broad potential of engineered immune cells as a leading therapeutic modality. However, expansion, persistence, armoring, and trafficking of allogeneic CAR-T cells are critical to achieving long-term efficacy. Caribou Biosciences is advancing a BCMA-specific allogeneic CAR-T cell product candidate, CB-011, with an immune cloaking approach that includes both the removal of the endogenous B2M protein and the insertion of a B2M-HLA-E-peptide (B2M-HLA-E) fusion protein transgene. This strategy is designed to blunt CAR-T cell rejection by both recipient T cells and NK cells, and CB-011 is in preclinical development for relapsed or refractory multiple myeloma (r/r MM). We used Cas12a chRDNA guides in the manufacture of CB-011 to make four edits, including site-specific insertion of a proprietary humanized anti-BCMA CAR into the TRAC locus with high specificity and efficiency, thus eliminating TCR expression to prevent graft-versus-host disease (GvHD). In addition, we inserted a gene encoding a B2M-HLA-E fusion protein into the native B2M gene locus. This method simultaneously prevents the expression of the native B2M protein and expresses a minor HLA class I antigen HLA-E to blunt both T- and NK-mediated rejection of the CAR-T cells by the recipient’s immune system. B2M is a protein that stabilizes all HLA class I antigens on the cell surface, therefore its knockout eliminates endogenous HLA class I presentation on the surface of the CAR-T cells. To demonstrate that the B2M-HLA-E fusion protein expression protects CB-011 from NK-mediated cell killing, we developed an in vitro competition assay in which CB-011 cells are co-incubated with NK cells. We observed that CAR-T cells expressing the B2M-HLA-E fusion have a survival advantage over cells that do not express the fusion in the presence of NK cells in vitro, indicating that they could resist killing by a recipient’s NK cells and potentially circulate longer. The BCMA-specific CAR leads to long-term survival in mice bearing established orthotopically-engrafted MM tumor cells. Furthermore, mice treated with high doses of CB-011 did not experience symptoms typical of GvHD. This strategy should enable CB-011 CAR-T cells to remain in circulation longer in recipients, providing for increased potential of antitumor activity. Citation Format: Elizabeth Garner, Emilie Degagne, Suparna Roy, Paul Donohoue, Tristan Fowler, Morena Stanaway, Vanina Vicena, Devin Mutha, Benjamin Schilling, McKay Shaw, Mara Bryan, Leslie Edwards, Stephen Smith, Bryan Kohrs, Lynda Banh, Kyle McSweeney, Justin Skoble, Steven Kanner. A BCMA-specific allogeneic CAR-T cell therapy (CB-011) genome-engineered to express an HLA-E fusion transgene to prevent immune cell rejection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB009.

Patients harboring mutations in the PI3K-AKT-MTOR pathway-encoding genes often develop a spectrum of neurodevelopmental disorders including epilepsy. A significant proportion remains unresponsive to conventional anti-seizure medications. Understanding mutation-specific pathophysiology is thus critical for molecularly targeted therapies. We previously determined that mouse models expressing a patient-related activating mutation in PIK3CA, encoding the p110α catalytic subunit of phosphoinositide-3-kinase (PI3K), are epileptic and acutely treatable by PI3K inhibition, irrespective of dysmorphology. Here we report the physiological mechanisms underlying this dysregulated neuronal excitability. In vivo, we demonstrate epileptiform events in the Pik3ca mutant hippocampus. By ex vivo analyses, we show that Pik3ca-driven hyperactivation of hippocampal pyramidal neurons is mediated by changes in multiple non-synaptic, cell-intrinsic properties. Finally, we report that acute inhibition of PI3K or AKT, but not MTOR activity, suppresses the intrinsic hyperactivity of the mutant neurons. These acute mechanisms are distinct from those causing neuronal hyperactivity in other AKT-MTOR epileptic models and define parameters to facilitate the development of new molecularly rational therapeutic interventions for intractable epilepsy.

Activity-dependent regulation of protein interactions enables synaptic scaling and stable neuronal excitability.

Repurposed drugs that block the interaction between the SARS-CoV-2 spike protein and its receptor ACE2 could offer a rapid route to novel COVID-19 treatments or prophylactics. Here, we screened 2,701 compounds from a commercial library of drugs approved by international regulatory agencies for their ability to inhibit the binding of recombinant, trimeric SARS-CoV-2 spike protein to recombinant human ACE2. We identified 56 compounds that inhibited binding in a concentration-dependent manner, measured the IC50 of binding inhibition, and computationally modeled the docking of the best inhibitors to the Spike-ACE2 binding interface. The best candidates were Thiostrepton, Oxytocin, Nilotinib, and Hydroxycamptothecin with IC50’s in the 4–9 μM range. These results highlight an effective screening approach to identify compounds capable of disrupting the Spike-ACE2 interaction, as well as identify several potential inhibitors of the Spike-ACE2 interaction.

A core network of ubiquitously expressed glutamate-synapse-associated proteins mediates activity-dependent synaptic plasticity throughout the brain, but the specific proteomic composition of synapses differs between brain regions. Here, we sought to classify the diversity of activity-dependent remodeling across brain regions using quantitative protein interaction network (PIN) analysis. We first compared the response of cultured neurons to distinct stimuli, and defined PIN parameters that differentiate input types. We next compared the response of three different brain regions maintained alive in vitro to an identical stimulus, and identified three qualitatively different PIN responses. Finally, we measured the PIN response following associative learning tasks, delay and trace eyeblink conditioning, in three brain regions, and found that the two forms of associative learning are distinguished from each other using brain-region-specific network mechanisms. We conclude that although the PIN of the glutamatergic post-synapse is expressed ubiquitously, its activity-dependent dynamics show remarkable stimulus-specific and brain-region-specific diversity.

Shank3 mutations contribute to intellectual disability. Because SHANK3 is a protein scaffold that helps organize the multiprotein network of the glutamatergic postsynaptic density (PSD), alterations in chemical synaptic transmission are implicated. Electrical synaptic transmission is a second form of synaptic transmission, enabled by intercellular channels comprised of connexin36 that support direct electrical communication among neurons, electrical brain rhythms, and neurocognitive states. Using multiplex proteomics, we report that two autism-related mutations of mouse Shank3 disrupt the glutamatergic PSD differently, but have in common the disruption of an association between NMDA-type glutamate-receptors (NMDARs) and connexin36. Mutation of Shank3 exons 13-16 most robustly dissociated connexin36 from NMDARs while impairing electrical synaptic transmission and the synchrony of an electrical rhythm in mouse inferior olive. We suggest that electrical synapses are a component of an “extended PSD” sensitive to Shank3 mutations that produce intellectual disability, at least in part, by impairing electrical synaptic transmission.

Huntington disease (HD) is a neurodegenerative disease caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. Therapeutics that lower HTT have shown preclinical promise and are being evaluated in clinical trials. However, clinical assessment of brain HTT lowering presents challenges. We have reported that mutant HTT (mHTT) in the CSF of HD patients correlates with clinical measures, including disease burden as well as motor and cognitive performance. We have also shown that lowering HTT in the brains of HD mice results in correlative reduction of mHTT in the CSF, prompting the use of this measure as an exploratory marker of target engagement in clinical trials. In this study, we investigate the mechanisms of mHTT clearance from the brain in adult mice of both sexes to elucidate the significance of therapy-induced CSF mHTT changes. We demonstrate that, although neurodegeneration increases CSF mHTT concentrations, mHTT is also present in the CSF of mice in the absence of neurodegeneration. Importantly, we show that secretion of mHTT from cells in the CNS followed by glymphatic clearance from the extracellular space contributes to mHTT in the CSF. Furthermore, we observe secretion of wild type HTT from healthy control neurons, suggesting that HTT secretion is a normal process occurring in the absence of pathogenesis. Overall, our data support both passive release and active clearance of mHTT into CSF, suggesting that its treatment-induced changes may represent a combination of target engagement and preservation of neurons.SIGNIFICANCE STATEMENT:Changes in CSF mutant huntingtin (mHTT) are being used as an exploratory endpoint in HTT lowering clinical trials for the treatment of Huntington disease (HD). Recently, it was demonstrated that intrathecal administration of a HTT lowering agent leads to dose-dependent reduction of CSF mHTT in HD patients. However, little is known about how HTT, an intracellular protein, reaches the extracellular space and ultimately the CSF. Our findings that HTT enters CSF by both passive release and active secretion followed by glymphatic clearance may have significant implications for interpretation of treatment-induced changes of CSF mHTT in clinical trials for HD.