De novo design of peptide binders to conformationally diverse targets with contrastive language modeling.

Designing binders to target undruggable proteins presents a formidable challenge in drug discovery. In this work, we provide an algorithmic framework to design short, target-binding linear peptides, requiring only the amino acid sequence of the target protein. To do this, we propose a process to generate naturalistic peptide candidates through Gaussian perturbation of the peptidic latent space of the ESM-2 protein language model and subsequently screen these novel sequences for target-selective interaction activity via a contrastive language-image pretraining (CLIP)-based contrastive learning architecture.

The Epilepsy-Aphasia Syndrome gene, Cnksr2, Plays a Critical Role in the Anterior Cingulate Cortex Mediating Vocal Communication.

Epilepsy Aphasia Syndrome (EAS) is a spectrum of childhood disorders that exhibit complex co-morbidities that include epilepsy and the emergence of cognitive and language disorders. CNKSR2 is an X-linked gene in which mutations are linked to EAS. We previously demonstrated Cnksr2 knockout (KO) mice model key phenotypes of EAS analogous to those present in clinical patients with mutations in the gene.

Astrocytic LRRK2 Controls Synaptic Connectivity via Regulation of ERM Phosphorylation.

Astrocytes, a major glial cell type of the brain, regulate synapse numbers and function. However, whether astrocyte dysfunction can cause synaptic pathologies in neurological disorders such as Parkinson's Disease (PD) is unknown. Here, we investigated the impact of the most common PD-linked mutation in the leucine-rich repeat kinase 2 () gene (G2019S) on the synaptic functions of astrocytes.

Design of Peptide Binders to Conformationally Diverse Targets with Contrastive Language Modeling.

Designing binders to target undruggable proteins presents a formidable challenge in drug discovery, requiring innovative approaches to overcome the lack of putative binding sites. Recently, generative models have been trained to design binding proteins via three-dimensional structures of target proteins, but as a result, struggle to design binders to disordered or conformationally unstable targets. In this work, we provide a generalizable algorithmic framework to design short, target-binding linear peptides, requiring only the amino acid sequence of the target protein.

Presynaptic Rac1 in the hippocampus selectively regulates working memory.

One of the most extensively studied members of the Ras superfamily of small GTPases, Rac1 is an intracellular signal transducer that remodels actin and phosphorylation signaling networks. Previous studies have shown that Rac1-mediated signaling is associated with hippocampal-dependent working memory and longer-term forms of learning and memory and that Rac1 can modulate forms of both pre- and postsynaptic plasticity. How these different cognitive functions and forms of plasticity mediated by Rac1 are linked, however, is unclear.

Identification of new ciliary signaling pathways in the brain and insights into neurological disorders.

Primary cilia are conserved sensory hubs essential for signaling transduction and embryonic development. Ciliary dysfunction causes a variety of developmental syndromes with neurological features and cognitive impairment, whose basis mostly remains unknown. Despite connections to neural function, the primary cilium remains an overlooked organelle in the brain.

A Ventromedial Prefrontal-to-Lateral Entorhinal Cortex Pathway Modulates the Gain of Behavioral Responding During Threat.

Background: The ability to correctly associate cues and contexts with threat is critical for survival, and the inability to do so can result in threat-related disorders such as posttraumatic stress disorder. The prefrontal cortex (PFC) and hippocampus are well known to play critical roles in cued and contextual threat memory processing. However, the circuits that mediate prefrontal-hippocampal modulation of context discrimination during cued threat processing are less understood.

Cnksr2 Loss in Mice Leads to Increased Neural Activity and Behavioral Phenotypes of Epilepsy-Aphasia Syndrome.

Epilepsy Aphasia Syndromes (EAS) are a spectrum of childhood epileptic, cognitive, and language disorders of unknown etiology. is a strong X-linked candidate gene implicated in EAS; however, there have been no studies of genetic models to dissect how its absence may lead to EAS. Here we develop a novel KO mouse line and show that male mice exhibit increased neural activity and have spontaneous electrographic seizures.

Action potential-coupled Rho GTPase signaling drives presynaptic plasticity.

In contrast to their postsynaptic counterparts, the contributions of activity-dependent cytoskeletal signaling to presynaptic plasticity remain controversial and poorly understood. To identify and evaluate these signaling pathways, we conducted a proteomic analysis of the presynaptic cytomatrix using in vivo biotin identification (iBioID). The resultant proteome was heavily enriched for actin cytoskeleton regulators, including Rac1, a Rho GTPase that activates the Arp2/3 complex to nucleate branched actin filaments.

Tripartite synaptomics: Cell-surface proximity labeling in vivo.

The astrocyte is a central glial cell and plays a critical role in the architecture and activity of neuronal circuits and brain functions through forming a tripartite synapse with neurons. Emerging evidence suggests that dysfunction of tripartite synaptic connections contributes to a variety of psychiatric and neurodevelopmental disorders. Furthermore, recent advancements with transcriptome profiling, cell biological and physiological approaches have provided new insights into the molecular mechanisms into how astrocytes control synaptogenesis in the brain.

Master Regulators and Cofactors of Human Neuronal Cell Fate Specification Identified by CRISPR Gene Activation Screens.

Technologies to reprogram cell-type specification have revolutionized the fields of regenerative medicine and disease modeling. Currently, the selection of fate-determining factors for cell reprogramming applications is typically a laborious and low-throughput process. Therefore, we use high-throughput pooled CRISPR activation (CRISPRa) screens to systematically map human neuronal cell fate regulators.

Chemico-genetic discovery of astrocytic control of inhibition in vivo.

Perisynaptic astrocytic processes are an integral part of central nervous system synapses; however, the molecular mechanisms that govern astrocyte-synapse adhesions and how astrocyte contacts control synapse formation and function are largely unknown. Here we use an in vivo chemico-genetic approach that applies a cell-surface fragment complementation strategy, Split-TurboID, and identify a proteome that is enriched at astrocyte-neuron junctions in vivo, which includes neuronal cell adhesion molecule (NRCAM). We find that NRCAM is expressed in cortical astrocytes, localizes to perisynaptic contacts and is required to restrict neuropil infiltration by astrocytic processes.

Dysregulation of the Synaptic Cytoskeleton in the PFC Drives Neural Circuit Pathology, Leading to Social Dysfunction.

Psychiatric disorders are highly heritable pathologies of altered neural circuit functioning. How genetic mutations lead to specific neural circuit abnormalities underlying behavioral disruptions, however, remains unclear. Using circuit-selective transgenic tools and a mouse model of maladaptive social behavior (ArpC3 mutant), we identify a neural circuit mechanism driving dysfunctional social behavior.

Essential role for InSyn1 in dystroglycan complex integrity and cognitive behaviors in mice.

Human mutations in the dystroglycan complex (DGC) result in not only muscular dystrophy but also cognitive impairments. However, the molecular architecture critical for the synaptic organization of the DGC in neurons remains elusive. Here, we report Inhibitory Synaptic protein 1 (InSyn1) is a critical component of the DGC whose loss alters the composition of the GABAergic synapses, excitatory/inhibitory balance in vitro and in vivo, and cognitive behavior.

Plug-and-Play Protein Modification Using Homology-Independent Universal Genome Engineering.

Analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, including the diversity of cell types in the brain. However, current approaches are often low throughput and resource intensive. Here, we describe a CRISPR-Cas9-based homology-independent universal genome engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application.

Identifying Synaptic Proteins by In Vivo BioID from Mouse Brain.

Two anatomically and functionally distinct types of synapses are present in the central nervous system, excitatory synapses, and inhibitory synapses. Purification and analysis of the protein complex at the excitatory postsynapses have led to fundamental insights into neurobiology. In contrast, the biochemical purification and analysis of the inhibitory postsynaptic density have been largely intractable.

Genetically Encoded Fluorescent Indicator GRAPHIC Delineates Intercellular Connections.

Intercellular contacts are essential for precise organ morphogenesis, function, and maintenance; however, spatiotemporal information of cell-cell contacts or adhesions remains elusive in many systems. We developed a genetically encoded fluorescent indicator for intercellular contacts with optimized intercellular GFP reconstitution using glycosylphosphatidylinositol (GPI) anchor, GRAPHIC (GPI anchored reconstitution-activated proteins highlight intercellular connections), which can be used for an expanded number of cell types. We observed a robust GFP signal specifically at the interface between cultured cells, without disrupting natural cell contact.

In vivo proximity proteomics of nascent synapses reveals a novel regulator of cytoskeleton-mediated synaptic maturation.

Excitatory synapse formation during development involves the complex orchestration of both structural and functional alterations at the postsynapse. However, the molecular mechanisms that underlie excitatory synaptogenesis are only partially resolved, in part because the internal machinery of developing synapses is largely unknown. To address this, we apply a chemicogenetic approach, in vivo biotin identification (iBioID), to discover aspects of the proteome of nascent synapses.

Restoring striatal WAVE-1 improves maze exploration performance of GluN1 knockdown mice.

NMDA receptors are important for cognition and are implicated in neuropsychiatric disorders. GluN1 knockdown (GluN1KD) mice have reduced NMDA receptor levels, striatal spine density deficits, and cognitive impairments. However, how NMDA depletion leads to these effects is unclear.

Thrombospondin receptor α2δ-1 promotes synaptogenesis and spinogenesis via postsynaptic Rac1.

Astrocytes control excitatory synaptogenesis by secreting thrombospondins (TSPs), which function via their neuronal receptor, the calcium channel subunit α2δ-1. α2δ-1 is a drug target for epilepsy and neuropathic pain; thus the TSP-α2δ-1 interaction is implicated in both synaptic development and disease pathogenesis. However, the mechanism by which this interaction promotes synaptogenesis and the requirement for α2δ-1 for connectivity of the developing mammalian brain are unknown.

Regulation of spine structural plasticity by Arc/Arg3.1.

Dendritic spines are actin-rich, postsynaptic protrusions that contact presynaptic terminals to form excitatory chemical synapses. These synaptic contacts are widely believed to be the sites of memory formation and information storage, and changes in spine shape are thought to underlie several forms of learning-related plasticity. Both membrane trafficking pathways and the actin cytoskeleton drive activity-dependent structural and functional changes in dendritic spines.