Biology subjects

Martin-de-Saavedra, M. D.

Publications and source records attributed to Martin-de-Saavedra, M. D..

6 recordsLinked to original sources

bioRxiv2024

Pro-cognitive reshaping of neuronal dynamics by a human CSF-based factor

Neuronal connection dysfunction is a convergent cause of cognitive deficits in mental disorders. Cognitive processes are finely regulated at the synaptic level by membrane proteins, some of which are shed and detectable in patients cerebrospinal fluid (CSF). However, whether these soluble synaptic proteins can harnessed as innovative pro-cognitive factors to treat brain disorders remains unclear. Here, we use quantitative proteomics to identify shed synaptic proteins dysregulated in the CSF of subjects with schizophrenia (SCZ), a mental disorder characterized by cognitive and synaptic dysfunction. The level of a yet uncharacterized soluble form of the voltage-gated calcium channel auxiliary subunit, 2{delta}-1, is robustly reduced in SCZ CSF. Remarkably, soluble 2{delta}-1 is convergently downregulated across several brain disorder CSF proteomes. We show that the brain releases soluble 2{delta}-1 in an activity-dependent manner, which can reorganize neuronal network dynamics by binding to synaptic targets and promoting inhibitory neuron plasticity. A single brain injection of a synthetic soluble 2{delta}-1 improved interneuron and cognitive deficits in a mutant mouse model of SCZ and cortical dysfunction. These findings underscore the potential of shed synaptic proteins as novel therapeutic agents capable of enhancing brain function in diverse brain disorders characterized by cognitive impairment.

neuroscience↗

bioRxiv2024

ASD-ASSOCIATED CNTNAP2 VARIANTS DISRUPT NEURONAL ARBORIZATION THROUGH IMPAIRED REGULATION BY ECTODOMAIN SHEDDING

Ectodomain shedding (ES) is a process by which a protease cleaves the extracellular portion of membrane-bound proteins, releasing soluble fragments that influence diverse cellular functions. ES is critical in neurodevelopment, plasticity, and neurodegenerative disorders, such as Alzheimers disease, and has recently been implicated in neurodevelopmental conditions, including autism spectrum disorders (ASD). Contactin-associated protein-like 2 (CNTNAP2) is an adhesion molecule regulated by ES, releasing a soluble ectodomain (sCNTNAP2) that enhances neuronal synchrony. CNTNAP2 is implicated in ASD, schizophrenia, and cortical dysplasia focal epilepsy syndrome (CDFE) and it is known to regulate neuronal arborization, as well as dendritic spine maturation and maintenance. However, little is known about how neuroplasticity impacts ES or the role of CNTNAP2 ES in dendritic arborization. Here, we show that the brain sheddome is enriched in shed ectodomains that regulate neuronal projections, and that its molecular and functional composition is modulated by sensory deprivation in a sex dependent manner, with a decrease in sCNTNAP2 levels observed only in male mice. Furthermore, we demonstrate that sCNTNAP2 promotes dendritic arborization, while ASD-associated CNTNAP2 variants present reduced sCNTNAP2 levels in culture and decreased neuronal branching. Together, these findings underscore the role of ES in neuroplasticity and ASD and reveal how CNTNAP2 genetic variations disrupt its regulation by ES, leading to altered dendritic branching.

neuroscience↗

bioRxiv2022

Rescue of neuropsychiatric phenotypes in a mouse model of 16p11.2 duplication syndrome by genetic correction of an epilepsy network hub

Neuropsychiatric disorders (NPDs) share genetic etiology and are frequently co-morbid with epilepsy, but the biological basis of this shared risk remains poorly understood. The 16p11.2 microduplication (16p11.2dup/+) is a highly pleiotropic copy number variant (CNV) conferring risk for multiple NPDs including autism spectrum disorder, schizophrenia and intellectual disability, and is associated with a high prevalence of seizures. We used a mouse model of the 16p11.2 duplication (16p11.2dup/+) to uncover molecular and circuit properties associated with this broad phenotypic spectrum, and examined genes within the locus capable of phenotype reversal. Quantitative proteomics of cortical membranes revealed alterations to synaptic protein networks and products of diverse NPD risk genes in 16p11.2dup/+ mice. Network analysis identified an epilepsy-associated protein subnetwork, which was dysregulated in 16p11.2dup/+ mice and proteomic datasets from human NPDs. We investigated circuit properties in 16p11.2dup/+ mice and found they exhibited hypersynchronous activity and enhanced network glutamate release, which increased susceptibility to seizures. We hypothesized that a regulator of the synaptic and epilepsy-associated protein network could have an important impact on pathophysiology. Human brain co-expression and interactome analysis revealed PRRT2 as a major hub in the dysregulated epilepsy subnetwork. Remarkably, restoring Prrt2 copy number to wild-type levels rescued aberrant circuit properties, seizure susceptibility and social interaction deficits in 16p11.2dup/+ mice. We show that proteomics and network biology can identify important disease hubs in multigenic CNVs, and reveal molecular and circuit phenotypes which may be relevant to the complex symptomatology of 16p11.2 duplication carriers.

neuroscience↗

bioRxiv2019

Rapid 3D enhanced resolution microscopy reveals the dynamics of cortical dendritic spinules

Dendritic spinules are thin, membranous protrusions formed by neuronal dendritic spines that are not adequately resolved by diffraction-limited light microscopy. Hence, our understanding of spinules is inferred predominantly from fixed-tissue electron microscopy (EM). Super-resolution modalities have enabled live-cell nanoscopic imaging, but their utility for fast, time-lapse, volumetric imaging has been restricted. Herein, we utilized rapid structured illumination microscopy (SIM) and enhanced resolution confocal microscopy to study spatiotemporal spinule dynamics in live cultured cortical pyramidal neurons. Spinules on mushroom spines typically recurred at the same topographical locations and most were short-lived, originating near simple post-synaptic densities (PSDs), while a subset was long-lived and elongated, emerging from complex PSDs. Ca2+ puncta within spinules synchronized with spine head transients and Ca2+ depletion drastically decreased spinule number. Moreover, we uncovered evidence of differential Ca2+-mediated regulation of short-lived and long-lived spinules. Thus, we identified unique spinule classes divergent in lifespan, dynamics, morphology, relationship to the PSD, and regulation. These data suggest distinct synaptic functions of spinule classes, informing future studies, while demonstrating a new application for enhanced resolution microscopy.

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↗

bioRxiv2019

CNTNAP2 is targeted to endosomes by the polarity protein Par3

A decade of genetic studies has established Contactin-associated protein-like 2 (CNTNAP2) as a prominent susceptibility gene associated with multiple neurodevelopmental disorders. The development and characterization of Cntnap2 knockout models in multiple species have bolstered this claim by establishing clear connections with certain endophenotypes. Despite these remarkable in vivo findings, CNTNAP2s molecular functions are relatively unexplored, highlighting the need to identify novel protein partners. Here, we characterized an interaction between CNTNAP2 and Partitioning-defective 3 (Par3) - a polarity molecule we isolated in a yeast-two hybrid screen with CNTNAP2s C-terminus. We provide evidence that the two proteins interact via PDZ domain-mediated binding, that CNTNAP2+/Par3+ complexes are largely associated with clathrin-coated endocytic vesicles, and that Par3 causes an enlargement of these structures. Live imaging and fluorescence recovery after photobleaching (FRAP) reveals that Par3 limits the mobility of CNTNAP2 at endosomes, thus stabilizing it at that location. Finally, expression of Par3 but not Par3{Delta}PDZ can cluster endogenous CNTNAP2 in primary neurons. Collectively, we conclude that Par3 regulates CNTNAP2 spatial localization to endocytic compartments.

neuroscience↗