Biology subjects

Claxton, S.

Publications and source records attributed to Claxton, S..

5 recordsLinked to original sources

bioRxiv2026

CDKL5 phosphorylates neuronal ELAVL proteins to promote mRNA binding, protein synthesis and visual cortex development

Loss-of-function mutations in the X-linked CDKL5 gene lead to a severe neurodevelopmental disorder characterized by early-onset epilepsy, known as CDKL5 Deficiency Disorder (CDD). Despite its clinical significance, the physiological substrates of the serine/threonine kinase CDKL5 and its roles in neuronal development remain poorly understood. To address this, we performed quantitative phosphoproteomics analysis in Cdkl5 knockout (KO) mouse brains, identifying 22 CDKL5 substrates involved in diverse cellular functions. Among these, we focused on the neuronal RNA-binding proteins (nELAVLs) ELAVL2, ELAVL3, and ELAVL4, as these represented the only evolutionarily conserved phosphorylation and are known regulators of neuronal differentiation. Through kinase assays and individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP), we found that CDKL5 phosphorylates S119/131 in ELAVL2/3/4, promoting their cytoplasmic localization and enhancing their binding to target mRNAs at 3UTRs. Loss of CDKL5 activity in neurons caused reduced new protein synthesis, as measured by puromycin incorporation; this phenotype was rescued by knockdown of the nELAVL inhibitor long non-coding RNA, RNY3, revealing an essential function of CDKL5 in enhancing protein synthesis via nELAVL phosphorylation. To investigate the in vivo functions of nELAVL phosphorylations, we generated Elavl2/3/4 phosphomutant mice and found that collectively nELAVL phosphorylations are required for viability. Proteomic and transcriptomic analyses of Elavl2/3 homozygous phosphomutants, which exhibited sub-viability, revealed compensatory upregulation of ELAVL4 and synaptic proteins. Functionally, in Elavl2/3/4 triple heterozygous mice Neuropixels recordings in the primary visual cortex showed deficits in receptive field properties and orientation tuning, revealing the role of nELAVL phosphorylation for accurate cortical circuit formation. Our study uncovers a crucial role for CDKL5 in regulating nELAVL-mediated protein synthesis and the development of cortical circuits.

neuroscience↗

bioRxiv2025

Tracking gene expression of single mitochondria in live neurons using nanotweezers

Neurons are highly polarised cells that depend on mitochondria for energy and signalling homeostasis. Importantly, energy and signalling requirements vary considerably across individual neurons both spatially and temporally. Therefore, to fully understand neuronal mitochondria, methods are needed to analyse mitochondria in live cells over time. The nanotweezer, a minimally invasive single-cell sampling technique, enables precise extraction a individual mitochondria from defined subcellular locations. Here, we combine single-mitochondrial extraction from live neurons with mitochondrial gene expression tracking and mtDNA profiling. By tracking mitochondrial gene expression in the same neurons over time, we reveal a downregulation of mitochondrial genes MT-ND1 and MT-ATP6 following exposure to -synuclein aggregates, independent of the proximity of the aggregates to the sampled mitochondria. Our approach provides precise, dynamic measurements of mitochondrial composition and gene expression in vivo at single-organelle resolution, enabling mechanistic studies of neuronal mitochondrial heterogeneity and its perturbation in models of neurodegeneration.

neuroscience↗

bioRxiv2024

CDKL5's role in microtubule-based transport and cognitive function

Cyclin-dependent kinase like 5 (CDKL5) is a serine-threonine kinase highly enriched in mammalian neurons. CDKL5 is located on the X-chromosome and its loss-of-function leads to a severe neurodevelopmental disorder called CDKL5 deficiency disorder (CDD). CDKL5 phosphorylates microtubule-associated protein MAP1S and regulates its binding to microtubules. How MAP1S phosphorylation affects microtubule function is not well understood. To address this question, we generated MAP1S phosphomutant mice, in which the CDKL5 phosphorylation sites S786 and S812 are mutated to Alanine (MAP1S S786/812A or MAP1S SA). Using a microtubule co-sedimentation assay, we showed that dynein binding to microtubules is severely reduced in CDKL5 knockout (KO) and MAP1S SA brains. Time-lapse imaging in primary neurons showed impaired dynein motility in both Cdkl5 KO and MAP1S SA. Dynein-driven cargo transport was affected in mutant neuron dendrites, including the delivery of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. We next studied tubulin tyrosination in Cdkl5 KO and MAP1S SA neurons and found that both mutants had a reduced tubulin tyrosination when compared to WT neurons. Since dynein-dynactin has a higher affinity for tyrosinated microtubules, we hypothesized that reduced tyrosination in MAP1S phosphomutant mice could be the mechanistic cause of impaired dynein motility. In support of this, we show that upon expression of tubulin tyrosine kinase TTL, we rescued dynein motility defects in MAP1S phosphomutant neurons. Hippocampal neurons derived from MAP1S SA mice revealed a significant reduction in spine density and synapses, and altered spine morphology. Finally, behavioral phenotyping of MAP1S phosphomutant mice showed increased anxiety, impaired motor performance, social and memory deficits, mirroring to some extent the clinical manifestations present in CDD patients. Our results reveal MAP1S phosphorylation to be an important contributor to dynein-mediated transport and synapse formation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/610038v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@12e50a2org.highwire.dtl.DTLVardef@1aff4b4org.highwire.dtl.DTLVardef@ea7b37org.highwire.dtl.DTLVardef@3f7826_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

Epilepsy-linked kinase CDKL5 phosphorylates voltage-gated calcium channel Cav2.3, altering inactivation kinetics and neuronal excitability

Developmental and epileptic encephalopathies (DEEs) are a group of rare childhood disorders characterized by severe epilepsy and cognitive deficits. Numerous DEE genes have been discovered thanks to advances in genomic diagnosis, yet putative molecular links between these disorders are unknown. CDKL5 deficiency disorder (CDD, DEE2), one of the most common genetic epilepsies, is caused by loss-of-function mutations in the brain-enriched kinase CDKL5. To elucidate CDKL5 function, we looked for CDKL5 substrates using a SILAC-based phosphoproteomic screen. We identified the voltage-gated Ca2+ channel Cav2.3 (encoded by CACNA1E) as a novel physiological target of CDKL5 in mice and humans. Recombinant channel electrophysiology and interdisciplinary characterization of Cav2.3 phosphomutant mice revealed that loss of Cav2.3 phosphorylation leads to channel gain-of-function via slower inactivation and enhanced cholinergic stimulation, resulting in increased neuronal excitability. Our results thus show that CDD is partly a channelopathy. The properties of unphosphorylated Cav2.3 closely resemble those described for CACNA1E gain-of-function mutations causing DEE69, a disorder sharing clinical features with CDD. We show that these two single-gene diseases are mechanistically related and could be ameliorated with Cav2.3 inhibitors.

neuroscience↗

bioRxiv2022

NDR1/2 kinases regulate membrane trafficking, enable efficient autophagy and prevent neurodegeneration

Autophagy is essential for neuronal development and its deregulation contributes to neurodegenerative diseases. NDR1 and NDR2 are highly conserved kinases implicated in neuronal development, mitochondrial health and autophagy, but how they affect mammalian brain development in vivo is not known. Using single and double Ndr1/2 knockout mouse models we show that, dual, but not individual loss of Ndr1/2 in neurons causes neurodegeneration during brain development, but also in adult mice. Proteomic and phosphoproteomic comparisons between Ndr1/2 knockout and control brains revealed novel kinase substrates and indicated that endocytosis is significantly affected in the absence of NDR1/2. We validated the endocytic protein, Raph1/Lpd1 as a novel NDR1/2 substrate and showed that both NDR1/2 and Raph1 are critical for endocytosis and membrane recycling. In NDR1/2 knockout brains, we observed prominent accumulation of transferrin receptor, p62 and ubiquitinated proteins, indicative of a major impairment of protein homeostasis. Furthermore, the levels of LC3-positive autophagosomes were reduced in knockout neurons, implying that reduced autophagy efficiency mediates p62 accumulation and neurotoxicity. Mechanistically, pronounced mislocalisation of the transmembrane autophagy protein ATG9A at the neuronal periphery, impaired axonal ATG9A trafficking and increased ATG9A surface levels further confirm defects in membrane trafficking and could underlie the impairment in autophagy. We provide novel insight into the roles of NDR1/2 kinases in maintaining neuronal health. HighlightsO_LIDual neuronal Ndr1 and Ndr2 knockout during development or in adult mice causes neurodegeneration. C_LIO_LIPhosphoproteomics comparison of Ndr1/2 knockouts with control littermates shows endocytosis and membrane trafficking to be affected and reveals novel substrates. C_LIO_LIRaph1/Lamellipodin is a novel NDR1/2 substrate that is required for TfR endocytosis. C_LIO_LINdr1/2 knockout brains exhibit a severe defect in ubiquitinated protein clearance and reduced autophagy. C_LIO_LINDR1/2 and Raph1 are required for the trafficking of the only transmembrane autophagy protein, ATG9A. C_LI

neuroscience↗