Biology subjects

Ultanir, S. K.

Publications and source records attributed to Ultanir, S. K..

11 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↗

bioRxiv2026

The phosphoproteomic landscape of the neurological manifestations in tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is a rare disease caused by mutations in TSC1 and TSC2, resulting in activation of mechanistic target of rapamycin complex 1 (mTORC1). Neurological manifestations in TSC patients include epilepsy, autism and intellectual disability. Two types of brain lesions, cortical tubers and subependymal giant cell astrocytomas (SEGAs), cause the majority of neurological manifestations in TSC. We have limited understanding of the molecular changes that occur in tubers and SEGAs and how these contribute to disease pathogenesis. To investigate this, we performed proteomic and phosphoproteomic analysis of TSC patient tuber and SEGA tissue. Tubers showed evidence of alterations in mitochondrial respiration, cytoskeleton organisation and neuronal function. However, we were unable to detect mTORC1 activation in tubers, likely due to the small number of cells with complete inactivation of TSC1 or TSC2. By contrast, SEGAs showed evidence of strong mTORC1 activation and large-scale changes in the proteome and phosphoproteome. SEGAs exhibited increased expression of ribosomal proteins and activation of a neuroinflammatory response. Phosphoproteomics identified 6060 phosphosites within 2154 proteins increased in SEGAs. Phosphorylation of multiple proteins involved in RNA-metabolism, including mRNA splicing, were increased in SEGAs. Consistent with this, we found evidence of extensive alterations in mRNA transcript splicing in SEGA tissue. These data greatly expand the repertoire of known mTORC1 target proteins in the human brain and reveal large-scale mis-regulation of mRNA splicing in SEGAs in TSC.

neuroscience↗

bioRxiv2024

Presynaptic filopodia form kinapses and modulate membrane mechanics for synchronous neurotransmission and seizure generation.

The structural stability of synapses directly contrasts with their functional plasticity. This conceptual dichotomy is explained by the assumption that all synaptic plasticity is generated via either electrical and/or biochemical signaling. Here, we challenge this dogma by revealing an activity-dependent presynaptic response that is physical in nature. We show that dynamic filopodia emerge during action potential discharge and transiently deform synaptic boutons to enhance connectivity. Filopodia generation requires neuronal activity, calcium and actin, and occurs in intact brain circuits and human brain. Mechanistically, their extension preserves synchronous neurotransmitter release by increasing presynaptic membrane tension. However, filopodia generation becomes maladaptive during dysregulated brain activity, exacerbating seizures in vivo. Therefore, we provide direct evidence that presynaptic mechanical forces determine the extent and timing of synaptic signals.

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↗

bioRxiv2024

Discovery and Characterization of a Chemical Probe for Cyclin-Dependent Kinase-Like 2

Acylaminoindazole-based inhibitors of CDKL2 were identified via analyses of cell-free binding and selectivity data. Compound 9 was selected as a CDKL2 chemical probe based on its potent inhibition of CDKL2 enzymatic activity, engagement of CDKL2 in cells, and excellent kinome-wide selectivity, especially when used in cells. Compound 16 was designed as a negative control to be used alongside compound 9 in experiments to interrogate CDKL2-mediated biology. A solved co-crystal structure of compound 9 bound to CDKL2 highlighted key interactions it makes within its ATP-binding site. Inhibition of downstream phosphorylation of EB2, a CDKL2 substrate, in rat primary neurons provided evidence that engagement of CDKL2 by compound 9 in cells resulted in inhibition of its activity. When used at relevant concentrations, compound 9 does not impact the viability of rat primary neurons or certain breast cancer cells nor elicit consistent changes in the expression of proteins involved in epithelial-mesenchymal transition.

cell biology↗

bioRxiv2023

LRRK2 kinase dependent and independent function on endolysosomal repair promotes macrophage cell death

LRRK2 is commonly mutated in Parkinsons disease and has cell type-specific mechanisms of activation and function. In macrophages, LRRK2 is associated with lysosomes and is activated following lysosomal damage. However, effects of pathogenic LRRK2-G2019S in macrophages are unknown. Here, using primary mouse and human iPSC-derived macrophage (iPSDM) models of LRRK2-G2019S, we defined the substrates of LRRK2 after lysosomal damage. Using phosphoproteomics we found that LRRK2-G2019S and wild-type macrophages showed similar levels of Rab phosphorylation after lysosomal damage, with the exceptions of Rab12 and Rab35, which were increased and decreased, respectively, in LRRK2-G2019S. LRRK2-G2019S macrophages showed a LRRK2 kinase activity-independent deficit in lysosomal membrane repair which resulted in more cell death and increased apoptosis. Importantly, we recapitulated this phenotype in iPSDM from patients carrying the G2019S mutation, but not in isogenic control iPSDM. Altogether, we define here the signaling downstream of G2019S in macrophages and identify susceptibility to cell death after lysosomal damage as an important phenotype of this mutation.

cell biology↗

bioRxiv2023

A Potent and Selective CDKL5/GSK3 Chemical Probe is Neuroprotective

Despite mediating several essential processes in the brain, including during development, cyclin-dependent kinase-like 5 (CDKL5) remains a poorly characterized human protein kinase. Accordingly, its substrates, functions, and regulatory mechanisms have not been fully described. We realized that availability of a potent and selective small molecule probe targeting CDKL5 could enable illumination of its roles in normal development as well as in diseases where it has become aberrant due to mutation. We prepared analogs of AT-7519, a known inhibitor of several cyclin dependent and cyclin-dependent kinase-like kinases that has been advanced into Phase II clinical trials. We identified analog 2 as a highly potent and cell-active chemical probe for CDKL5/GSK3 (glycogen synthase kinase 3). Evaluation of its kinome-wide selectivity confirmed that analog 2 demonstrates excellent selectivity and only retains GSK3/{beta} affinity. As confirmation that our chemical probe is a high-quality tool to use in directed biological studies, we demonstrated inhibition of downstream CDKL5 and GSK3/{beta} signaling and solved a co-crystal structure of analog 2 bound to CDKL5. A structurally similar analog (4) proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3/{beta}. Finally, we used our chemical probe pair (2 and 4) to demonstrate that inhibition of CDKL5 and/or GSK3/{beta} promotes the survival of human motor neurons exposed to endoplasmic reticulum (ER) stress. We have demonstrated a neuroprotective phenotype elicited by our chemical probe pair and exemplified the utility of our compounds to characterize the role of CDKL5/GSK3 in neurons and beyond.

cell biology↗

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

Phosphorylation of the novel mTOR substrate Unkempt regulates cellular morphogenesis

Mechanistic target of rapamycin (mTOR) is a protein kinase that integrates multiple inputs to regulate anabolic cellular processes. mTOR complex I (mTORC1) has key functions in growth control, autophagy and metabolism. Much less is known about the signalling components that act downstream of mTORC1 that regulate cellular morphology, a vital determinant of cellular function. Here we show that the RNA-binding protein Unkempt, a key regulator of cellular morphogenesis, is a novel substrate mTORC1. We find that Unkempt phosphorylation is regulated by nutrient levels and growth factors via mTORC1. Furthermore, Unkempt physically interacts with and is directly phosphorylated by mTORC1 through binding to the regulatory-associated protein of mTOR, Raptor. Phosphorylation of Unkempt, which we find is mTORC1-dependent in cultured mammalian cell lines as well as in primary tissues, occurs largely within the highly serine-rich intrinsically disordered region of Unkempt. Importantly, mutation analysis of this region indicates that phosphorylation inhibits the ability of Unkempt to induce a bipolar morphology. Our findings reveal a novel molecular link between mTORC1 signalling and cellular morphogenesis.

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↗

bioRxiv2021

The master energy homeostasis regulator PGC-1alpha couples transcriptional co-activation and mRNA nuclear export

PGC-1 plays a central role in maintaining the mitochondrial and energy metabolism homeostasis, linking external stimuli to the transcriptional co-activation of genes involved in adaptive and age-related pathways. The carboxyl-terminus encodes a serine/arginine-rich (RS) region and a putative RNA recognition motif, however potential RNA-processing role(s) have remained elusive for the past 20 years. Here, we show that the RS domain of human PGC-1 directly interacts with RNA and the nuclear RNA export factor NXF1. Inducible depletion of endogenous PGC-1 and expression of RNAi-resistant RS-deleted PGC-1 further demonstrate that the RNA-binding activity is required for nuclear export of co-activated transcripts and mitochondrial homeostasis. Moreover, a quantitative proteomics approach confirmed PGC-1-dependent RNA transport and mitochondrial-related functions, identifying also novel mRNA nuclear export targets in age-related telomere maintenance. Discovering a novel function for a major cellular homeostasis regulator provides new directions to further elucidate the roles of PGC-1 in gene expression, metabolic disorders, ageing and neurodegenerative diseases.

molecular biology↗