Mutations in the postsynaptic density signaling hub TNIK disrupt PSD signaling in human models of neurodevelopmental disorders.

A large number of synaptic proteins have been recurrently associated with complex brain disorders. One of these proteins, the Traf and Nck interacting kinase (TNIK), is a postsynaptic density (PSD) signaling hub, with many variants reported in neurodevelopmental disorder (NDD) and psychiatric disease. While rodent models of TNIK dysfunction have abnormal spontaneous synaptic activity and cognitive impairment, the role of mutations found in patients with TNIK protein deficiency and TNIK protein kinase activity during early stages of neuronal and synapse development has not been characterized. Here, using hiPSC-derived excitatory neurons, we show that TNIK mutations dysregulate neuronal activity in human immature synapses. Moreover, the lack of TNIK protein kinase activity impairs MAPK signaling and protein phosphorylation in structural components of the PSD. We show that the TNIK interactome is enriched in NDD risk factors and TNIK lack of function disrupts signaling networks and protein interactors associated with NDD that only partially overlap to mature mouse synapses, suggesting a differential role of TNIK in immature synapsis in NDD.

Cell type-specific expression, regulation and compensation of CDKL5 activity in mouse brain.

CDKL5 is a brain-enriched serine/threonine kinase, associated with a profound developmental and epileptic encephalopathy called CDKL5 deficiency disorder (CDD). To design targeted therapies for CDD, it is essential to determine where CDKL5 is expressed and is active in the brain and test if compensatory mechanisms exist at cellular level. We generated conditional Cdkl5 knockout mice in excitatory neurons, inhibitory neurons and astrocytes. To assess CDKL5 activity, we utilized a phosphospecific antibody for phosphorylated EB2, a well-known substrate of CDKL5. We found that CDKL5 and EB2 pS222 were prominent in excitatory and inhibitory neurons but were not detected in astrocytes. We observed that approximately 15-20% of EB2 pS222 remained in Cdkl5 knockout brains and primary neurons. Surprisingly, the remaining phosphorylation was modulated by NMDA and PP1/PP2A in neuronal CDKL5 knockout cultures, indicating the presence of a compensating kinase. Using a screen of candidate kinases with highest homology to the CDKL5 kinase domain, we found that CDKL2 and ICK can phosphorylate EB2 S222 in HEK293T cells and in primary neurons. We then generated Cdkl5/Cdkl2 dual knockout mice to directly test if CDKL2 phosphorylates EB2 in vivo and found that CDKL2 phosphorylates CDKL5 substrates in the brain. This study is the first indication that CDKL2 could potentially replace CDKL5 functions in the brain, alluding to novel therapeutic possibilities.

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 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.

The master energy homeostasis regulator PGC-1α exhibits an mRNA nuclear export function.

PGC-1α plays a central role in maintaining mitochondrial and energy metabolism homeostasis, linking external stimuli to transcriptional co-activation of genes involved in adaptive and age-related pathways. The carboxyl-terminus encodes a serine/arginine-rich (RS) region and an RNA recognition motif, however the RNA-processing function(s) were poorly investigated over the past 20 years. Here, we show that the RS domain of human PGC-1α directly interacts with RNA and the nuclear RNA export receptor NXF1. Inducible depletion of PGC-1α and expression of RNAi-resistant RS-deleted PGC-1α further demonstrate that its RNA/NXF1-binding activity is required for the nuclear export of some canonical mitochondrial-related mRNAs and mitochondrial homeostasis. Genome-wide investigations reveal that the nuclear export function is not strictly linked to promoter-binding, identifying in turn novel regulatory targets of PGC-1α in non-homologous end-joining and nucleocytoplasmic transport. These findings provide new directions to further elucidate the roles of PGC-1α in gene expression, metabolic disorders, aging and neurodegeneration.

Loss of NDR1/2 kinases impairs endomembrane trafficking and autophagy leading to 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 only dual loss of Ndr1/2 in neurons causes neurodegeneration. This phenotype was present when NDR kinases were deleted both during embryonic development, as well as 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.

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. For example, mTOR complex 1 (mTORC1) has key functions in growth control, autophagy, and metabolism. However, much less is known about the signaling components that act downstream of mTORC1 to regulate cellular morphogenesis. Here, we show that the RNA-binding protein Unkempt, a key regulator of cellular morphogenesis, is a novel substrate of mTORC1. We show that Unkempt phosphorylation is regulated by nutrient levels and growth factors via mTORC1. To analyze Unkempt phosphorylation, we immunoprecipitated Unkempt from cells in the presence or the absence of the mTORC1 inhibitor rapamycin and used mass spectrometry to identify mTORC1-dependent phosphorylated residues. This analysis showed that mTORC1-dependent phosphorylation is concentrated in a serine-rich intrinsically disordered region in the C-terminal half of Unkempt. We also found that Unkempt physically interacts with and is directly phosphorylated by mTORC1 through binding to the regulatory-associated protein of mTOR, Raptor. Furthermore, analysis in the developing brain of mice lacking TSC1 expression showed that phosphorylation of Unkempt is mTORC1 dependent in vivo. Finally, mutation analysis of key serine/threonine residues in the serine-rich region indicates that phosphorylation inhibits the ability of Unkempt to induce a bipolar morphology. Phosphorylation within this serine-rich region thus profoundly affects the ability of Unkempt to regulate cellular morphogenesis. Taken together, our findings reveal a novel molecular link between mTORC1 signaling and cellular morphogenesis.

The zinc finger/RING domain protein Unkempt regulates cognitive flexibility.

Correct orchestration of nervous system development is a profound challenge that involves coordination of complex molecular and cellular processes. Mechanistic target of rapamycin (mTOR) signaling is a key regulator of nervous system development and synaptic function. The mTOR kinase is a hub for sensing inputs including growth factor signaling, nutrients and energy levels. Activation of mTOR signaling causes diseases with severe neurological manifestations, such as tuberous sclerosis complex and focal cortical dysplasia. However, the molecular mechanisms by which mTOR signaling regulates nervous system development and function are poorly understood. Unkempt is a conserved zinc finger/RING domain protein that regulates neurogenesis downstream of mTOR signaling in Drosophila. Unkempt also directly interacts with the mTOR complex I component Raptor. Here we describe the generation and characterisation of mice with a conditional knockout of Unkempt (UnkcKO) in the nervous system. Loss of Unkempt reduces Raptor protein levels in the embryonic nervous system but does not affect downstream mTORC1 targets. We also show that nervous system development occurs normally in UnkcKO mice. However, we find that Unkempt is expressed in the adult cerebellum and hippocampus and behavioural analyses show that UnkcKO mice have improved memory formation and cognitive flexibility to re-learn. Further understanding of the role of Unkempt in the nervous system will provide novel mechanistic insight into the role of mTOR signaling in learning and memory.

Proteomic and cellular localisation studies suggest non-tight junction cytoplasmic and nuclear roles for occludin in astrocytes.

Occludin is a component of tight junctions, which are essential structural components of the blood-brain barrier. However, occludin is expressed in cells without tight junctions, implying additional functions. We determined the expression and localisation of occludin in astrocytes in cell culture and in human brain tissue, and sought novel binding partners using a proteomic approach. Expression was investigated by immunocytochemistry and immunoblotting in the 1321N1 astrocytoma cell line and ScienCell human primary astrocytes, and by immunohistochemistry in human autopsy brain tissue. Recombinant N- and C-terminal occludin was used to pull-down proteins from 1321N1 cell lysates and protein-binding partners identified by mass spectrometry analysis. Occludin was expressed in both the cytoplasm and nucleus of astrocytes in vitro and in vivo. Mass spectrometry identified binding to nuclear and cytoplasmic proteins, particularly those related to RNA metabolism and nuclear function. Occludin is expressed in several subcellular compartments of brain cell-types that do not form tight junctions and the expression patterns in cell culture reflect those in human brain tissue, indicating they are suitable model systems. Proteomic analysis suggests that occludin has novel functions in neuroepithelial cells that are unrelated to tight junction formation. Further research will establish the roles of these functions in both cellular physiology and in disease states.

Arginine methylation and citrullination of splicing factor proline- and glutamine-rich (SFPQ/PSF) regulates its association with mRNA.

Splicing factor proline- and glutamine-rich (SFPQ) also commonly known as polypyrimidine tract-binding protein-associated-splicing factor (PSF) and its binding partner non-POU domain-containing octamer-binding protein (NONO/p54nrb), are highly abundant, multifunctional nuclear proteins. However, the exact role of this complex is yet to be determined. Following purification of the endogeneous SFPQ/NONO complex, mass spectrometry analysis identified a wide range of interacting proteins, including those involved in RNA processing, RNA splicing, and transcriptional regulation, consistent with a multifunctional role for SFPQ/NONO. In addition, we have identified several sites of arginine methylation in SFPQ/PSF using mass spectrometry and found that several arginines in the N-terminal domain of SFPQ/PSF are asymmetrically dimethylated. Furthermore, we find that the protein arginine N-methyltransferase, PRMT1, catalyzes this methylation in vitro and that this is antagonized by citrullination of SFPQ. Arginine methylation and citrullination of SFPQ/PSF does not affect complex formation with NONO. However, arginine methylation was shown to increase the association with mRNA in mRNP complexes in mammalian cells. Finally we show that the biochemical properties of the endogenous complex from cell lysates are significantly influenced by the ionic strength during purification. At low ionic strength, the SFPQ/NONO complex forms large heterogeneous protein assemblies or aggregates, preventing the purification of the SFPQ/NONO complex. The ability of the SFPQ/NONO complex to form varying protein assemblies, in conjunction with the effect of post-translational modifications of SFPQ modulating mRNA binding, suggests key roles affecting mRNP dynamics within the cell.

Invited review: decoding the pathophysiological mechanisms that underlie RNA dysregulation in neurodegenerative disorders: a review of the current state of the art.

Altered RNA metabolism is a key pathophysiological component causing several neurodegenerative diseases. Genetic mutations causing neurodegeneration occur in coding and noncoding regions of seemingly unrelated genes whose products do not always contribute to the gene expression process. Several pathogenic mechanisms may coexist within a single neuronal cell, including RNA/protein toxic gain-of-function and/or protein loss-of-function. Genetic mutations that cause neurodegenerative disorders disrupt healthy gene expression at diverse levels, from chromatin remodelling, transcription, splicing, through to axonal transport and repeat-associated non-ATG (RAN) translation. We address neurodegeneration in repeat expansion disorders [Huntington's disease, spinocerebellar ataxias, C9ORF72-related amyotrophic lateral sclerosis (ALS)] and in diseases caused by deletions or point mutations (spinal muscular atrophy, most subtypes of familial ALS). Some neurodegenerative disorders exhibit broad dysregulation of gene expression with the synthesis of hundreds to thousands of abnormal messenger RNA (mRNA) molecules. However, the number and identity of aberrant mRNAs that are translated into proteins - and how these lead to neurodegeneration - remain unknown. The field of RNA biology research faces the challenge of identifying pathophysiological events of dysregulated gene expression. In conclusion, we discuss current research limitations and future directions to improve our characterization of pathological mechanisms that trigger disease onset and progression.