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.

TDP-43 causes neurotoxicity and cytoskeletal dysfunction in primary cortical neurons.

TDP-43-mediated proteinopathy is a key factor in the pathology of amyotrophic lateral sclerosis (ALS). A potential underlying mechanism is dysregulation of the cytoskeleton. Here we investigate the effects of expressing TDP-43 wild-type and M337V and Q331K mutant isoforms on cytoskeletal integrity and function, using rat cortical neurons in vitro. We find that TDP-43 protein becomes mislocalised in axons over 24-72 hours in culture, with protein aggregation occurring at later timepoints (144 hours). Quantitation of cell viability showed toxicity of both wild-type and mutant constructs which increased over time, especially of the Q331K mutant isoform. Analysis of the effects of TDP-43 on axonal integrity showed that TDP-43-transfected neurons had shorter axons than control cells, and that growth cone sizes were smaller. Axonal transport dynamics were also impaired by transfection with TDP-43 constructs. Taken together these data show that TDP-43 mislocalisation into axons precedes cell death in cortical neurons, and that cytoskeletal structure and function is impaired by expression of either TDP-43 wild-type or mutant constructs in vitro. These data suggest that dysregulation of cytoskeletal and neuronal integrity is an important mechanism for TDP-43-mediated proteinopathy.

C9orf72 poly GA RAN-translated protein plays a key role in amyotrophic lateral sclerosis via aggregation and toxicity.

An intronic GGGGCC (G4C2) hexanucleotide repeat expansion inC9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of G4C2 RNA can result in five different dipeptide repeat proteins (DPR: poly GA, poly GP, poly GR, poly PA, and poly PR), which aggregate into neuronal cytoplasmic and nuclear inclusions in affected patients, however their contribution to disease pathogenesis remains controversial. We show that among the DPR proteins, expression of poly GA in a cell culture model activates programmed cell death and TDP-43 cleavage in a dose-dependent manner. Dual expression of poly GA together with other DPRs revealed that poly GP and poly PA are sequestered by poly GA, whereas poly GR and poly PR are rarely co-localised with poly GA. Dual expression of poly GA and poly PA ameliorated poly GA toxicity by inhibiting poly GA aggregation both in vitro and in vivo in the chick embryonic spinal cord. Expression of alternative codon-derived DPRs in chick embryonic spinal cord confirmed in vitro data, revealing that each of the dipeptides caused toxicity, with poly GA being the most toxic. Further, in vivo expression of G4C2 repeats of varying length caused apoptotic cell death, but failed to generate DPRs. Together, these data demonstrate that C9-related toxicity can be mediated by either RNA or DPRs. Moreover, our findings provide evidence that poly GA is a key mediator of cytotoxicity and that cross-talk between DPR proteins likely modifies their pathogenic status in C9ALS/FTD.

Tar DNA-binding protein-43 (TDP-43) regulates axon growth in vitro and in vivo.

Intracellular inclusions of the TAR-DNA binding protein 43 (TDP-43) have been reported in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD-TDP). Rare mutations in TARDBP have been linked to both ALS and FTD-TDP suggesting that TDP-43 dysfunction is mechanistic in causing disease. TDP-43 is a predominantly nuclear protein with roles in regulating RNA transcription, splicing, stability and transport. In ALS, TDP-43 aberrantly accumulates in the cytoplasm of motor neurons where it forms aggregates. However it has until recently been unclear whether the toxic effects of TDP-43 involve recruitment to motor axons, and what effects this might have on axonal growth and integrity. Here we use chick embryonic motor neurons, in vivo and in vitro, to model the acute effects of TDP-43. We show that wild-type and two TDP-43 mutant proteins cause toxicity in chick embryonic motor neurons in vivo. Moreover, TDP-43 is increasingly mislocalised to axons over time in vivo, axon growth to peripheral targets is truncated, and expression of neurofilament-associated antigen is reduced relative to control motor neurons. In primary spinal motor neurons in vitro, a progressive translocation of TDP-43 to the cytoplasm occurs over time, similar to that observed in vivo. This coincides with the appearance of cytoplasmic aggregates, a reduction in the axonal length, and cellular toxicity, which was most striking for neurons expressing TDP-43 mutant forms. These observations suggest that the capacity of spinal motor neurons to produce and maintain an axon is compromised by dysregulation of TDP-43 and that the disruption of cytoskeletal integrity may play a role in the pathogenesis of ALS and FTD-TDP.

Axon guidance in the developing ocular motor system and Duane retraction syndrome depends on Semaphorin signaling via alpha2-chimaerin.

Eye movements depend on correct patterns of connectivity between cranial motor axons and the extraocular muscles. Despite the clinical importance of the ocular motor system, little is known of the molecular mechanisms underlying its development. We have recently shown that mutations in the Chimaerin-1 gene encoding the signaling protein α2-chimaerin (α2-chn) perturb axon guidance in the ocular motor system and lead to the human eye movement disorder, Duane retraction syndrome (DRS). The axon guidance cues that lie upstream of α2-chn are unknown; here we identify candidates to be the Semaphorins (Sema) 3A and 3C, acting via the PlexinA receptors. Sema3A/C are expressed in and around the developing extraocular muscles and cause growth cone collapse of oculomotor neurons in vitro. Furthermore, RNAi knockdown of α2-chn or PlexinAs in oculomotor neurons abrogates Sema3A/C-dependent growth cone collapse. In vivo knockdown of endogenous PlexinAs or α2-chn function results in stereotypical oculomotor axon guidance defects, which are reminiscent of DRS, whereas expression of α2-chn gain-of-function constructs can rescue PlexinA loss of function. These data suggest that α2-chn mediates Sema3-PlexinA repellent signaling. We further show that α2-chn is required for oculomotor neurons to respond to CXCL12 and hepatocyte growth factor (HGF), which are growth promoting and chemoattractant during oculomotor axon guidance. α2-chn is therefore a potential integrator of different types of guidance information to orchestrate ocular motor pathfinding. DRS phenotypes can result from incorrect regulation of this signaling pathway.