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Mutations in MAPT (microtubule-associated protein tau) cause frontotemporal dementia (FTD). MAPT mutations are associated with abnormal tau phosphorylation levels and accumulation of misfolded tau protein that can propagate between neurons ultimately leading to cell death (tauopathy). Recently, a p.A152T tau variant was identified as a risk factor for FTD, Alzheimer's disease, and synucleinopathies. Here we used induced pluripotent stem cells (iPSC) from a patient carrying this p.A152T variant to create a robust, functional cellular assay system for probing pathophysiological tau accumulation and phosphorylation. Using stably transduced iPSC-derived neural progenitor cells engineered to enable inducible expression of the pro-neural transcription factor Neurogenin 2 (Ngn2), we generated disease-relevant, cortical-like glutamatergic neurons in a scalable, high-throughput screening compatible format. Utilizing automated confocal microscopy, and an advanced image-processing pipeline optimized for analysis of morphologically complex human neuronal cultures, we report quantitative, subcellular localization-specific effects of multiple kinase inhibitors on tau, including ones under clinical investigation not previously reported to affect tau phosphorylation. These results demonstrate the potential for using patient iPSC-derived ex vivo models of tauopathy as genetically accurate, disease-relevant systems to probe tau biochemistry and support the discovery of novel therapeutics for tauopathies.

Tuberous sclerosis is a neurological disorder associated with seizures and cognitive dysfunction. Alcino Silva and his colleagues find that rapamycin, an inhibitor of the mTOR signaling pathway, can ameliorate cognitive deficits in a mouse model of the disease. Tuberous sclerosis is a single-gene disorder caused by heterozygous mutations in the TSC1 (9q34) or TSC2 (16p13.3) gene 1 , 2 and is frequently associated with mental retardation, autism and epilepsy. Even individuals with tuberous sclerosis and a normal intelligence quotient (approximately 50%) 3 , 4 , 5 are commonly affected with specific neuropsychological problems, including long-term and working memory deficits 6 , 7 . Here we report that mice with a heterozygous, inactivating mutation in the Tsc2 gene ( Tsc2 +/− mice) 8 show deficits in learning and memory. Cognitive deficits in Tsc2 +/− mice emerged in the absence of neuropathology and seizures, demonstrating that other disease mechanisms are involved 5 , 9 , 10 , 11 . We show that hyperactive hippocampal mammalian target of rapamycin (mTOR) signaling led to abnormal long-term potentiation in the CA1 region of the hippocampus and consequently to deficits in hippocampal-dependent learning. These deficits included impairments in two spatial learning tasks and in contextual discrimination. Notably, we show that a brief treatment with the mTOR inhibitor rapamycin in adult mice rescues not only the synaptic plasticity, but also the behavioral deficits in this animal model of tuberous sclerosis. The results presented here reveal a biological basis for some of the cognitive deficits associated with tuberous sclerosis, and they show that treatment with mTOR antagonists ameliorates cognitive dysfunction in a mouse model of this disorder.

Neurofibromatosis type 2 (NF2) is an inherited disorder characterized by bilateral vestibular schwannomas (VS) that arise from neoplastic Schwann cells (SCs). NF2-associated VSs are often accompanied by meningioma (MN), and the majority of NF2 patients show loss of the NF2 tumor suppressor. mTORC1 and mTORC2-specific serum/glucocorticoid-regulated kinase 1 (SGK1) are constitutively activated in MN with loss of NF2 . In a recent high-throughput kinome screen in NF2 -null human arachnoidal and meningioma cells, we showed activation of EPH RTKs, c-KIT, and SFK members independent of mTORC1/2 activation. Subsequently, we demonstrated in vitro and in vivo efficacy of combination therapy with the dual mTORC1/2 inhibitor AZD2014 and the multi-kinase inhibitor dasatinib. For these reasons, we investigated activated mTORC1/2 and EPH receptor-mediated signaling in sporadic and NF2-associated VS. Using primary human VS cells and a mouse allograft model of schwannoma, we evaluated the dual mTORC1/2 inhibitor AZD2014 and the tyrosine kinase inhibitor dasatinib as monotherapies and in combination. Escalating dose-response experiments on primary VS cells grown from 15 human tumors show that combination therapy with AZD2014 and dasatinib is more effective at reducing metabolic activity than either drug alone and exhibits a therapeutic effect at a physiologically reasonable concentration (~0.1 µM). In vivo , while AZD2014 and dasatinib each inhibit tumor growth alone, the effect of combination therapy exceeds that of either drug. Co-targeting the mTOR and EPH receptor pathways with these or similar compounds may constitute a novel therapeutic strategy for VS, a condition for which there is no FDA-approved pharmacotherapy.

Key Points Merlin (the neurofibromatosis 2 tumour suppressor protein) and the ERM proteins (ezrin, radixin and moesin) link membrane proteins with the cortical cytoskeleton. They are involved in various signalling pathways and have important functions in the PNS and CNS. All four proteins are members of the band 4.1 superfamily and have a FERM (4.1, ezrin, radixin, moesin) domain — a ∼300-amino-acid module at the amino terminus that is involved in localizing proteins to the plasma membrane and mediating interactions between the membrane and the cytoskeleton. Merlin is the protein that is mutated in neurofibromatosis 2 (NF2), which predisposes patients to schwannomas and meningiomas. Although merlin is lacking in tumours derived from Schwann cells and arachnoidal cells of NF2 patients, they still express the ERM proteins. It is unclear how the absence of merlin alone results in these tumours. ERM proteins are negatively regulated by an intramolecular interaction between their N and C termini. In the 'closed' state, their ligand-binding sites are masked. Cellular signals such as Rho-mediated signalling and phospholipids such as phosphatidylinositol-4,5-bisphosphate (PIP 2 ) can remove the intramolecular interaction and allow the proteins to take on an 'open', active state. Merlin is also conformationally regulated, although this process is less well understood. Merlin is expressed in both glia and neurons, and is found in cellular processes. The other ERM proteins are also expressed in the nervous system, but there is less information about their distribution. All three ERM proteins are expressed in the microvilli of Schwann cells, and are enriched in the nodal processes. Activation of the ERM proteins seems to be important in the formation of myelin and the nodes of Ranvier. Although the function of merlin in Schwann cells is unclear, it acts as a tumour suppressor protein and influences cell adhesion and the cytoskeleton. Radixin is enriched in the growth cones of many neurons and, together with moesin, seems to be important for their motility. ERM proteins interact with the axonal adhesion molecule L1, which has been implicated in the control of growth cone motility. Tuberous sclerosis complex is a neurocutaneous syndrome that is caused by mutations in the genes for hamartin or tuberin. Hamartin interacts with the ERM proteins and with neurofilament light polypeptide (NFL), indicating that it is important for cytoskeletal regulation. Together with another protein, Pam, the ERM proteins, hamartin and NFL are proposed to form a complex that influences neuronal development and synaptic plasticity. Merlin — the neurofibromatosis 2 (NF2) tumour suppressor protein — and members of the ezrin–radixin–moesin (ERM) family of proteins link membrane proteins with the cortical cytoskeleton and participate in various signalling pathways. They also carry out important functions in both the CNS and PNS. Schwann cells express the ERM proteins and merlin, but the absence of merlin alone results in NF2-related schwannomas, indicating that merlin has a distinct function in these cells. In neurons, the ERM proteins regulate key aspects of actin-dependent growth-cone motility. With emerging evidence of localized protein synthesis and proteolysis in growth cones, it remains a challenge to understand the role(s) of these proteins in growth cone guidance.

Background Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with frequent occurrence of epilepsy, autism spectrum disorder (ASD), intellectual disability (ID), and tumors in multiple organs. The aberrant activation of mTORC1 in TSC has led to treatment with mTORC1 inhibitor rapamycin as a lifelong therapy for tumors, but TSC-associated neurocognitive manifestations remain unaffected by rapamycin. Methods Here, we generated patient-specific, induced pluripotent stem cells (iPSCs) from a TSC patient with a heterozygous, germline, nonsense mutation in exon 15 of TSC1 and established an isogenic set of heterozygous (Het), null and corrected wildtype (Corr-WT) iPSCs using CRISPR/Cas9-mediated gene editing. We differentiated these iPSCs into neural progenitor cells (NPCs) and examined neurodevelopmental phenotypes, signaling and changes in gene expression by RNA-seq. Results Differentiated NPCs revealed enlarged cell size in TSC1-Het and Null NPCs, consistent with mTORC1 activation. TSC1-Het and Null NPCs also revealed enhanced proliferation and altered neurite outgrowth in a genotype-dependent manner, which was not reversed by rapamycin. Transcriptome analyses of TSC1-NPCs revealed differentially expressed genes that display a genotype-dependent linear response, i.e., genes upregulated/downregulated in Het were further increased/decreased in Null. In particular, genes linked to ASD, epilepsy, and ID were significantly upregulated or downregulated warranting further investigation. In TSC1-Het and Null NPCs, we also observed basal activation of ERK1/2, which was further activated upon rapamycin treatment. Rapamycin also increased MNK1/2-eIF4E signaling in TSC1-deficient NPCs. Conclusion MEK-ERK and MNK-eIF4E pathways regulate protein translation, and our results suggest that aberrant translation distinct in TSC1/2-deficient NPCs could play a role in neurodevelopmental defects. Our data showing upregulation of these signaling pathways by rapamycin support a strategy to combine a MEK or a MNK inhibitor with rapamycin that may be superior for TSC-associated CNS defects. Importantly, our generation of isogenic sets of NPCs from TSC patients provides a valuable platform for translatome and large-scale drug screening studies. Overall, our studies further support the notion that early developmental events such as NPC proliferation and initial process formation, such as neurite number and length that occur prior to neuronal differentiation, represent primary events in neurogenesis critical to disease pathogenesis of neurodevelopmental disorders such as ASD.

Neurofibromatosis 2 (NF2) is a rare tumor suppressor syndrome that manifests with multiple schwannomas and meningiomas. There are no effective drug therapies for these benign tumors and conventional therapies have limited efficacy. Various model systems have been created and several drug targets have been implicated in NF2-driven tumorigenesis based on known effects of the absence of merlin, the product of the NF2 gene. We tested priority compounds based on known biology with traditional dose-concentration studies in meningioma and schwann cell systems. Concurrently, we studied functional kinome and gene expression in these cells pre- and post-treatment to determine merlin deficient molecular phenotypes. Cell viability results showed that three agents (GSK2126458, Panobinostat, CUDC-907) had the greatest activity across schwannoma and meningioma cell systems, but merlin status did not significantly influence response. In vivo, drug effect was tumor specific with meningioma, but not schwannoma, showing response to GSK2126458 and Panobinostat. In culture, changes in both the transcriptome and kinome in response to treatment clustered predominantly based on tumor type. However, there were differences in both gene expression and functional kinome at baseline between meningioma and schwannoma cell systems that may form the basis for future selective therapies. This work has created an openly accessible resource (www.synapse.org/SynodosNF2) of fully characterized isogenic schwannoma and meningioma cell systems as well as a rich data source of kinome and transcriptome data from these assay systems before and after treatment that enables single and combination drug discovery based on molecular phenotype.

Background Tuberous sclerosis complex (TSC) is an inherited neurocutaneous disorder caused by mutations in the TSC1 or TSC2 genes, with patients often exhibiting neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD) and intellectual disability. Hamartin (TSC1) and tuberin (TSC2) proteins form a complex inhibiting mechanistic target of rapamycin complex 1 (mTORC1) signaling. Loss of TSC1 or TSC2 activates mTORC1 that, among several targets, controls protein synthesis by inhibiting translational repressor eIF4E-binding proteins. Using TSC1 patient-derived neural progenitor cells (NPCs), we recently reported early ND phenotypic changes, including increased cell proliferation and altered neurite outgrowth in TSC1 -null NPCs, which were unaffected by the mTORC1 inhibitor rapamycin. Methods Here, we used polysome profiling, which quantifies changes in mRNA abundance and translational efficiencies at a transcriptome-wide level, to compare CRISPR-edited TSC1 -null with CRISPR-corrected TSC1 -WT NPCs generated from one TSC donor (one clone/genotype). To assess the relevance of identified gene expression alterations, we performed polysome profiling in postmortem brains from ASD donors and age-matched controls. We further compared effects on translation of a subset of transcripts and rescue of early ND phenotypes in NPCs following inhibition of mTORC1 using the allosteric inhibitor rapamycin versus a third-generation bi-steric, mTORC1-selective inhibitor RMC-6272. Results Polysome profiling of NPCs revealed numerous TSC1-associated alterations in mRNA translation that were largely recapitulated in human ASD brains. Moreover, although rapamycin treatment partially reversed the TSC1-associated alterations in mRNA translation, most genes related to neural activity/synaptic regulation or ASD were rapamycin-insensitive. In contrast, treatment with RMC-6272 inhibited rapamycin-insensitive translation and reversed TSC1-associated early ND phenotypes including proliferation and neurite outgrowth that were unaffected by rapamycin. Conclusions Our work reveals ample mRNA translation alterations in TSC1 patient-derived NPCs that recapitulate mRNA translation in ASD brain samples. Further, suppression of TSC1-associated but rapamycin-insensitive translation and ND phenotypes by RMC-6272 unveils potential implications for more efficient targeting of mTORC1 as a superior treatment strategy for TAND.

The multi-subunit mammalian Mediator complex acts as an integrator of transcriptional regulation by RNA Polymerase II, and has emerged as a master coordinator of development and cell fate determination. We previously identified the Mediator subunit, MED28, as a cytosolic binding partner of merlin, the Neurofibromatosis 2 (NF2) tumor suppressor, and thus MED28 is distinct in having a cytosolic role as an NF2 interacting protein as well as a nuclear role as a Mediator complex subunit. Although limited in vitro studies have been performed on MED28, its in vivo function remains unknown. Employing a knockout mouse model, we describe for the first time the requirement for Med28 in the developing mouse embryo. Med28-deficiency causes peri-implantation lethality resulting from the loss of pluripotency of the inner cell mass accompanied by reduced expression of key pluripotency transcription factors Oct4 and Nanog. Further, overexpression of Med28 in mouse embryonic fibroblasts enhances the efficiency of their reprogramming to pluripotency. Cre-mediated inactivation of Med28 in induced pluripotent stem cells shows that Med28 is required for their survival. Intriguingly, heterozygous loss of Med28 results in differentiation of induced pluripotent stem cells into extraembryonic trophectoderm and primitive endoderm lineages. Our findings document the essential role of Med28 in the developing embryo as well as in acquisition and maintenance of pluripotency during reprogramming.

Saltpan is an extreme environment, which inhabit organisms that survive at very high salinities, high temperatures and withstand severe solar radiations. Halophilic bacteria have been isolated from different hypersaline conditions like salt lakes, natural and artificial solar saltpans. In the present study, a total of two overlaying saltpan water samples were collected from different sites of saltpans from Kovalam in Chennai, of which 8 distinct halophilic bacterial isolates were obtained. Optimization of growth parameters of the isolated halophilic bacteria was done in order to determine the optimum NaCl, temperature, pH and LDPE source required for their growth. The optimum NaCl, temperature, pH, and LDPE source required for their growth were as follows: 20%, 40°C, pH 9-10 and 0.5% LDPE source. Out of the 8 isolates which were tested, only two of them showed some measure of hydrophobicity. Clear zone assay was done to detect the biodegradation of LDPE (Low Density Polyethylene) by halophilic bacteria. Of the 8 isolates 2/8 (28%) isolates showed clearance around the colony showing their potential to degrade LDPE. SEM analysis of LDPE film treated with the halophilic bacterial isolate showed that there were several cracks and pits on the surface which developed after 60 days of treatment in comparison to the control film. The halophilic bacteria Nesiotobacter exalbescens and Bacillus vietnamensis were perhaps for the first time reported from the hypersaline lakes of Chennai in this study.