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Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have distinct clinical features but a common pathology-cytoplasmic inclusions rich in transactive response element DNA-binding protein of 43 kDa (TDP43). Rare TDP43 mutations cause ALS o

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have distinct clinical features but a common pathology - cytoplasmic inclusions rich in transactive response element DNA-binding protein of 43 kDa (TDP43). Rare TDP43 mutations cause ALS or FTD, but abnormal TDP43 levels and localization may cause disease even if TDP43 lacks a mutation. Here we show that individual neurons vary in their ability to clear TDP43 and are exquisitely sensitive to TDP43 levels. To measure TDP43 clearance, we developed and validated a single-cell optical method that overcomes the confounding effects of aggregation and toxicity and discovered that pathogenic mutations shorten TDP43 half-life. New compounds that stimulate autophagy improved TDP43 clearance and localization and enhanced survival in primary murine neurons and in human stem cell-derived neurons and astrocytes harboring mutant TDP43. These findings indicate that the levels and localization of TDP43 critically determine neurotoxicity and show that autophagy induction mitigates neurodegeneration by acting directly on TDP43 clearance. © 2014 Nature America, Inc. All rights reserved.

In vitro stem cell systems traditionally employ oxygen levels that are far removed from the in vivo situation. This study investigates whether an ambient environment containing a physiological oxygen level of 3% (normoxia) enables the generation of neural precursor cells (NPCs) from human embryonic stem cells (hESCs) and whether the resultant NPCs can undergo regional specification and functional maturation. We report robust and efficient neural conversion at 3% O2, demonstration of tri-lineage potential of resultant NPCs and the subsequent electrophysiological maturation of neurons. We also show that NPCs derived under 3% O2 can be differentiated long term in the absence of neurotrophins and can be readily specified into both spinal motor neurons and midbrain dopaminergic neurons. Finally, modelling the oxygen stress that occurs during transplantation, we demonstrate that in vitro transfer of NPCs from a 20 to 3% O2 environment results in significant cell death, while maintenance in 3% O2 is protective. Together these findings support 3% O2 as a physiologically relevant system to study stem cell-derived neuronal differentiation and function as well as to model neuronal injury. [ABSTRACT FROM AUTHOR]

Transactive response DNA-binding (TDP-43) protein is the dominant disease protein in amyotrophic lateral sclerosis (ALS) and a sub-group of frontotemporal lobar degeneration (FTLD-TDP). Identification of TARDBP mutations in familial ALS confirms a mechanistic link between misaccumulation of TDP-43 and neurodegeneration and provides an opportunity to study TDP-43 proteinopathies in human neurons generated from patient fibroblasts using induced pluripotent stem cells (iPSC). Here, we report the generation of iPSCs that carry the TDP-43 M337V mutation, and their differentiation into neurons and functional motor neurons. Mutant neurons had elevated levels of soluble and detergent- resistant TDP-43 protein, decreased survival in longitudinal studies, and increased vulnerability to antagonism of the phosphoinositide 3-kinase pathway. We conclude that expression of physiological levels of TDP-43 in human neurons is sufficient to reveal a mutation-specific cell autonomous phenotype and strongly supports this approach for the study of disease mechanisms and for drug screening., Molecular and Cellular Biology

Induced pluripotent stem cell (iPSC) derived endothelial cells (iECs) have emerged as a promising tool for studying vascular biology and providing a platform for modelling various vascular diseases, including those with genetic origins. Currently, primary ECs are the main source for disease modelling in this field. However, they are difficult to edit and have a limited lifespan. To study the effects of targeted mutations on an endogenous level, we generated and characterized an iPSC derived model for venous malformations (VMs). CRISPR-Cas9 technology was used to generate a novel human iPSC line with an amino acid substitution L914F in the TIE2 receptor, known to cause VMs. This enabled us to study the differential effects of VM causative mutations in iECs in multiple in vitro models and assess their ability to form vessels in vivo. The analysis of TIE2 expression levels in TIE2 L914F iECs showed a significantly lower expression of TIE2 on mRNA and protein level, which has not been observed before due to a lack of models with endogenous edited TIE2 L914F and sparse patient data. Interestingly, the TIE2 pathway was still significantly upregulated and TIE2 showed high levels of phosphorylation. TIE2 L914F iECs exhibited dysregulated angiogenesis markers and upregulated migration capability, while proliferation was not affected. Under shear stress TIE2 L914F iECs showed reduced alignment in the flow direction and a larger cell area than TIE2 WT iECs. In summary, we developed a novel TIE2 L914F iPSC-derived iEC model and characterized it in multiple in vitro models. The model can be used in future work for drug screening for novel treatments for VMs., (© 2024. The Author(s).)

This study focuses on the utilization of linden mucilage, extracted from the linden tree, as a potential natural polymer source for the production of composite films. The films, which incorporating linden water extract, essential oil, and oil, exhibited improved thermal stability, surface morphology, and water resistance. Biodegradability assessments, particularly for films using essential oil and oil, showed promising outcomes by maintaining structural integrity. Antimicrobial assays demonstrated significant resistance against pathogens, indicating potential applications requiring microbial resistance. Mechanical analyses revealed a trade-off between tensile strength and elongation at break with addition of components. Composite films exhibited reduced water vapor permeability which correlate with water solubility and contact angle measurements. Soil biodegradation studies highlighted the films' potential to mitigate environmental impact. Cytotoxicity tests confirmed the safety of these films for potential food applications. Additionally, antioxidant assays showed increased radical scavenging activity in films with added components. In conclusion, linden-based composite films exhibit promising characteristics, suggesting their potential as sustainable and functional materials, particularly for use in food packaging., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The pathogenesis and clinical heterogeneity of Parkinson's disease (PD) have been evaluated from molecular, pathophysiological, and clinical perspectives. High-throughput proteomic analysis of cerebrospinal fluid (CSF) opened new opportunities for scrutinizing this heterogeneity. To date, this is the most comprehensive CSF-based proteomics profiling study in PD with 569 patients (350 idiopathic patients, 65 GBA + mutation carriers and 154 LRRK2 + mutation carriers), 534 controls, and 4135 proteins analyzed. Combining CSF aptamer-based proteomics with genetics we determined protein quantitative trait loci (pQTLs). Analyses of pQTLs together with summary statistics from the largest PD genome wide association study (GWAS) identified 68 potential causal proteins by Mendelian randomization. The top causal protein, GPNMB, was previously reported to be upregulated in the substantia nigra of PD patients. We also compared the CSF proteomes of patients and controls. Proteome differences between GBA + patients and unaffected GBA + controls suggest degeneration of dopaminergic neurons, altered dopamine metabolism and increased brain inflammation. In the LRRK2 + subcohort we found dysregulated lysosomal degradation, altered alpha-synuclein processing, and neurotransmission. Proteome differences between idiopathic patients and controls suggest increased neuroinflammation, mitochondrial dysfunction/oxidative stress, altered iron metabolism and potential neuroprotection mediated by vasoactive substances. Finally, we used proteomic data to stratify idiopathic patients into "endotypes". The identified endotypes show differences in cognitive and motor disease progression based on previously reported protein-based risk scores.Our findings not only contribute to the identification of new therapeutic targets but also to shape personalized medicine in CNS neurodegeneration., (© 2023. The Author(s).)

We performed collapsing analyses on 454,796 UK Biobank (UKB) exomes to detect gene-level associations with diabetes. Recessive carriers of nonsynonymous variants in MAP3K15 were 30% less likely to develop diabetes ( P = 5.7 × 10 -10 ) and had lower glycosylated hemoglobin (β = -0.14 SD units, P = 1.1 × 10 -24 ). These associations were independent of body mass index, suggesting protection against insulin resistance even in the setting of obesity. We replicated these findings in 96,811 Admixed Americans in the Mexico City Prospective Study ( P < 0.05)Moreover, the protective effect of MAP3K15 variants was stronger in individuals who did not carry the Latino-enriched SLC16A11 risk haplotype ( P = 6.0 × 10 -4 ). Separately, we identified a Finnish-enriched MAP3K15 protein-truncating variant associated with decreased odds of both type 1 and type 2 diabetes ( P < 0.05) in FinnGen. No adverse phenotypes were associated with protein-truncating MAP3K15 variants in the UKB, supporting this gene as a therapeutic target for diabetes.

Hydrogels are widely used materials in biomedical, pharmaceutical, cosmetic, and agricultural fields. However, these hydrogels are usually formed synthetically via a long and complicated process involving crosslinking natural polymers. Herein, we describe a natural hydrogel isolated using a 'gentle' acid treatment from the girdle of a chiton species ( Chiton articulatus ). This novel hydrogel is shown to have a proliferative effect on mouse fibroblast cells (cell line, L929). The swelling capacity of this natural hydrogel was recorded as approximately 1,200% in distilled water, which is within desired levels for hydrogels. Detailed characterizations reveal that the hydrogel consists predominantly (83.93%) of protein. Considering its non-toxicity, proliferative effect and swelling properties, this natural hydrogel is an important discovery for material sciences, with potential for further applications in industry. Whether the girdle has some hydrogel activity in the living animal is unknown, but we speculate that it may enable the animal to better survive extreme environmental conditions by preventing desiccation., Competing Interests: Suzanne T. Williams is an Academic Editor for PeerJ., (© 2022 Çakmak et al.)

Commercially available types of chitin or chitin isolate are usually in powder form and are nanofibrous in microstructure. However, the surface characteristics of natural chitin in the body of insects are currently understudied. Herein, natural chitin film was successfully produced from bio-waste of insect pupae of the Japanese giant silkworm. Two different surface morphologies of the chitin film were observed. We report for the first time a micropapillary surface structure of chitin which was observed on the dorsal side of the film. To further potential of the micropapillary structured natural chitin in sensing applications, we develop a protocol for generating a nanoscopic film of Ag using thermal evaporation. The Ag-deposited natural chitin films exhibited surface-enhanced Raman scattering (SERS) activity to an extent depending on the structure of the film. In conclusion, materials science has been expanded by addition of a natural, three-dimensional chitin film with utilizable properties., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In the literature, the produced β-chitin samples are in powder or flake forms but there is no natural β-chitin based film. Also, the commercially available transdermal patches are produced from synthetic polymers. In this regard, we produced natural β-chitin-protein complex (CPC) film from the waste shells of Ensis spp. The obtained natural film was characterized by FTIR, TGA and SEM. Additionally, swelling, thickness, contact angle and antioxidant tests were done to learn more about the films. After production and characterization of the film, capsaicin, which is commonly used for pain relief was loaded into the film. The loading capacity was recorded as 5.79%. The kinetic models were studied in three different pH, then the results were fitted with Higuchi model with high correlation at pH 7.4. After considering all the obtained results, the capsaicin loaded CPC film may be an alternative candidate for transdermal patch instead of the synthetic ones., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Chitosan is an indispensable biopolymer for use as a drug carrier thanks to its non-toxic, biodegradable, biocompatible, antimicrobial, and anti-oxidative nature. In previous studies, chitosan was first dissolved into weak acids and formed into gel, then used for carrying pharmaceutically active compounds such as nanoparticles, capsules, composites, and films. Using the produced chitosan gel after dissolving it in weak acids has advantages, such as ease of processing for loading the required amount of active substance and making the desired shape and size. However, dissolved chitosan loses some of its natural properties such as fibrous structure, crystallinity, and thermal stability. In this study, for the first time, three-dimensional chitosan lenses obtained from an insect's (Tabanus bovinus) compound eyes, with the original shape intact, were tested as a drug carrier. A model drug, quercetin, was loaded into chitosan membrane, and its release profile was examined. Also, a point-of-care test was conducted for both chitin and chitosan membranes. Chitin and chitosan membranes obtained from insect corneal lenses were characterized by using FTIR, TGA, elemental analysis, and surface wettability analysis as well as stereo, binocular, and scanning electron microscopies. It was observed that chitosan membrane could be used as a drug carrier material. Both chitin and chitosan membranes will be improved for lateral flow assay, and these membranes can be tested for other bioengineering applications in further studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2018. Published by Elsevier B.V.)

We develop CellSIUS (Cell Subtype Identification from Upregulated gene Sets) to fill a methodology gap for rare cell population identification for scRNA-seq data. CellSIUS outperforms existing algorithms for specificity and selectivity for rare cell types and their transcriptomic signature identification in synthetic and complex biological data. Characterization of a human pluripotent cell differentiation protocol recapitulating deep-layer corticogenesis using CellSIUS reveals unrecognized complexity in human stem cell-derived cellular populations. CellSIUS enables identification of novel rare cell populations and their signature genes providing the means to study those populations in vitro in light of their role in health and disease.

Zika virus (ZIKV) can cross the placental barrier, resulting in infection of the fetal brain and neurological defects including microcephaly. The cellular tropism of ZIKV and the identity of attachment factors used by the virus to gain access to key cell types involved in pathogenesis are under intense investigation. Initial studies suggested that ZIKV preferentially targets neural progenitor cells (NPCs), providing an explanation for the developmental phenotypes observed in some pregnancies. The AXL protein has been nominated as a key attachment factor for ZIKV in several cell types including NPCs. However, here we show that genetic ablation of AXL has no effect on ZIKV entry or ZIKV-mediated cell death in human induced pluripotent stem cell (iPSC)-derived NPCs or cerebral organoids. These findings call into question the utility of AXL inhibitors for preventing birth defects after infection and suggest that further studies of viral attachment factors in NPCs are needed., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Evolutionary differences in gene regulation between humans and lower mammalian experimental systems are incompletely understood, a potential translational obstacle that is challenging to surmount in neurons, where primary tissue availability is poor. Rodent-based studies show that activity-dependent transcriptional programs mediate myriad functions in neuronal development, but the extent of their conservation in human neurons is unknown. We compared activity-dependent transcriptional responses in developing human stem cell-derived cortical neurons with those induced in developing primary- or stem cell-derived mouse cortical neurons. While activity-dependent gene-responsiveness showed little dependence on developmental stage or origin (primary tissue vs. stem cell), notable species-dependent differences were observed. Moreover, differential species-specific gene ortholog regulation was recapitulated in aneuploid mouse neurons carrying human chromosome-21, implicating promoter/enhancer sequence divergence as a factor, including human-specific activity-responsive AP-1 sites. These findings support the use of human neuronal systems for probing transcriptional responses to physiological stimuli or indeed pharmaceutical agents., Competing Interests: The authors declare that no competing interests exist.

We have assessed, using whole-cell patch-clamp recording and RNA-sequencing (RNA-seq), the properties and composition of GABAA receptors (GABAARs) and strychnine-sensitive glycine receptors (GlyRs) expressed by excitatory cortical neurons derived from human embryonic stem cells (hECNs). The agonists GABA and muscimol gave EC50 values of 278 μm and 182 μm, respectively, and the presence of a GABAAR population displaying low agonist potencies is supported by strong RNA-seq signals for α2 and α3 subunits. GABAAR-mediated currents, evoked by EC50 concentrations of GABA, were blocked by bicuculline and picrotoxin with IC50 values of 2.7 and 5.1 μm, respectively. hECN GABAARs are predominantly γ subunit-containing as assessed by the sensitivity of GABA-evoked currents to diazepam and insensitivity to Zn(2+), together with the weak direct agonist action of gaboxadol; RNA-seq indicated a predominant expression of the γ2 subunit. Potentiation of GABA-evoked currents by propofol and etomidate and the lack of inhibition of currents by salicylidine salycylhydrazide (SCS) indicate expression of the β2 or β3 subunit, with RNA-seq analysis indicating strong expression of β3 in hECN GABAARs. Taken together our data support the notion that hECN GABAARs have an α2/3β3γ2 subunit composition - a composition that also predominates in immature rodent cortex. GlyRs expressed by hECNs were activated by glycine with an EC50 of 167 μm. Glycine-evoked (500 μm) currents were blocked by strychnine (IC50 = 630 nm) and picrotoxin (IC50 = 197 μm), where the latter is suggestive of a population of heteromeric receptors. RNA-seq indicates GlyRs are likely to be composed of α2 and β subunits., (© 2014 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

TDP-43 is found in cytoplasmic inclusions in 95% of amyotrophic lateral sclerosis (ALS) and 60% of frontotemporal lobar degeneration (FTLD). Approximately 4% of familial ALS is caused by mutations in TDP-43. The majority of these mutations are found in the glycine-rich domain, including the variant M337V, which is one of the most common mutations in TDP-43. In order to investigate the use of allele-specific RNA interference (RNAi) as a potential therapeutic tool, we designed and screened a set of siRNAs that specifically target TDP-43(M337V) mutation. Two siRNA specifically silenced the M337V mutation in HEK293T cells transfected with GFP-TDP-43(wt) or GFP-TDP-43(M337V) or TDP-43 C-terminal fragments counterparts. C-terminal TDP-43 transfected cells show an increase of cytosolic inclusions, which are decreased after allele-specific siRNA in M337V cells. We then investigated the effects of one of these allele-specific siRNAs in induced pluripotent stem cells (iPSCs) derived from an ALS patient carrying the M337V mutation. These lines showed a two-fold increase in cytosolic TDP-43 compared to the control. Following transfection with the allele-specific siRNA, cytosolic TDP-43 was reduced by 30% compared to cells transfected with a scrambled siRNA. We conclude that RNA interference can be used to selectively target the TDP-43(M337V) allele in mammalian and patient cells, thus demonstrating the potential for using RNA interference as a therapeutic tool for ALS.

Rodent-based studies have shown that neurons undergo major developmental changes to ion channel expression and ionic gradients that determine their excitation-inhibition balance. Neurons derived from human pluripotent stem cells theoretically offer the potential to study classical developmental processes in a human-relevant system, although this is currently not well explored. Here, we show that excitatory cortical-patterned neurons derived from multiple human pluripotent stem cell lines exhibit native-like maturation changes in AMPAR composition such that there is an increase in the expression of GluA2(R) subunits. Moreover, we observe a dynamic shift in intracellular Cl- levels, which determines the reversal potential of GABAAR-mediated currents and is influenced by neurotrophic factors. The shift is concomitant with changes in KCC2 and NKCC1 expression. Because some human diseases are thought to involve perturbations to AMPAR GluA2 content and others in the chloride reversal potential, human stem-cell-derived neurons represent a valuable tool for studying these fundamental properties.

The RNA-binding protein TDP-43 regulates RNA metabolism at multiple levels, including transcription, RNA splicing, and mRNA stability. TDP-43 is a major component of the cytoplasmic inclusions characteristic of amyotrophic lateral sclerosis and some types of frontotemporal lobar degeneration. The importance of TDP-43 in disease is underscored by the fact that dominant missense mutations are sufficient to cause disease, although the role of TDP-43 in pathogenesis is unknown. Here we show that TDP-43 forms cytoplasmic mRNP granules that undergo bidirectional, microtubule-dependent transport in neurons in vitro and in vivo and facilitate delivery of target mRNA to distal neuronal compartments. TDP-43 mutations impair this mRNA transport function in vivo and in vitro, including in stem cell-derived motor neurons from ALS patients bearing any one of three different TDP-43 ALS-causing mutations. Thus, TDP-43 mutations that cause ALS lead to partial loss of a novel cytoplasmic function of TDP-43., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Widespread use of human pluripotent stem cells (hPSCs) to study neuronal physiology and function is hindered by the ongoing need for specialist expertise in converting hPSCs to neural precursor cells (NPCs). Here, we describe a new methodology to generate cryo-preservable hPSC-derived NPCs that retain an anterior identity and are propagatable long-term prior to terminal differentiation, thus abrogating regular de novo neuralization. Key to achieving passagable NPCs without loss of identity is the combination of both absence of EGF and propagation in physiological levels (3%) of O2. NPCs generated in this way display a stable long-term anterior forebrain identity and importantly retain developmental competence to patterning signals. Moreover, compared to NPCs maintained at ambient O2 (21%), they exhibit enhanced uniformity and speed of functional maturation, yielding both deep and upper layer cortical excitatory neurons. These neurons display multiple attributes including the capability to form functional synapses and undergo activity-dependent gene regulation. The platform described achieves long-term maintenance of anterior neural precursors that can give rise to forebrain neurones in abundance, enabling standardised functional studies of neural stem cell maintenance, lineage choice and neuronal functional maturation for neurodevelopmental research and disease-modelling.

We have established and efficient system to specify NG2/PDGF-Rα/OLIG2+ oligodendrocyte precursor cells (OPCs) from human embryonic stem cells (hESCs) at low, physiological (3%) oxygen levels. This was achieved via both forebrain and spinal cord origins, with up to 98% of cells expressing NG2. Developmental insights reveal a critical role for fibroblast growth factor 2 (FGF-2) in OLIG2 induction via ventral forebrain pathways. The OPCs mature in vitro to express O4 (46%) and subsequently become galactocerebroside (GALC), O1, and myelin basic protein-positive (MBP+) multibranching oligodendrocytes. These were cultured alongside hESC-derived neurons. The electrophysiological properties of human OPCs are similar to those of rat OPCs, with large voltage-gated sodium currents and the ability to fire action potentials. Exposure to a selective retinoid X receptor agonist increased the proportion of O4+ oligodendrocytes that express MBP from 5% to 30%. Thus, we have established a developmentally engineered system to investigate the biological properties of human OPCs and test the effects of putative remyelinating agents prior to clinical application.

Traditionally, in vitro stem cell systems have used oxygen tensions that are far removed from the in vivo situation. This is particularly true for the central nervous system, where oxygen (O2) levels range from 8% at the pia to 0.5% in the midbrain, whereas cells are usually cultured in a 20% O2 environment. Cell transplantation strategies therefore typically introduce a stress challenge at the time of transplantation as the cells are switched from 20% to 3% O2 (the average in adult organs). We have modeled the oxygen stress that occurs during transplantation, demonstrating that in vitro transfer of neonatal rat cortical neural precursor cells (NPCs) from a 20% to a 3% O2 environment results in significant cell death, whereas maintenance at 3% O2 is protective. This survival benefit translates to the in vivo environment, where culture of NPCs at 3% rather than 20% O2 approximately doubles survival in the immediate post-transplantation phase. Furthermore, NPC fate is affected by culture at low, physiological O2 tensions (3%), with particularly marked effects on the oligodendrocyte lineage, both in vitro and in vivo. We propose that careful consideration of physiological oxygen environments, and particularly changes in oxygen tension, has relevance for the practical approaches to cellular therapies.

Glial proliferation and activation are associated with disease progression in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia. In this study, we describe a unique platform to address the question of cell autonomy in transactive response DNA-binding protein (TDP-43) proteinopathies. We generated functional astroglia from human induced pluripotent stem cells carrying an ALS-causing TDP-43 mutation and show that mutant astrocytes exhibit increased levels of TDP-43, subcellular mislocalization of TDP-43, and decreased cell survival. We then performed coculture experiments to evaluate the effects of M337V astrocytes on the survival of wild-type and M337V TDP-43 motor neurons, showing that mutant TDP-43 astrocytes do not adversely affect survival of cocultured neurons. These observations reveal a significant and previously unrecognized glial cell-autonomous pathological phenotype associated with a pathogenic mutation in TDP-43 and show that TDP-43 proteinopathies do not display an astrocyte non-cell-autonomous component in cell culture, as previously described for SOD1 ALS. This study highlights the utility of induced pluripotent stem cell-based in vitro disease models to investigate mechanisms of disease in ALS and other TDP-43 proteinopathies.

Transactive response DNA-binding (TDP-43) protein is the dominant disease protein in amyotrophic lateral sclerosis (ALS) and a subgroup of frontotemporal lobar degeneration (FTLD-TDP). Identification of mutations in the gene encoding TDP-43 (TARDBP) in familial ALS confirms a mechanistic link between misaccumulation of TDP-43 and neurodegeneration and provides an opportunity to study TDP-43 proteinopathies in human neurons generated from patient fibroblasts by using induced pluripotent stem cells (iPSCs). Here, we report the generation of iPSCs that carry the TDP-43 M337V mutation and their differentiation into neurons and functional motor neurons. Mutant neurons had elevated levels of soluble and detergent-resistant TDP-43 protein, decreased survival in longitudinal studies, and increased vulnerability to antagonism of the PI3K pathway. We conclude that expression of physiological levels of TDP-43 in human neurons is sufficient to reveal a mutation-specific cell-autonomous phenotype and strongly supports this approach for the study of disease mechanisms and for drug screening.

Human pluripotent stem cells from embryonic origins and those generated from reprogrammed somatic cells share many characteristics, including indefinite proliferation and a sustained capacity to differentiate into a wide variety of cell types. However, it remains to be demonstrated whether both cell types rely on similar mechanisms to maintain their pluripotent status and to control their differentiation. Any differences in such mechanisms would suggest that reprogramming of fibroblasts to generate induced pluripotent stem cells (iPSCs) results in novel states of pluripotency. In that event, current methods for expanding and differentiating human embryonic stem cells (ESCs) might not be directly applicable to human iPSCs. However, we show here that human iPSCs rely on activin/nodal signaling to control Nanog expression and thereby maintain pluripotency, thus revealing their mechanistic similarity to human ESCs. We also show that growth factors necessary and sufficient for achieving specification of human ESCs into extraembryonic tissues, neuroectoderm, and mesendoderm also drive differentiation of human iPSCs into the same tissues. Importantly, these experiments were performed in fully chemically defined medium devoid of factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. Together these data reveal that human iPSCs rely on mechanisms similar to human ESCs to maintain their pluripotency and to control their differentiation, showing that these pluripotent cell types are functionally equivalent.

During normal development oligodendrocyte precursors (OPCs) are generated in the ventral spinal cord in response to Sonic hedgehog (Shh) signalling. There is also a second, late wave of oligodendrogenesis in the dorsal spinal cord independent of Shh activity. Two signalling pathways, controlled by bone morphogenetic protein and fibroblast growth factor (FGF), are active players in dorsal spinal cord specification. In particular, BMP signalling from the roof plate has a crucial role in setting up dorsal neural identity and its inhibition is sufficient to generate OPCs both in vitro and in vivo. In contrast, FGF signalling can induce OPC production from dorsal spinal cord cultures in vitro. In this study, we examined the cross-talk between mitogen-activated protein kinase (MAPK) and BMP signalling in embryonic dorsal spinal cord cultures at the SMAD1/5/8 (SMAD1) transcription factor level, the main effectors of BMP activity. We have previously shown that FGF2 treatment of neural precursor cells (NPCs) derived from rat E14 dorsal spinal cord is sufficient to generate OPCs in vitro. Utilising the same system, we now show that FGF prevents BMP-induced nuclear localisation of SMAD1-phosphorylated at the C-terminus (C-term-pSMAD1). This nuclear exclusion of C-term-pSMAD1 is dependent on MAPK activity and correlates with OLIG2 upregulation, the obligate transcription factor for oligodendrogenesis. Furthermore, inhibition of the MAPK pathway abolishes OLIG2 expression. We also show that SMAD4, which acts as a common partner for receptor-regulated Smads including SMAD1, associates with a Smad binding site in the Olig2 promoter and dissociates from it upon differentiation. Taken together, these results suggest that FGF can promote OPC generation from embryonic NPCs by counteracting BMP signalling at the Smad1 transcription factor level and that Smad-containing transcriptional complexes may be involved in direct regulation of the Olig2 promoter.

T-box transcription factors play a crucial role in development where they are implicated in patterning and cell fate decisions. Tbx2 and Tbx3 have also been implicated in several cancers including melanoma, and can act as antisenescence factors through their ability to repress p19(ARF) and p21(CIP1) expression. Although several target genes for T-box factors have been identified, it is unknown whether this family of proteins can bind chromatin, a property that would facilitate the epigenetic reprogramming that occurs in both development and cancer progression. Here, we show that Tbx2 has the potential to recognize mitotic chromatin in a DNA-dependent fashion, can interact specifically with the histone H3 N-terminal tail, a property shared with Tbx4, Tbx5 and Tbx6, and can also recognize nucleosomal DNA, with binding to nucleosomes being antagonized by the presence of the histone tails. Strikingly, in vivo Tbx2 co-localization with pericentric heterochromatin appears to be regulated and ectopic expression of Tbx2 leads to severe mitotic defects. Taken together our results suggest that Tbx2, and most likely other members of the T-box family, are able to target chromatin and may indicate a role for the T-box factors in epigenetic reprogramming events.

T-box transcription factors play key roles in development and in particular the determination or maintenance of cell fate. Tbx2 is a transcriptional repressor implicated in several developmental processes and which has also been implicated in cancer through its ability to suppress senescence via repression of the p19(ARF) and p21(Cip1) (CDKN1A) promoters. However, despite its importance, little is known about how Tbx2 may be regulated. Here, we show that Tbx2 protein expression is tightly regulated during cell cycle progression, with levels being low in G1, increasing in mid-S-phase and persisting at high levels though G2 until finally undergoing a dramatic reduction at the onset of mitosis. Moreover, in S-phase, Tbx2 is present at a subset of late, but not early, replication foci and a significant fraction of Tbx2 is tightly associated with the nucleus in small DNA-associated foci that do not correspond with telomeres, PML or cajal bodies. The results are consistent with Tbx2 playing a role in cell cycle progression and organization of subnuclear compartments.

C1D is a gamma-irradiation inducible nuclear matrix protein that interacts with and activates the DNA-dependent protein kinase (DNA-PK) that is essential for the repair of the DNA double-strand breaks and V(D)J recombination. Recently, it was demonstrated that C1D can also interact with TRAX and prevent the association of TRAX with Translin, a factor known to bind DNA break-point junctions, and that over expression of C1D can induce p53-dependent apoptosis. Taken together, these findings suggest that mammalian C1D could be involved in maintenance of genome integrity by regulating the activity of proteins involved in DNA repair and recombination. To obtain direct evidence for the biological function of C1D that we show is highly conserved between diverse species, we have analysed the Saccharomyces cerevisiae C1D homologue. We report that the disruption of the YC1D gene results in a temperature sensitivity and that yc1d mutant strains exhibit defects in non-homologous DNA end joining (NHEJ) and accurate DNA repair. In addition, using a novel plasmid-based in vivo recombination assay, we show that yc1d mutant strains are also defective in homologous recombination. These results indicate that YC1D is implicated in both homologous recombination and NHEJ pathways for the repair of DNA double-strand breaks.

The nuclear matrix protein C1D is an activator of the DNA-dependent protein kinase (DNA-PK), which is essential for the repair of DNA double-strand breaks (DSBs) and V(D)J recombination. C1D is phosphorylated very efficiently by DNA-PK, and its mRNA and protein levels are induced upon gamma-irradiation, suggesting that C1D may play a role in repair of DSBs in vivo. In an attempt to identify the biological function of C1D, we have employed the yeast two-hybrid system and found that C1D interacts specifically with Translin-associated factor X, TRAX. Although the biological function of TRAX remains unknown, its bipartite nuclear targeting sequences suggest a role for TRAX in the movement of associated proteins, including Translin, into the nucleus. We show that C1D and TRAX interact specifically in both yeast and mammalian cells. Interestingly, however, interaction of these two proteins in mammalian cells only occur following gamma-irradiation, raising the possibility of involvement of TRAX in DNA double-strand break repair and providing evidence for biological functions of the nuclear matrix protein C1D and TRAX. Moreover, we show, using fluorescently tagged proteins, that the relative expression levels of TRAX and Translin affect their subcellular localization. These results suggest that one role for C1D may be to regulate TRAX/Translin complex formation.