Mutations in FUS lead to synaptic dysregulation in ALS-iPSC derived neurons.

Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset neurodegenerative disorder characterized by progressive muscular weakness due to the selective loss of motor neurons. Mutations in the gene Fused in Sarcoma (FUS) were identified as one cause of ALS. Here, we report that mutations in FUS lead to upregulation of synaptic proteins, increasing synaptic activity and abnormal release of vesicles at the synaptic cleft. Consequently, FUS-ALS neurons showed greater vulnerability to glutamate excitotoxicity, which raised neuronal swellings (varicose neurites) and led to neuronal death. Fragile X mental retardation protein (FMRP) is an RNA-binding protein known to regulate synaptic protein translation, and its expression is reduced in the FUS-ALS lines. Collectively, our data suggest that a reduction of FMRP levels alters the synaptic protein dynamics, leading to synaptic dysfunction and glutamate excitotoxicity. Here, we present a mechanistic hypothesis linking dysregulation of peripheral translation with synaptic vulnerability in the pathogenesis of FUS-ALS.

Generation of an Open-Access Patient-Derived iPSC Biobank for Amyotrophic Lateral Sclerosis Disease Modelling.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting the upper and lower motor neurons, causing patients to lose control over voluntary movement, and leading to gradual paralysis and death. There is no cure for ALS, and the development of viable therapeutics has proved challenging, demonstrated by a lack of positive results from clinical trials. One strategy to address this is to improve the tool kit available for pre-clinical research. Here, we describe the creation of an open-access ALS iPSC biobank generated from patients carrying mutations in the TARDBP, FUS, ANXA11, ARPP21, and C9ORF72 genes, alongside healthy controls. To demonstrate the utilisation of these lines for ALS disease modelling, a subset of FUS-ALS iPSCs were differentiated into functionally active motor neurons. Further characterisation revealed an increase in cytoplasmic FUS protein and reduced neurite outgrowth in FUS-ALS motor neurons compared to the control. This proof-of-principle study demonstrates that these novel patient-derived iPSC lines can recapitulate specific and early disease-related ALS phenotypes. This biobank provides a disease-relevant platform for discovery of ALS-associated cellular phenotypes to aid the development of novel treatment strategies.

Psychosis Risk Candidate ZNF804A Localizes to Synapses and Regulates Neurite Formation and Dendritic Spine Structure.

BACKGROUND: Variation in the gene encoding zinc finger binding protein 804A (ZNF804A) is associated with schizophrenia and bipolar disorder. Evidence suggests that ZNF804A is a regulator of gene transcription and is present in nuclear and extranuclear compartments. However, a detailed examination of ZNF804A distribution and its neuronal functions has yet to be performed. METHODS: The localization of ZNF804A protein was examined in neurons derived from human neural progenitor cells, human induced pluripotent stem cells, or in primary rat cortical neurons. In addition, small interfering RNA-mediated knockdown of ZNF804A was conducted to determine its role in neurite formation, maintenance of dendritic spine morphology, and responses to activity-dependent stimulations. RESULTS: Endogenous ZNF804A protein localized to somatodendritic compartments and colocalized with the putative synaptic markers in young neurons derived from human neural progenitor cells and human induced pluripotent stem cells. In mature rat neurons, Zfp804A, the homolog of ZNF804A, was present in a subset of dendritic spines and colocalized with synaptic proteins in specific nanodomains, as determined by super-resolution microscopy. Interestingly, knockdown of ZNF804A attenuated neurite outgrowth in young neurons, an effect potentially mediated by reduced neuroligin-4 expression. Furthermore, knockdown of ZNF804A in mature neurons resulted in the loss of dendritic spine density and impaired responses to activity-dependent stimulation. CONCLUSIONS: These data reveal a novel subcellular distribution for ZNF804A within somatodendritic compartments and a nanoscopic organization at excitatory synapses. Moreover, our results suggest that ZNF804A plays an active role in neurite formation, maintenance of dendritic spines, and activity-dependent structural plasticity.

Erasure and reestablishment of random allelic expression imbalance after epigenetic reprogramming.

Clonal level random allelic expression imbalance and random monoallelic expression provides cellular heterogeneity within tissues by modulating allelic dosage. Although such expression patterns have been observed in multiple cell types, little is known about when in development these stochastic allelic choices are made. We examine allelic expression patterns in human neural progenitor cells before and after epigenetic reprogramming to induced pluripotency, observing that loci previously characterized by random allelic expression imbalance (0.63% of expressed genes) are generally reset to a biallelic state in induced pluripotent stem cells (iPSCs). We subsequently neuralized the iPSCs and profiled isolated clonal neural stem cells, observing that significant random allelic expression imbalance is reestablished at 0.65% of expressed genes, including novel loci not found to show allelic expression imbalance in the original parental neural progenitor cells. Allelic expression imbalance was associated with altered DNA methylation across promoter regulatory regions, with clones characterized by skewed allelic expression being hypermethylated compared to their biallelic sister clones. Our results suggest that random allelic expression imbalance is established during lineage commitment and is associated with increased DNA methylation at the gene promoter.

Resveratrol: A Potential Hippocampal Plasticity Enhancer.

The search for molecules capable of restoring altered hippocampal plasticity in psychiatric and neurological conditions is one of the most important tasks of modern neuroscience. It is well established that neural plasticity, such as the ability of the postnatal hippocampus to continuously generate newly functional neurons throughout life, a process called adult hippocampal neurogenesis (AHN), can be modulated not only by pharmacological agents, physical exercise, and environmental enrichment, but also by "nutraceutical" agents. In this review we focus on resveratrol, a phenol and phytoalexin found in the skin of grapes and red berries, as well as in nuts. Resveratrol has been reported to have antioxidant and antitumor properties, but its effects as a neural plasticity inducer are still debated. The current review examines recent evidence implicating resveratrol in regulating hippocampal neural plasticity and in mitigating the effects of various disorders and diseases on this important brain structure. Overall, findings show that resveratrol can improve cognition and mood and enhance hippocampal plasticity and AHN; however, some studies report opposite effects, with resveratrol inhibiting aspects of AHN. Therefore, further investigation is needed to resolve these controversies before resveratrol can be established as a safe coadjuvant in preventing and treating neuropsychiatric conditions.

Utilizing induced pluripotent stem cells (iPSCs) to understand the actions of estrogens in human neurons.

This article is part of a Special Issue "Estradiol and Cognition". Over recent years tremendous progress has been made towards understanding the molecular and cellular mechanism by which estrogens exert enhancing effects on cognition, and how they act as a neuroprotective or neurotrophic agent in disease. Currently, much of this work has been carried out in animal models with only a limited number of studies using native human tissue or cells. Recent advances in stem cell technology now make it possible to reprogram somatic cells from humans into induced pluripotent stem cells (iPSCs), which can subsequently be differentiated into neurons of specific lineages. Importantly, the reprogramming of cells allows for the generation of iPSCs that retain the genetic "makeup" of the donor. Therefore, it is possible to generate iPSC-derived neurons from patients diagnosed with specific diseases, that harbor the complex genetic background associated with the disorder. Here, we review the iPSC technology and how it's currently being used to model neural development and neurological diseases. Furthermore, we explore whether this cellular system could be used to understand the role of estrogens in human neurons, and present preliminary data in support of this. We further suggest that the use of iPSC technology offers a novel system to not only further understand estrogens' effects in human cells, but also to investigate the mechanism by which estrogens are beneficial in disease. Developing a greater understanding of these mechanisms in native human cells will also aid in the development of safer and more effective estrogen-based therapeutics.

Hippocampal biomarkers of fear memory in an animal model of generalized anxiety disorder.

Generalized anxiety disorder (GAD) is highly prevalent and incapacitating. Here we used the Carioca High-Conditioned Freezing (CHF) rats, a previously validated animal model for GAD, to identify biomarkers and structural changes in the hippocampus that could be part of the underlying mechanisms of their high-anxiety profile. Spatial and fear memory was assessed in the Morris water maze and passive avoidance test. Serum corticosterone levels, immunofluorescence for glucocorticoid receptors (GR) in the dentate gyrus (DG), and western blotting for hippocampal brain derived neurotrophic factor (BDNF) were performed. Immunohistochemistry for markers of cell proliferation (bromodeoxiuridine/Ki-67), neuroblasts (doublecortin), and cell survival were undertaken in the DG, along with spine staining (Golgi) and dendritic arborization tracing. Hippocampal GABA release was assessed by neurochemical assay. Fear memory was higher among CHF rats whilst spatial learning was preserved. Serum corticosterone levels were increased, with decreased GR expression. No differences were observed in hippocampal cell proliferation/survival, but the number of newborn neurons was decreased, along with their number and length of tertiary dendrites. Increased expression of proBDNF and dendritic spines was observed; lower ratio of GABA release in the hippocampus was also verified. These findings suggest that generalized anxiety/fear could be associated with different hippocampal biomarkers, such as increased spine density, possibly as a compensatory mechanism for the decreased hippocampal number of neuroblasts and dendritic arborization triggered by high corticosterone. Disruption of GABAergic signaling and BDNF impairment are also proposed as part of the hippocampal mechanisms possibly underlying the anxious phenotype of this model.

The utility of patient specific induced pluripotent stem cells for the modelling of Autistic Spectrum Disorders.

Until now, models of psychiatric diseases have typically been animal models. Whether they were to be used to further understand the pathophysiology of the disorder, or as drug discovery tools, animal models have been the choice of preference in mimicking psychiatric disorders in an experimental setting. While there have been cellular models, they have generally been lacking in validity. This situation is changing with the advent of patient-specific induced pluripotent stem cells (iPSCs). In this article, we give a methodological evaluation of the current state of the iPS technology with reference to our own work in generating patient-specific iPSCs for the study of autistic spectrum disorder (ASD). In addition, we will give a broader perspective on the validity of this technology and to what extent it can be expected to complement animal models of ASD in the coming years.

Translational findings on brain-derived neurotrophic factor and anxiety: contributions from basic research to clinical practice.

BACKGROUND/AIMS: Anxious responses are evolutionarily adaptive, but excessive fear can become disabling and lead to anxiety disorders. Translational models of anxiety might be useful sources for understanding the neurobiology of fear and anxiety and can contribute to future proposals of therapeutic intervention for the disorders studied. Brain-derived neurotrophic factor (BDNF), which is known for its importance on neuroplasticity and contextual memory, has emerged as a relevant element for emotional memory. Recent studies show that the Val(66)Met BDNF polymorphism correlates with various psychiatric disorders, including anxiety, but there are several differences between experimental and clinical studies. METHODS: In this work, we review the literature focused on the BDNF Val(66)Met polymorphism and anxiety, and discuss biological findings from animal models to clinical studies. RESULTS: As occurs with other psychiatric disorders, anxiety correlates with anatomical, behavioral and physiological changes related to the BDNF polymorphism. In animal studies, it has been shown that a significant decrease in regulated secretion from both BDNFVal/Met and BDNFMet/Met neurons represented a significant decrease in available BDNF. CONCLUSION: These studies suggest that developing pharmacological strategies facilitating the release of BDNF from synapses or prolongation of the half-life of secreted BDNF may improve the therapeutic responses of humans expressing the BDNF polymorphism.

Human conditionally immortalized neural stem cells improve locomotor function after spinal cord injury in the rat.

INTRODUCTION: A growing number of studies have highlighted the potential of stem cell and more-differentiated neural cell transplantation as intriguing therapeutic approaches for neural repair after spinal cord injury (SCI). METHODS: A conditionally immortalized neural stem cell line derived from human fetal spinal cord tissue (SPC-01) was used to treat a balloon-induced SCI. SPC-01 cells were implanted into the lesion 1 week after SCI. To determine the feasibility of tracking transplanted stem cells, a portion of the SPC-01 cells was labeled with poly-L-lysine-coated superparamagnetic iron-oxide nanoparticles, and the animals grafted with labeled cells underwent magnetic resonance imaging. Functional recovery was evaluated by using the BBB and plantar tests, and lesion morphology, endogenous axonal sprouting and graft survival, and differentiation were analyzed. Quantitative polymerase chain reaction (qPCR) was used to evaluate the effect of transplanted SPC-01 cells on endogenous regenerative processes. RESULTS: Transplanted animals displayed significant motor and sensory improvement 2 months after SCI, when the cells robustly survived in the lesion and partially filled the lesion cavity. qPCR revealed the increased expression of rat and human neurotrophin and motor neuron genes. The grafted cells were immunohistologically positive for glial fibrillary acidic protein (GFAP); however, we found 25% of the cells to be positive for Nkx6.1, an early motor neuron marker. Spared white matter and the robust sprouting of growth-associated protein 43 (GAP43)(+) axons were found in the host tissue. Four months after SCI, the grafted cells matured into Islet2(+) and choline acetyltransferase (ChAT)(+) neurons, and the graft was grown through with endogenous neurons. Grafted cells labeled with poly-L-lysine-coated superparamagnetic nanoparticles before transplantation were detected in the lesion on T2-weighted images as hypointense spots that correlated with histologic staining for iron and the human mitochondrial marker MTCO2. CONCLUSIONS: The transplantation of SPC-01 cells produced significant early functional improvement after SCI, suggesting an early neurotrophic action associated with long-term restoration of the host tissue, making the cells a promising candidate for future cell therapy in patients with SCI.

Conditionally immortalized stem cell lines from human spinal cord retain regional identity and generate functional V2a interneurons and motorneurons.

INTRODUCTION: The use of immortalized neural stem cells either as models of neural development in vitro or as cellular therapies in central nervous system (CNS) disorders has been controversial. This controversy has centered on the capacity of immortalized cells to retain characteristic features of the progenitor cells resident in the tissue of origin from which they were derived, and the potential for tumorogenicity as a result of immortalization. Here, we report the generation of conditionally immortalized neural stem cell lines from human fetal spinal cord tissue, which addresses these issues. METHODS: Clonal neural stem cell lines were derived from 10-week-old human fetal spinal cord and conditionally immortalized with an inducible form of cMyc. The derived lines were karyotyped, transcriptionally profiled by microarray, and assessed against a panel of spinal cord progenitor markers with immunocytochemistry. In addition, the lines were differentiated and assessed for the presence of neuronal fate markers and functional calcium channels. Finally, a clonal line expressing eGFP was grafted into lesioned rat spinal cord and assessed for survival, differentiation characteristics, and tumorogenicity. RESULTS: We demonstrate that these clonal lines (a) retain a clear transcriptional signature of ventral spinal cord progenitors and a normal karyotype after extensive propagation in vitro, (b) differentiate into relevant ventral neuronal subtypes with functional T-, L-, N-, and P/Q-type Ca(2+) channels and spontaneous calcium oscillations, and (c) stably engraft into lesioned rat spinal cord without tumorogenicity. CONCLUSIONS: We propose that these cells represent a useful tool both for the in vitro study of differentiation into ventral spinal cord neuronal subtypes, and for examining the potential of conditionally immortalized neural stem cells to facilitate functional recovery after spinal cord injury or disease.

Treating spinal cord injury in rats with a combination of human fetal neural stem cells and hydrogels modified with serotonin.

Currently, there is no effective strategy for the treatment of spinal cord injury (SCI). A combination of biomaterials and stem cell therapy seems to be a promising approach to increase regenerative potential after SCI. We evaluated the use of a cellpolymer construct based on a combination of the conditionally immortalized spinal progenitor cell line SPC-01_GFP3, derived from human fetal spinal cord tissue, with a serotonin-modified poly(2-hydroxyethyl methacrylate) hydrogel (pHEMA-5HT). We compared the effect of treatment with a pHEMA-5HT hydrogel seeded with SPC-01_GFP3 cells, treatment with a pHEMA-5HT only and no treatment on functional outcome and tissue reconstruction in hemisected rats. Prior to transplantation the cell-polymer construct displayed a high potential to support the growth, proliferation and differentiation of SPC-01 cells in vitro. One month after surgery, combined hydrogel-cell treatment reduced astrogliosis and tissue atrophy and increased axonal and blood vessel ingrowth into the implant; however, two months later only the ingrowth of blood vessels remained increased. SPC-01_GFP3 cells survived well in vivo and expressed advanced markers of neuronal differentiation. However, a majority of the transplanted cells migrated out of the lesion and only rarely remained in the hydrogel. No differences among the groups in motor or sensory recovery were observed. Despite the support of the hydrogel as a cell carrier in vitro, and good results in vivo one month postsurgery, there was only a small effect on long term recovery, mainly due to the limited ability of the hydrogels to support the in vivo growth and differentiation of cells within the implant. Further modifications will be necessary to achieve stable long term improvement in functional outcome.

The use of superporous Ac-CGGASIKVAVS-OH-modified PHEMA scaffolds to promote cell adhesion and the differentiation of human fetal neural precursors.

Modifications of poly(2-hydroxyethyl methacrylate) (PHEMA) with laminin-derived Ac-CGGASIKVAVS-OH peptide sequences have been developed to construct scaffolds that promote cell adhesion and neural differentiation. Radical copolymerization of 2-hydroxyethyl methacrylate with 2-aminoethyl methacrylate (AEMA) and ethylene dimethacrylate in the presence of ammonium oxalate crystals resulted in the formation of superporous P(HEMA-AEMA) hydrogels. They were reacted with gamma-thiobutyrolactone to yield 2-(4-sulfanylbutanamido)ethyl methacrylate (P(HEMA-AEMA)-SH) unit. The Ac-CGGASIKVAVS-OH peptide was immobilized to the sulfhydryl groups of P(HEMA-AEMA)-SH by 2,2'-dithiodipyridine linking reagent via 2-[4-(2-pyridyldisulfanyl)butanamido]ethyl methacrylate (P(HEMA-AEMA)-TPy). The adhesion and morphology of rat mesenchymal stem cells were investigated on the Ac-CGGASIKVAVS-OH-modified P(HEMA-AEMA) as well as on PHEMA, P(HEMA-AEMA)-SH and P(HEMA-AEMA)-TPy hydrogels. Superporous Ac-CGGASIKVAVS-OH-modified PHEMA scaffolds significantly increased the number of attached cells and their growth area on the hydrogel surface in the absence and in the presence of serum in the culture medium. Additionally, the Ac-CGGASIKVAVS-OH peptide supported the attachment, proliferation, differentiation and process spreading of human fetal neural stem cells during the first two weeks of expansion and contributed to the formation of a high percentage of more mature neural cells after four weeks of expansion. The Ac-CGGASIKVAVS-OH modification of superporous P(HEMA-AEMA) hydrogels improves cell adhesive properties and promotes neural stem cell differentiation.

The thiazolidinedione pioglitazone increases cholesterol biosynthetic gene expression in primary cortical neurons by a PPARgamma-independent mechanism.

In a recent clinical study, the thiazolidinedione (TZD) pioglitazone (Actos was reported to preserve cognitive function in patients with mild to moderate Alzheimer's disease and type II diabetes mellitus. TZDs are agonists of the nuclear hormone receptor peroxisome proliferator-activated receptor-gamma (PPARgamma), are peripheral insulin sensitizers, and have recently been reported to increase mitochondrial biogenesis in the central nervous system and dendritic spine density. We report a transcriptional profile of the TZD pioglitazone and the non-TZD PPARgamma agonist GW347845 in primary cortical culture. We observed that pioglitazone, but not GW347845, increased cholesterol biosynthetic and lipogenic gene expression after 6 h, and the expression of the cholesterol efflux transporters Abca1 and Abcg1 after 24 h. Co-treatment of pioglitazone with the PPARgamma antagonist GW9662 did not significantly reduce these effects, suggesting a PPARgamma-independent mechanism. These findings suggest a novel effect of TZDs in neurons that may be of relevance as a novel approach against Alzheimer's disease.