Graham Cocks

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

IntroductionA 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).MethodsA 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.ResultsTransplanted 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.ConclusionsThe 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.

Single nucleotide polymorphism in the neuroplastin locus associates with cortical thickness and intellectual ability in adolescents

Despite the recognition that cortical thickness is heritable and correlates with intellectual ability in children and adolescents, the genes contributing to individual differences in these traits remain unknown. We conducted a large-scale association study in 1583 adolescents to identify genes affecting cortical thickness. Single-nucleotide polymorphisms (SNPs; n=54 837) within genes whose expression changed between stages of growth and differentiation of a human neural stem cell line were selected for association analyses with average cortical thickness. We identified a variant, rs7171755, associating with thinner cortex in the left hemisphere (P=1.12 × 10−7), particularly in the frontal and temporal lobes. Localized effects of this SNP on cortical thickness differently affected verbal and nonverbal intellectual abilities. The rs7171755 polymorphism acted in cis to affect expression in the human brain of the synaptic cell adhesion glycoprotein-encoding gene NPTN. We also found that cortical thickness and NPTN expression were on average higher in the right hemisphere, suggesting that asymmetric NPTN expression may render the left hemisphere more sensitive to the effects of NPTN mutations, accounting for the lateralized effect of rs7171755 found in our study. Altogether, our findings support a potential role for regional synaptic dysfunctions in forms of intellectual deficits.

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.

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.

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

IntroductionThe 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.MethodsClonal 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.ResultsWe 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 Ca2+ channels and spontaneous calcium oscillations, and (c) stably engraft into lesioned rat spinal cord without tumorogenicity.ConclusionsWe 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.

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.

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.

Neural Plasticity and Neurogenesis in Mental Disorders

Adult neurogenesis, the continuous generation of newborn neurons in discrete regions of the brain throughout life, is now widely regarded as a fundamental mechanism of neural plasticity. This phenomenon, and in particular the integration of new neurons into the dentate gyrus of the hippocampus, has been associated with the regulation of important but quite subtle and complex aspects of cognition and memory formation. In addition to its important role in the healthy adult brain, adult neurogenesis has also been of considerable interest to the research community because of a growing body of literature implicating its deregulation in mental disorders. Perhaps the most high-profile example of this is the putative role of reduced neurogenesis in the adult hippocampus in the pathogenesis of major depression. However, despite the fact that adult neurogenesis is confined to very discrete regions of the brain, and the established role of adult neurogenesis in major depression notwithstanding, it is becoming increasingly apparent that the deregulation of neurogenesis may impact a much wider range of mental disorders. In this special issue on neural plasticity and neurogenesis in mental disorders, we are pleased to present a series of articles that reflect the broad scope of psychiatric and neurological conditions that are potentially impacted by abnormalities in neurogenesis and neuroplasticity. L. Varela-Nallar et al. (“Andrographolide Stimulates Neurogenesis in the Adult Hippocampus”) report new data in this issue demonstrating the effects of Andrographolide (ANDRO) on adult hippocampal neurogenesis, a compound the authors have previously identified as a GSK3beta inhibitor. The regulation of beta-catenin by GSK3beta, which is modulated by the Wnt signalling pathway, is well established in playing an important role in regulating neural stem cell proliferation. The authors demonstrate that ANDRO increases adult hippocampal neurogenesis in young and aged mice and in a transgenic mouse model of Alzheimers disease. Impaired neurogenesis has been associated with early pathological changes in Alzheimers disease and is also reduced in normal aging. Novel small molecules such as ANDRO that can upregulate adult neurogenesis are therefore of potentially important therapeutic interest in reducing cognitive decline. D. Feldman et al. (“Developmental Dynamics of Rett Syndrome”) review the role of MeCP2 in abnormal developmental neurogenesis and neural plasticity in Rett syndrome across the lifespan. Rett syndrome is a disorder that has until recently been largely characterized as arising from abnormalities in neural plasticity in postnatal development. In their review article in this issue, D. Feldman et al. also provide insight into the more recent identification of earlier pathological events involving developmental neurogenesis. These earlier effects of MeCP2 loss of function on the generation and integration of neurons in the developing brain are also in line with recent research on other closely related neurodevelopmental conditions such as Autistic Spectrum Disorder where a growing body of literature has also begun to identify aberrant developmental neurogenesis to be an important pathological process, in addition to abnormalities in activity-dependent synaptic plasticity postnatally. Such insights will be important for developing therapeutic strategies to target abnormalities in different aspects of neuronal dysfunction in Rett syndrome arising from these different stages of development. Defects in synaptic plasticity have been implicated in the pathogenesis of Schizophrenia. J. Gonzalez-Heydrich et al. (“N100 Repetition Suppression Indexes Neuroplastic Defects in Clinical High Risk and Psychotic Youth”) present original data to begin to validate auditory N100 adaptation as a biomarker of clinical high risk and progression to psychosis individuals. Developing biomarkers such as this as an indication of abnormal neural plasticity, particularly in the prodromal stage of psychosis, will be of great value in assessing therapeutic strategies in clinical trial settings in the future. Despite the considerable progress being made in understanding the role of adult neurogenesis and neuroplasticity in mental disorders, in some areas, there is also a great deal of uncertainty about the nature of such associations. These articles therefore reflect upon and elucidate what is currently known but also often highlight the uncertainty that exists and the continuing work that needs to be done to understand these associations. In this respect, G. Perna et al. (“Are Anxiety Disorders Associated with Accelerated Aging? A Focus on Neuroprogression”) present a valuable novel review of the literature looking at the association of anxiety disorders and aging. This review examines a wide range of potential consequences of anxiety disorder from reduced adult neurogenesis and altered neuroplasticity through to increased beta-amyloid production, telomere shortening, oxidative stress, and chronic inflammation. The paper highlights the need for more work to be undertaken to establish these potentially very serious consequences of the already debilitating condition of anxiety disorder. Finally, A. A. Marques et al. (“Gender Differences in the Neurobiology of Anxiety: Focus on Adult Hippocampal Neurogenesis”) present an insightful review of gender differences in anxiety and potential differences in adult hippocampal neurogenesis between the sexes. This review provides a useful insight into the neurobiological processes that influence these differences and the important implications for the use of animal models of anxiety disorders. The contributors to this special issue provide a valuable snapshot of the range of mental disorders associated with abnormalities in neural plasticity and neurogenesis. These articles provide important insights into our current understanding of the role of neural plasticity and neurogenesis in mental disorders, highlighting the current gaps in our knowledge and providing a valuable perspective on the future directions of the field. Graham Cocks Mauro G. Carta Oscar Arias-Carrion Antonio E. Nardi