Induced pluripotent stem cells (iPSCs) have previously been shown to retain some of the epigenetic features associated with the tissues from which they were derived, which in turn can help the iPSCs to differentiate towards similar cell fates. Here we investigate whether human iPSCs (hiPSC) derived from the lateral ganglionic eminence (LGE) retain sufficient epigenetic features of their original tissue to enhance their capacity to differentiate into striatal medium spiny neurons (MSN) compared to isogenic controls. We identify a subtle methylation signature within these isogenic hiPSCs, and observe line specific capacities to produce MSN-like cells and their subtypes. We directly compare these cells to authentic LGE derived MSNs and identify large differences in DNA methylation between these populations. Using single cell RNA sequencing, we also find subpopulations that do not reflect the transcriptional profile of authentic MSNs. While we observe some potential epigenetic “benefits” within our LGE derived hiPSC-MSNs (e.g. reduced hypomethylation of off-target pathways), we did not observe an improved propensity to differentiate towards an MSN-like fate. Overall, this work highlights that pluripotent stem cells are unique in their individual capacity for differentiation, that subtle differences between cell lines can have far reaching effects on the final cell product, and that while hPSC-MSNs can model many aspects of MSN development, there are still epigenetic and transcriptional differences that limit the full recapitulation of the authentic LGE-derived MSN phenotype. ### Competing Interest Statement The authors have declared no competing interest.

The Stroop test is a widely used neuropsychological test measuring attention and conflict resolution, which shows sensitivity across a range of diseases, including Alzheimer's, Parkinson's and Huntington's diseases. A rodent analogue of the Stroop test, the Response-Conflict task (rRCT), allows for systematic investigation of the neural systems underpinning performance in this test. Little is known about the involvement of the basal ganglia in this neural process. The aim of this study was to use the rRCT to determine whether striatal subregions are recruited during conflict resolution processing. To achieve this, rats were exposed to Congruent or Incongruent stimuli in the rRCT and the expression patterns of the immediate early gene Zif268 were analysed throughout cortical, hippocampal and basal ganglia subregions. The results confirmed the previously reported involvement of prefrontal cortical and hippocampal regions, as well as identifying a specific role for the dysgranular (but not granular) retrosplenial cortex in conflict resolution. Finally, performance accuracy correlated significantly with reduced neural activation in the dorsomedial striatum. Involvement of the basal ganglia in this neural process has not previously been reported. These data demonstrate that the cognitive process of conflict resolution requires not only prefrontal cortical regions, but also recruits the dysgranular retrosplenial cortex and the medial region of the neostriatum. These data have implications for understanding the neuroanatomical changes that underpin impaired Stroop performance in people with neurological disorders.

AbstractWhite matter (WM) volume loss has been reported in people with Huntington’s disease (HD), but the cellular basis of this deficit remains to be elucidated. To address this, we assessed ex vivo WM microstructure in the transgenic R6/1 mouse model of HD with magnetic resonance imaging (MRI) and studied the neurobiological basis of the MRI brain signals with histological and electron microscopy analyses in a separate cohort of age- and sex-matched mice. Differences in the macromolecular proton fraction (MPF) from quantitative magnetization transfer (qMT) as a proxy myelin measure, and the intra-axonal signal fraction (FR) from the composite hindered and restricted model of diffusion (CHARMED) as a proxy marker of axon density, were assessed alongside diffusion tensor imaging (DTI) parameters. A tractometry approach was employed to inspect region-specific differences across the corpus callosum (CC). Furthermore, voxel-based morphometry (VBM) and tract-based spatial statistics (TBSS) were used to explore brain-wise WM macro- and microstructure abnormalities. To gain insight into disease-associated impairments in attentional and visuospatial processing, a third cohort of age-matched mice was assessed with the 5-choice serial reaction time task (5-CSRTT). We report cognitive impairments in R6/1 mice and, by evaluating MRI and light and electron microscopy results, we show that this HD mouse model presents disruptions in axonal morphology (i.e. less complex, thinner axons) and organization (i.e. more densely packed axons). Furthermore, we show that, at least early in disease progression, R6/1 mice present a reduction in the expression or content of myelin-associated proteins without significant alterations in the structure of myelin sheaths. Finally, our findings indicate that neuroinflammation-driven glial and axonal swelling might also affect this mouse model early in disease progression. Crucially, we demonstrate the potential of FR, an in vivo estimate of axon density, as a novel MRI biomarker of HD-associated changes in WM microstructure.

Objectives Clinical trials of intracerebral cell replacement therapy (CRT) yield inconsistent results owing to poor graft survival and ectopic graft placement. A paucity of available CE marked specialised delivery systems and a lack of reliable delivery protocols could be major contributing factors to both phenomena. Here, we aimed to investigate current needle delivery strategies in-vitro and in a large animal model in pigs. Design In-vitro laboratory and in-vivo pigs experiments. Subjects In-vitro Agarose gel In-vivo: 4 white Landracer pigs. Methods In-vitro: Human Embryonic Kidney cells expressing luciferase and 0.6% agarose gel were used to test 3 delivery strategies: Bracelet deposit, Large deposit in a pre–formed tract, Multiple deposits in a pre–formed tract. In-vivo: Pigs underwent MRI- guided Human Foetal Luciferase-transduced cell transplantation into the putamen and thalamus. Post-operative MRI, Bioluminescence imaging (BLI) and histology were used to identify graft location and viability. Results Using a commercially available needle delivery system, significant reflux of deposits was noted in all 3 delivery strategies during in-vitro testing. Depositing into a preformed tract yielded the best delivery, and was therefore used for in-vivo testing. Studies in pigs using MRI and BLI confirmed significant reflux and ectopic deposition of grafts. Conclusions Simple needle delivery systems appear to suffer from significant reflux and ectopic cell deposition. This may adversely affect the outcomes of CRT trials in humans.

Background Limited data suggests that an altered metabolic and cardiorespiratory exercise response may affect exercise performance in individuals with Huntington's disease (HD). There is no clear exploration of the response in individuals at different stages of the disease or in relation to genetic markers. This study aimed to examine the exercise response and recovery of HD participants, and the relationship to genetic and clinical markers. Method HD gene-positive participants (n = 31; 9 pre-manifest; 22 manifest HD) and a healthy control group (n = 29) performed an incremental exercise test until exhaustion. Performance, cardiorespiratory, metabolic and perceptual responses to exercise were determined from a maximal cycle ergometer test throughout the exercise test and during a recovery period. Results During sub-maximal exercise, metabolic (lactate levels, oxygen uptake) and cardiorespiratory markers (heart rate) were elevated in HD participants compared to controls. Lactate elevation was specific to pre-manifest HD participants. Work capacity was reduced in both pre-manifest and manifest HD participants with tests terminated with no difference in metabolic, perceptual or cardiorespiratory markers. Submaximal oxygen uptake was correlated with motor score, whilst peak measures were unrelated to genetic or clinical markers. Heart rate recovery was attenuated in pre-manifest and manifest HD participants. Conclusions Our findings confirm metabolic and cardiorespiratory deficits reduce exercise performance and affect recovery from an early stage in HD, with submaximal deficits related to phenotypic expression. Exercise capacity appears to be limited by an altered movement economy, thus clinicians should consider an altered exercise response and recovery may affect prescription in HD.

Background and purposeHuntington's disease (HD) is an autosomal dominant, neurodegenerative movement disorder, typically characterized by chorea. Dystonia is also recognized as part of the HD motor phenotype, although little work detailing its prevalence, distribution, severity and impact on functional capacity has been published to date.MethodsPatients (>18 years of age) were recruited from the Cardiff (UK) HD clinic, each undergoing a standardized videotaped clinical examination and series of functional assessment questionnaires (Unified Huntington's Disease Rating Scale, Burke-Fahn-Marsden Dystonia Rating Scale and modified version of the Toronto Western Spasmodic Torticollis Rating Scale). The presence and severity of dystonia were scored by four independent neurologists using the Burke-Fahn-Marsden Dystonia Rating Scale and Unified Huntington's Disease Rating Scale. Statistical analysis included Fisher's exact test, Wilcoxon test, anova and calculation of correlation coefficients where appropriate.ResultsForty-eight patients [91% (48/53)] demonstrated evidence of dystonia, with the highest prevalence in the left upper limb (n = 44, 83%), right upper limb most severely affected and eyes least affected. Statistically significant positive correlations (P < 0.05) were observed between dystonia severity and increasing HD disease stage and motor disease duration. Deterioration in functional capacity also correlated with increasing dystonia severity. No significant relationship was observed with age at motor symptom onset or CAG repeat length.ConclusionsWe report a high prevalence of dystonia in adult patients with HD, with worsening dystonia severity with increasing HD disease stage and motor disease duration. The recognition and management of dystonic symptoms in routine clinical practice will aid overall symptomatic treatment and functional improvement.

Background: Patients suffering from Parkinson's disease (PD) display cognitive and neuropsychiatric dysfunctions, especially with disease progression. Although these impairments have been reported to impact more heavily upon a patient's quality of life than any motor dysfunctions, there are currently no interventions capable of adequately targeting these non-motor deficits.Objectives: Utilizing a rodent model of PD, we investigated whether cell replacement therapy, using intrastriatal transplants of human-derived ventral mesencephalic (hVM) grafts, could alleviate cognitive and neuropsychiatric, as well as motor, dysfunctions.Methods: Rats with unilateral 6-hydroxydopamine lesions to the medial forebrain bundle were tested on a complex operant task that dissociates motivational, visuospatial and motor impairments sensitive to the loss of dopamine. A subset of lesioned rats received intrastriatal hVM grafts of similar to 9 weeks gestation. Post-graft, rats underwent repeated drug-induced rotation tests and were tested on two versions of the complex operant task, before post-mortem analysis of the hVM tissue grafts.Results: Post-graft behavioural testing revealed that hVM grafts improved non-motor aspects of task performance, specifically visuospatial function and motivational processing, as well as alleviating motor dysfunctions.Conclusions: We report the first evidence of human VM cell grafts alleviating both non-motor and motor dysfunctions in an animal model of PD. This intervention, therefore, is the first to improve cognitive and neuropsychiatric symptoms long-term in a model of PD. (C) 2016 The Authors. Published by Elsevier Inc.

Identifying the steps involved in striatal development is important both for understanding the striatum in health and disease, and for generating protocols to differentiate striatal neurons for regenerative medicine. The most prominent neuronal subtype in the adult striatum is the medium spiny projection neuron (MSN), which constitutes more than 85% of all striatal neurons and classically expresses DARPP-32. Through a microarray study of genes expressed in the whole ganglionic eminence (WGE: the developing striatum) in the mouse, we identified the gene encoding the transcription factor Forkhead box protein P1 (FoxP1) as the most highly up-regulated gene, thus providing unbiased evidence for the association of FoxP1 with MSN development. We also describe the expression of FoxP1 in the human fetal brain over equivalent gestational stages. FoxP1 expression persisted through into adulthood in the mouse brain, where it co-localised with all striatal DARPP-32 positive projection neurons and a small population of DARPP-32 negative cells. There was no co-localisation of FoxP1 with any interneuron markers. FoxP1 was detectable in primary fetal striatal cells following dissection, culture, and transplantation into the adult lesioned striatum, demonstrating its utility as an MSN marker for transplantation studies. Furthermore, DARPP-32 expression was absent from FoxP1 knock-out mouse WGE differentiated in vitro, suggesting that FoxP1 is important for the development of DARPP-32-positive MSNs. In summary, we show that FoxP1 labels MSN precursors prior to the expression of DARPP-32 during normal development, and in addition suggest that FoxP1 labels a sub-population of MSNs that are not co-labelled by DARPP-32. We demonstrate the utility of FoxP1 to label MSNs in vitro and following neural transplantation, and show that FoxP1 is required for DARPP-32 positive MSN differentiation in vitro.

Background The course of Huntington’s disease (HD) is slowly progressive, typically over 20–30 years with little in the way of acute fluctuations, so any acute deterioration in either cognitive or motor symptoms justifies a search for an inter current illness. Here we describe three cases in which serious comorbidities were mistakenly ascribed to deterioration of HD. Case histories Case 1: A 64 year old lady with moderately advanced HD was still able to mobilise independently. Following a fall, she was admitted to hospital with increased chorea and some weakness, and a urinary tract infection was initially suspected. She went on to develop further weakness and urinary retention and eventually turned out to have a C2-7 spinal haematoma causing cord compression. She underwent decompression with some improvement. Case 2: A 58 year old man developed progressive weakness, reduced chorea and urinary retention following a fall. This was initially ascribed to progression of his HD and he was treated conservatively, but a subsequent MRI demonstrated complete dislocation of his cervical cord at C5. Surgery was not possible and he was treated palliatively. Case 3: A 42 year old woman presented with vomiting, reduced oral intake, severe constipation, confusion, restlessness and insomnia. Symptoms were attributed to her HD, but turned out to be secondary to primary hyperparathyroidism and improved with treatment. Conclusions All three cases demonstrate the diagnostic difficulties of clinical deterioration in a chronic progressive condition like HD. In all cases the initial symptoms did not trigger any suspicion of comorbidity amongst admitting physicians until further deterioration had taken place and concerns were raised by a specialist HD team. This led to delayed diagnosis which may have been associated with poor outcomes in two of the cases. Strategies for better recognising comorbidities in HD are discussed.

Background Diffusion MRI is a non-invasive imaging technique to investigate white matter microstructure and has previously shown microstructural changes in the corpus callosum of Huntington’s disease (HD) patients. However, as HD is associated with grey and white matter atrophy, cerebrospinal fluid (CSF) may cause partial volume artefacts in the diffusion MRI metrics, leading to misleading results. CSF contamination is most problematic in regions close to the ventricles, such as the corpus callosum. Aim To model the effects of partial volume contamination on diffusion MRI metrics in the corpus callosum of HD patients, and mice. Methods The effect of CSF contamination was tested in diffusion MRI images (30 DW / 3 B0 directions, b = 1000 s/mm2) acquired in both a patient cohort (12 pre- and early-symptomatic HD patients, 8 healthy age, gender and education matched controls) and a mouse model of HD (21 HdhQ150 knock-in male mice / 23 age-matched wild-type mice). Partial volume correction was performed post-hoc (Pasternak et al, 2009), tractography was performed and mean diffusion tensor-based parameters obtained for the corpus callosum. Results In the mice, there were no differences in the diffusion MRI metrics at 7 months old prior to symptom onset. At 19-months, there was a significant difference in mean diffusivity in the corpus callosum between the HD and wild-type mice, suggesting white matter microstructural changes occur after symptom onset. Crucially, this difference was no longer significant after correcting for CSF contamination. Analysis of the patient data is on-going. Conclusions These findings suggest researchers should be cautious when interpreting diffusion MRI results in HD, and should perform appropriate corrections for CSF contamination.

Connexin43 (Cx43) is the most widely and abundantly expressed gap junction (GJ) protein and it is strongly associated with the regulation of cell cycle progression. Emerging roles for Cx43 in cell adhesion and migration during neural differentiation have also been recently recognized, and this has emphasized the involvement of Cx43 in different physiological process beyond its role as a GJ protein. In this study, we explore the function of Cx43 in the differentiation of human neural progenitor cells (hNPCs) using viral vectors that mediate the overexpression or knockdown of the protein. Results showed that in the absence of this protein fetal cortex-derived hNPCs differentiated toward a neuronal phenotype at expenses of a glial phenotype. Furthermore, the silencing of Cx43 did not affect hNPC proliferation rate or numbers of apoptotic cells. The increase in the number of neurons was not recapitulated when GJ intercellular communications were pharmacologically blocked, and this suggested that Cx43 was influencing hNPCs differentiation with a GJ-independent effect. In addition, Cx43 knockdown significantly increased β -catenin signaling, which has been shown to regulate the transcription of pro-neuronal genes during embryonic neural development. Our results add further support to the hypothesis that Cx43 protein itself regulates key signaling pathways during development and neurogenesis beyond its role as GJ protein.

Huntington’s disease (HD) is a neurodegenerative disease caused by a mutation in the huntingtin gene (HTT). The extended CAG repeat ultimately leads to loss of medium spiny neurons (MSNs) in the striatum of the HD brain. Cell replacement therapy using primary human fetal tissue as a source of “genuine” MSNs has shown ‘proof of principle’ as a strategy to treat this genetically inherited disease1. However, renewable cell sources need to be identified to overcome the ethical and logistical issues that are associated with using human fetuses. Here we attempted to generate iPS cells by introducing reprogramming factors using the piggyBac Transposon2 transduction system in human fetal fibroblasts and fetal neural stem cells. We wish to test the hypothesis that these cells are more easily reprogrammable and/or are more readily directed towards an MSN phenotype. The established iPS cell lines were similar to human embryonic stem (ES) cells in terms of their morphology, surface antigen, and proliferation. These iPS cells lines have been successfully manipulated to differentiate into MSNs in culture according to their expression of standard molecular markers of premature and mature MSNs - Ctip2 and Darrp32. Differentiation following transplantation into the quinolinic acid (QA) lesion model showed that grafts of these striatal progenitors derived from human fetal iPS cells could differentiate into neural progenitors according to expression of human nuclei marker (HuNu) and nestin.

Aberrant striatal function results in an array of physiological symptoms, including impaired consummatory and regulatory behaviours, which can lead to weight loss and dehydration. It was hypothesised, therefore, that cell loss in the neostriatum may contribute to altered fluid intake by regulating physiological signals related to dehydration status. To test this theory, rats with lesions of the lateral neostriatum and sham controls underwent a series of physiological challenges, including the experimental induction of intracellular and intravascular dehydration. No baseline differences in prandial or non-prandial drinking were observed, nor were differences in locomotor activity evident between groups. Furthermore, intracellular dehydration increased water intake in lesion rats in a manner comparable to sham rats. Interestingly, a specific impairment was evident in lesion rats after subcutaneous injection of poly-ethylene glycol was used to induce intravascular dehydration, such that lesion rats failed to adapt their water intake to this physiological change. The results suggest that the striatal lesions resulted in regulatory dysfunction by impairing motivational control over compensatory ingestive behaviour after intravascular hydration, while the physiological signals related to dehydration remain intact. Loss of these cells in neurodegenerative disorders, such Huntington's disease, may contribute to regulatory changes evident in the course of the disease.

The central nervous system is composed of the brain and the spinal cord. The brain is a complex organ that processes and coordinates activities of the body in bilaterian, higher-order animals. The development of the brain mirrors its complex function as it requires intricate genetic signalling at specific times, and deviations from this can lead to brain malformations such as anencephaly. Research into how the CNS is specified and patterned has been studied extensively in chick, fish, frog, and mice, but findings from the latter will be emphasised here as higher-order mammals show most similarity to the human brain. Specifically, we will focus on the embryonic development of an important forebrain structure, the striatum (also known as the dorsal striatum or neostriatum). Over the past decade, research on striatal development in mice has led to an influx of new information about the genes involved, but the precise orchestration between the genes, signalling molecules, and transcription factors remains unanswered. We aim to summarise what is known to date about the tightly controlled network of interacting genes that control striatal development. This paper will discuss early telencephalon patterning and dorsal ventral patterning with specific reference to the genes involved in striatal development.

"Proof-of-principle" that cell replacement therapy works for neurodegeneration has been reported, but only using donor cells collected from fetal brain tissue obtained from surgical terminations of pregnancy. Surgical terminations of pregnancy represent an increasingly limited supply of donor cells due to the tendency towards performing medical termination in much of Europe. This imposes a severe constraint on further experimental and clinical cell transplantation research. Therefore, we explore here the feasibility of using medical termination tissue as a donor source. Products of conception were retrieved from surgical terminations over the last 7 years and from medical terminations over the last 2.5 years. The number of collections that yielded fetal tissue, viable brain tissue, and identifiable brain regions (ganglionic eminence, ventral mesencephalon, and neocortex) were recorded. We studied cell viability, cell physiological properties, and differentiation potential both in vitro and following transplantation into the central nervous system of rodent models of neurodegenerative disease. Within equivalent periods, we were able to collect substantially greater numbers of fetal remains from medical than from surgical terminations of pregnancy, and the medical terminations yielded a much higher proportion of identifiable and dissectible brain tissue. Furthermore, we demonstrate that harvested cells retain the capacity to differentiate into neurons with characteristics appropriate to the region from which they are dissected. We show that, contrary to widespread assumption, medical termination of pregnancy-derived fetal brain cells represent a feasible and more readily available source of human fetal tissue for experimental cell transplantation with the potential for use in future clinical trials in human neurodegenerative disease.