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Human pluripotent stem cells (hPSCs) have generated unprecedented interest in the scientific community, given their potential applications in regenerative medicine, disease modeling, toxicology and drug screening. However, hPSCs are prone to acquire genomic alterations in vitro, mainly due to suboptimal culture conditions and inappropriate routines to monitor genome integrity. This poses a challenge to both the safety of clinical applications and the reliability of basic and translational hPSC research. In this study, we aim to investigate if the implementation of a Quality Management System (QMS) such as ISO9001:2015 to ensure reproducible and standardized cell culture conditions and genomic screening strategies can decrease the prevalence of genomic alterations affecting hPSCs used for research applications. To this aim, we performed a retrospective analysis of G-banding karyotype and Comparative Genomic Hybridization array (aCGH) data generated by our group over a 5-year span of different hESC and hiPSC cultures. This work demonstrates that application of a QMS to standardize cell culture conditions and genomic monitoring routines leads to a striking improvement of genomic stability in hPSCs cultured in vitro, as evidenced by a reduced probability of potentially pathogenic chromosomal aberrations and subchromosomal genomic alterations. These results support the need to implement QMS in academic laboratories performing hPSC research.

Human pluripotent stem cells (hPSCs) are a powerful tool for modelling human development. In recent years, hPSCs have become central in cell-based therapies for neurodegenerative diseases given their potential to replace affected neurons. However, directing hPSCs into specific neuronal types is complex and requires an accurate protocol that mimics endogenous neuronal development. Here we describe step-by-step a fast feeder-free neuronal differentiation protocol to direct hPSCs to mature forebrain neurons in 37 days in vitro (DIV). The protocol is based upon a combination of specific morphogens, trophic and growth factors, ions, neurotransmitters and extracellular matrix elements. A human-induced PSC line (Ctr-Q33) and a human embryonic stem cell line (GEN-Q18) were used to reinforce the potential of the protocol. Neuronal activity was analysed by single-cell calcium imaging. At 8 DIV, we obtained a homogeneous population of hPSC-derived neuroectodermal progenitors which self-arranged in bi-dimensional neural tube-like structures. At 16 DIV, we generated hPSC-derived neural progenitor cells (NPCs) with mostly a subpallial identity along with a subpopulation of pallial NPCs. Terminal in vitro neuronal differentiation was confirmed by the expression of microtubule associated protein 2b (Map 2b) by almost 100% of hPSC-derived neurons and the expression of specific-striatal neuronal markers including GABA, CTIP2 and DARPP-32. HPSC-derived neurons showed mature and functional phenotypes as they expressed synaptic markers, voltage-gated ion channels and neurotransmitter receptors. Neurons displayed diverse spontaneous activity patterns that were classified into three major groups, namely “high”, “intermediate” and “low” firing neurons. Finally, transplantation experiments showed that the NPCs survived and differentiated within mouse striatum for at least 3 months. NPCs integrated host environmental cues and differentiated into striatal medium-sized spiny neurons (MSNs), which successfully integrated into the endogenous circuitry without teratoma formation. Altogether, these findings demonstrate the potential of this robust human neuronal differentiation protocol, which will bring new opportunities for the study of human neurodevelopment and neurodegeneration, and will open new avenues in cell-based therapies, pharmacological studies and alternative in vitro toxicology. Electronic supplementary material The online version of this article (10.1007/s12035-020-01907-4) contains supplementary material, which is available to authorized users.

Changes in the transcription factor (TF) expression are critical for brain development, and they may also underlie neurodevelopmental disorders. Indeed, T-box brain1 (Tbr1) is a TF crucial for the formation of neocortical layer VI, and mutations and microdeletions in that gene are associated with malformations in the human cerebral cortex, alterations that accompany autism spectrum disorder (ASD). Interestingly, Tbr1 upregulation has also been related to the occurrence of ASD-like symptoms, although limited studies have addressed the effect of increased Tbr1 levels during neocortical development. Here, we analysed the impact of Tbr1 misexpression in mouse neural progenitor cells (NPCs) at embryonic day 14.5 (E14.5), when they mainly generate neuronal layers II–IV. By E18.5, cells accumulated in the intermediate zone and in the deep cortical layers, whereas they became less abundant in the upper cortical layers. In accordance with this, the proportion of Sox5+ cells in layers V–VI increased, while that of Cux1+ cells in layers II–IV decreased. On postnatal day 7, fewer defects in migration were evident, although a higher proportion of Sox5+ cells were seen in the upper and deep layers. The abnormal neuronal migration could be partially due to the altered multipolar-bipolar neuron morphologies induced by Tbr1 misexpression, which also reduced dendrite growth and branching, and disrupted the corpus callosum. Our results indicate that Tbr1 misexpression in cortical NPCs delays or disrupts neuronal migration, neuronal specification, dendrite development and the formation of the callosal tract. Hence, genetic changes that provoke ectopic Tbr1 upregulation during development could provoke cortical brain malformations.

There has been substantial progress in the development of regenerative medicine strategies for CNS disorders over the last decade, with progression to early clinical studies for some conditions. However, there are multiple challenges along the translational pipeline, many of which are common across diseases and pertinent to multiple donor cell types. These include defining the point at which the preclinical data are sufficiently compelling to permit progression to the first clinical studies; scaling-up, characterization, quality control and validation of the cell product; design, validation and approval of the surgical device; and operative procedures for safe and effective delivery of cell product to the brain. Furthermore, clinical trials that incorporate principles of efficient design and disease-specific outcomes are urgently needed (particularly for those undertaken in rare diseases, where relatively small cohorts are an additional limiting factor), and all processes must be adaptable in a dynamic regulatory environment. Here we set out the challenges associated with the clinical translation of cell therapy, using Huntington's disease as a specific example, and suggest potential strategies to address these challenges. Huntington's disease presents a clear unmet need, but, importantly, it is an autosomal dominant condition with a readily available gene test, full genetic penetrance and a wide range of associated animal models, which together mean that it is a powerful condition in which to develop principles and test experimental therapeutics. We propose that solving these challenges in Huntington's disease would provide a road map for many other neurological conditions. This white paper represents a consensus opinion emerging from a series of meetings of the international translational platforms Stem Cells for Huntington's Disease and the European Huntington's Disease Network Advanced Therapies Working Group, established to identify the challenges of cell therapy, share experience, develop guidance, and highlight future directions, with the aim to expedite progress towards therapies for clinical benefit in Huntington's disease.Rosser et al. discuss the challenges associated with clinical translation of cell therapies, using Huntington's disease as the primary example. They suggest strategies to address these challenges, based on the consensus view emerging from international workshops and discussion within the platform 'Stem Cells for Huntington's Disease'.

We evaluated the administration of ARI-0001 cells (chimeric antigen receptor T cells targeting CD19) in adult and pediatric patients with relapsed/refractory CD19(+) malignancies. Patients received cyclophosphamide and fludarabine followed by ARI-0001 cells at a dose of 0.4–5 × 10(6) ARI-0001 cells/kg, initially as a single dose and later split into 3 fractions (10%, 30%, and 60%) with full administration depending on the absence of cytokine release syndrome (CRS). 58 patients were included, of which 47 received therapy: 38 with acute lymphoblastic leukemia (ALL), 8 with non-Hodgkin’s lymphoma, and 1 with chronic lymphocytic leukemia. In patients with ALL, grade ≥3 CRS was observed in 13.2% (26.7% before versus 4.3% after the amendment), grade ≥3 neurotoxicity was observed in 2.6%, and the procedure-related mortality was 7.9% at day +100, with no procedure-related deaths after the amendment. The measurable residual disease-negative complete response rate was 71.1% at day +100. Progression-free survival was 47% (95% IC 27%–67%) at 1 year: 51.3% before versus 39.5% after the amendment. Overall survival was 68.6% (95% IC 49.2%–88%) at 1 year. In conclusion, the administration of ARI-0001 cells provided safety and efficacy results that are comparable with other academic or commercially available products. This trial was registered as ClinicalTrials.gov: NCT03144583.

Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal subtypes and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time courses of functional maturation that are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to postsynaptic receptor maturation. Here, we describe a simple protocol that employs the sequential addition of just two supplemented media that have been formulated to separate the two key phases of neural differentiation, the neurogenesis and synaptogenesis, each characterized by different signaling requirements. Employing these media, this new protocol synchronized neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials; moreover, they exhibited augmented: 1) spontaneous electrical activity; 2) regenerative induced action potential train activity; 3) Na+ current availability, and 4) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAA receptor phenotype that was demonstrated by Ca2+ imaging and the ability of GABAA receptor blockers to evoke seizurogenic network activity in multielectrode array recordings. Furthermore, since this protocol can exploit expanded and frozen prepatterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens.

In Huntington’s disease (HD), striatal medium spiny neurons (MSNs) are particularly sensitive to the presence of a CAG repeat in the huntingtin (HTT) gene. However, there are many evidences that cells from the peripheral immune system and central nervous system (CNS) immune cells, namely microglia, play an important role in the etiology and the progression of HD. However, it remains unclear whether MSNs neurodegeneration is mediated by a non-cell autonomous mechanism. The homeostasis in the healthy CNS is maintained by several mechanisms of interaction between all brain cells. Neurons can control microglia activation through several inhibitory mechanisms, such as the CD200–CD200R1 interaction. Due to the complete lack of knowledge about the CD200–CD200R1 system in HD, we determined the temporal patterns of CD200 and CD200R1 expression in the neocortex, hippocampus and striatum in the HD mouse models R6/1 and HdhQ111/7 from pre-symptomatic to manifest stages. In order to explore any alteration in the peripheral immune system, we also studied the levels of expression of CD200 and CD200R1 in whole blood. Although CD200R1 expression was not altered, we observed and increase in CD200 gene expression and protein levels in the brain parenchyma of all the regions we examined, along with HD pathogenesis in R6/1 mice. Interestingly, the expression of CD200 mRNA was also up-regulated in blood following a similar temporal pattern. These results suggest that canonical neuronal–microglial communication through CD200–CD200R1 interaction is not compromised, and CD200 up-regulation in R6/1 brain parenchyma could represent a neurotrophic signal to sustain or extend neuronal function in the latest stages of HD as pro-survival mechanism., This study was supported by grants from the Ministerio de Ciencia, Innovación y Universidades (Spain), (RTI2018-099001-B-I00 to JMC); Instituto de Salud Carlos III, Ministerio de Ciencia, Innovacio´n y Universidades and European Regional Development Fund (ERDF) [CIBERNED and RETICS (Red de Terapia Celular, RD16/0011/ 0012 to JMC)], Spain; Generalitat de Catalunya (2017SGR-1408 to J. M. C.), Spain; the CHDI Foundation (A12076 to JMC), USA; and ADVANCE (CAT) with the support of ACCIO´ (Catalonia Trade & Investment; Generalitat de Catalunya) and the European Community under the Catalonian ERDF operational program 2014-2020], Spain. T

Kv7 channels determine the resting membrane potential of neurons and regulate their excitability. Even though dysfunction of Kv7 channels has been linked to several debilitating childhood neuronal disorders, the ontogeny of the constituent genes, which encode Kv7 channels (KNCQ), and expression of their subunits have been largely unexplored. Here, we show that developmentally regulated expression of specific KCNQ mRNA and Kv7 channel subunits in mouse and human striatum is crucial to the functional maturation of mouse striatal neurons and human-induced pluripotent stem cell-derived neurons. This demonstrates their pivotal role in normal development and maturation, the knowledge of which can now be harnessed to synchronise and accelerate neuronal differentiation of stem cell-derived neurons, enhancing their utility for disease modelling and drug discovery.

et al., Coenzyme Q (CoQ) plays a crucial role in mitochondria as an electron carrier within the mitochondrial respiratory chain (MRC) and is an essential antioxidant. Mutations in genes responsible for CoQ biosynthesis (COQ genes) cause primary CoQ deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype–phenotype association, mainly affecting tissues with high-energy demand including brain and skeletal muscle (SkM). Here, we report a four-year-old girl diagnosed with minor mental retardation and lethal rhabdomyolysis harboring a heterozygous mutation (c.483G > C (E161D)) in COQ4. The patient's fibroblasts showed a decrease in [CoQ], CoQ biosynthesis, MRC activity affecting complexes I/II + III, and respiration defects. Bona fide induced pluripotent stem cell (iPSCs) lines carrying the COQ4 mutation (CQ4-iPSCs) were generated, characterized and genetically edited using the CRISPR-Cas9 system (CQ4-iPSCs). Extensive differentiation and metabolic assays of control-iPSCs, CQ4-iPSCs and CQ4-iPSCs demonstrated a genotype association, reproducing the disease phenotype. The COQ4 mutation in iPSC was associated with CoQ deficiency, metabolic dysfunction, and respiration defects. iPSC differentiation into SkM was compromised, and the resulting SkM also displayed respiration defects. Remarkably, iPSC differentiation in dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ deficiency-associated functional and metabolic phenotypes caused by COQ4 mutation., This work was supported by the ISCIII/FEDER (E-Rare-2 Call PI12/03112 to P.M.), FIS/ISCIII/FEDER project (PI14/01962 to P.N.) and the European Research Council (ERC-2014-CoG646903 to P.M.). D.R.M. and C.P. are supported by PFIS scholarships (FI11/0511 and FI12/00468, respectively). C.B is supported by a Miguel Servet II contract (CPII13/00011). P.M. also acknowledges the financial support from The Obra Social La Caixa-Fundacio Josep Carreras and The Generalitat de Catalunya (SGR330). P.M. and J.L.-B. are investigators of the Spanish Cell Therapy cooperative network (TERCEL). A.G. is supported by Ramon y Cajal Program (RyC-2013–13221).

Primary human fetal cells have been used in clinical trials of cell replacement therapy for the treatment of neurodegenerative disorders such as Huntington's disease (HD). However, human fetal primary cells are scarce and difficult to work with and so a renewable source of cells is sought. Human fetal neural stem cells (hfNSCs) can be generated from human fetal tissue, but little is known about the differences between hfNSCs obtained from different developmental stages and brain areas. In the present work we characterized hfNSCs, grown as neurospheres, obtained from three developmental stages: 4–5, 6–7 and 8–9 weeks post conception (wpc) and four brain areas: forebrain, cortex, whole ganglionic eminence (WGE) and cerebellum. We observed that, as fetal brain development proceeds, the number of neural precursors is diminished and post-mitotic cells are increased. In turn, primary cells obtained from older embryos are more sensitive to the dissociation process, their viability is diminished and they present lower proliferation ratios compared to younger embryos. However, independently of the developmental stage of derivation proliferation ratios were very low in all cases. Improvements in the expansion rates were achieved by mechanical, instead of enzymatic, dissociation of neurospheres but not by changes in the seeding densities. Regardless of the developmental stage, neurosphere cultures presented large variability in the viability and proliferation rates during the initial 3–4 passages, but stabilized achieving significant expansion rates at passage 5 to 6. This was true also for all brain regions except cerebellar derived cultures that did not expand. Interestingly, the brain region of hfNSC derivation influences the expansion potential, being forebrain, cortex and WGE derived cells the most expandable compared to cerebellar. Short term expansion partially compromised the regional identity of cortical but not WGE cultures. Nevertheless, both expanded cultures were multipotent and kept the ability to differentiate to region specific mature neuronal phenotypes.

RTP801 expression is induced by cellular stress and has a pro-apoptotic function in non-proliferating differentiated cells such as neurons. In several neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease, elevated levels of RTP801 have been observed, which suggests a role for RTP801 in neuronal death. Neuronal death is also a pathological hallmark in Huntington's disease (HD), an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Currently, the exact mechanisms underlying mutant huntingtin (mhtt)-induced toxicity are still unclear. Here, we investigated whether RTP801 is involved in (mhtt)-induced cell death. Ectopic exon-1 mhtt elevated RTP801 mRNA and protein levels in nerve growth factor (NGF)-differentiated PC12 cells and in rat primary cortical neurons. In neuronal PC12 cells, mhtt also contributed to RTP801 protein elevation by reducing its proteasomal degradation rate, in addition to promoting RTP801 gene expression. Interestingly, silencing RTP801 expression with short hairpin RNAs (shRNAs) blocked mhtt-induced cell death in NGF-differentiated PC12 cells. However, RTP801 protein levels were not altered in the striatum of Hdh(Q7/Q111) and R6/1 mice, two HD models that display motor deficits but not neuronal death. Importantly, RTP801 protein levels were elevated in both neural telencephalic progenitors differentiated from HD patient-derived induced pluripotent stem cells and in the putamen and cerebellum of human HD postmortem brains. Taken together, our results suggest that RTP801 is a novel downstream effector of mhtt-induced toxicity and that it may be relevant to the human disease.

Human gliomas are malignant brain tumours that carry a poor prognosis and are composed of a heterogeneous population of cells. There is a paucity of animal models available for study of these tumours and most have been created by genetic modification. Spontaneously arising canine gliomas may provide a model for the characterization of the human tumours. The present study shows that canine gliomas form a range of immunohistochemical patterns that are similar to those described for human gliomas. The in-vitro sphere assay was used to analyze the expansion and differentiation potential of glioma cells taken from the periphery and centre of canine tumours. Samples from the subventricular zone (SVZ) and contralateral parenchyma were used as positive and negative controls, respectively. The expansion potential for all of these samples was low and cells from only three cultures were expanded for six passages. These three cultures were derived from high-grade gliomas and the cells had been cryopreserved. Most of the cells obtained from the centre of the tumours formed spheres and were expanded, in contrast to samples taken from the periphery of the tumours. Spheres were also formed and expanded from two areas of apparently unaffected brain parenchyma. The neurogenic SVZ contralateral samples also contained progenitor proliferating cells, since all of them were expanded for three to five passages. Differentiation analysis showed that all cultured spheres were multipotential and able to differentiate towards both neurons and glial cells. Spontaneously arising canine gliomas might therefore constitute an animal model for further characterization of these tumours.

The epigenomic landscape of Parkinson's disease (PD) remains unknown. We performed a genomewide DNA methylation and a transcriptome studies in induced pluripotent stem cell (iPSC)‐derived dopaminergic neurons (DAn) generated by cell reprogramming of somatic skin cells from patients with monogenic LRRK2‐associated PD (L2PD) or sporadic PD (sPD), and healthy subjects. We observed extensive DNA methylation changes in PD DAn, and of RNA expression, which were common in L2PD and sPD. No significant methylation differences were present in parental skin cells, undifferentiated iPSCs nor iPSC‐derived neural cultures not‐enriched‐in‐DAn. These findings suggest the presence of molecular defects in PD somatic cells which manifest only upon differentiation into the DAn cells targeted in PD. The methylation profile from PD DAn, but not from controls, resembled that of neural cultures not‐enriched‐in‐DAn indicating a failure to fully acquire the epigenetic identity own to healthy DAn in PD. The PD‐associated hypermethylation was prominent in gene regulatory regions such as enhancers and was related to the RNA and/or protein downregulation of a network of transcription factors relevant to PD (FOXA1, NR3C1, HNF4A, and FOSL2). Using a patient‐specific iPSC‐based DAn model, our study provides the first evidence that epigenetic deregulation is associated with monogenic and sporadic PD.

Summary Neurons obtained directly from human somatic cells hold great promise for disease modeling and drug screening. Available protocols rely on overexpression of transcription factors using integrative vectors and are often slow, complex, and inefficient. We report a fast and efficient approach for generating induced neural cells (iNCs) directly from human hematopoietic cells using Sendai virus. Upon SOX2 and c-MYC expression, CD133-positive cord blood cells rapidly adopt a neuroepithelial morphology and exhibit high expansion capacity. Under defined neurogenic culture conditions, they express mature neuronal markers and fire spontaneous action potentials that can be modulated with neurotransmitters. SOX2 and c-MYC are also sufficient to convert peripheral blood mononuclear cells into iNCs. However, the conversion process is less efficient and resulting iNCs have limited expansion capacity and electrophysiological activity upon differentiation. Our study demonstrates rapid and efficient generation of iNCs from hematopoietic cells while underscoring the impact of target cells on conversion efficiency., Graphical Abstract, Highlights • Expandable iNCs are generated from CB cells by SeV at high efficiency • CB-iNCs differentiate into functional neurons in vitro • CB stem/progenitor cells are more amenable to neural conversion than PB-MNCs, Giorgetti, Sanchez-Pernaute and colleagues report a fast and efficient approach for generating functional induced neurons from both cord blood and peripheral blood cells using SOX2 and c-MYC Sendai virus. However, the reprogramming of adult peripheral blood cells is less efficient. This study provides additional evidence that the origin and the nature of target cells may impact the outcome of the conversion process.

Huntington's disease (HD) is a neurodegenerative disorder caused by an abnormal expansion of a CAG repeat encoding a polyglutamine tract in the huntingtin (Htt) protein. The mutation leads to neuronal death through mechanisms which are still unknown. One hypothesis is that mitochondrial defects may play a key role. In support of this, the activity of mitochondrial complex II (C-II) is preferentially reduced in the striatum of HD patients. Here, we studied C-II expression in different genetic models of HD expressing N-terminal fragments of mutant Htt (mHtt). Western blot analysis showed that the expression of the 30 kDa Iron–Sulfur (Ip) subunit of C-II was significantly reduced in the striatum of the R6/1 transgenic mice, while the levels of the FAD containing catalytic 70 kDa subunit (Fp) were not significantly changed. Blue native gel analysis showed that the assembly of C-II in mitochondria was altered early in N171-82Q transgenic mice. Early loco-regional reduction in C-II activity and Ip protein expression was also demonstrated in a rat model of HD using intrastriatal injection of lentiviral vectors encoding mHtt. Infection of the rat striatum with a lentiviral vector coding the C-II Ip or Fp subunits induced a significant overexpression of these proteins that led to significant neuroprotection of striatal neurons against mHtt neurotoxicity. These results obtained in vivo support the hypothesis that structural and functional alterations of C-II induced by mHtt may play a critical role in the degeneration of striatal neurons in HD and that mitochondrial-targeted therapies may be useful in its treatment.

Activating transcription factor 5 (ATF5) is a basic-leucine-zipper transcription factor of the ATF/CREB family. The Atf5 gene generates two transcripts, Atf5α and Atf5β, of which Atf5α is known to be selectively translated upon endoplasmic reticulum stress response in non-neuronal cells. ATF5 is highly expressed in the developing brain where it modulates proliferation of neural progenitor cells. These cells show a high level of ATF5 that has to decrease to allow them to differentiate into mature neurons or glial cells. This has led to the extended notion that differentiated neural cells do not express ATF5 unless they undergo tumourigenic transformation. However, no systematic analysis of the distribution of ATF5 in adult brain or of its potential role in neuronal endoplasmic reticulum stress response has been reported. By immunostaining here we confirm highest ATF5 levels in neuroprogenitor cells of the embryonic and adult subventricular zone but also found ATF5 in a large variety of neurons in adult mouse brain. By combining Atf5 in situ hybridization and immunohistochemistry for the neuronal marker NeuN we further confirmed Atf5 messenger RNA in adult mouse neurons. Quantitative reverse transcriptase polymerase chain reaction demonstrated that Atf5α is the most abundant transcript in adult mouse encephalon and injection of the endoplasmic reticulum stress inducer tunicamycin into adult mouse brain increased neuronal ATF5 levels. Accordingly, ATF5 levels increased in hippocampal neurons of a mouse model of status epilepticus triggered by intra-amygdala injection of kainic acid, which leads to abnormal hippocampal neuronal activity and endoplasmic reticulum stress. Interestingly, ATF5 upregulation occurred mainly in hippocampal neuronal fields that do not undergo apoptosis in this status epilepticus model such as CA1 and dentate gyrus, thus suggesting a neuroprotective role. This was confirmed in a primary neuronal culture model in which ATF5 overexpression resulted in decreased endoplasmic reticulum stress-induced apoptosis and the opposite result was achieved by Atf5 RNA interference. Furthermore, in vivo administration of the eIF2α phosphatase inhibitor salubrinal resulted in increased ATF5 hippocampal levels and attenuated status epilepticus-induced neuronal death in the vulnerable CA3 subfield. In good agreement with the neuroprotective effect of increased ATF5, we found that apoptosis-resistant epileptogenic foci from patients with temporal lobe epilepsy also showed increased levels of ATF5. Thus, our results demonstrate that adult neurons express ATF5 and that they increase its levels upon endoplasmic reticulum stress as a pro-survival mechanism, thus opening a new field for neuroprotective strategies focused on ATF5 modulation. © 2013 The Author (2013).

Recent findings suggest that altered cholesterol homeostasis may contribute to the pathophysiology of Huntington's disease (HD). To understand the underlying mechanisms, here we used a combination of two-photon microscopy, epifluorescence, and biochemical methods to visualize and quantify lipid distribution in cell cultures expressing mutant huntingtin. Such expression promotes lipid imbalance, and cholesterol accumulation in cellular and murine models and in HD-affected human brains. Interestingly, cells expressing mutant huntingtin also showed higher content of ordered domains in their plasma membranes. These findings correlated with high levels of caveolin-1 and glycosphingolipid GM1, two well-defined markers of cholesterol-enriched domains, at the cell surface. In addition, cells expressing mutant huntingtin showed increased localization of NMDA receptors with cholesterol-enriched domains, contributing to increased NMDA receptor susceptibility to excitotoxic insults. Treatment with simvastatin or β-cyclodextrin, two cholesterol-lowering drugs, reduced the content of ordered domains at the cell surface, which in turn, protected cells against NMDA-mediated excitotoxicity. Taken together, our results indicate that mutant huntingtin produces accumulation of cholesterol and alters its cellular distribution that contributes to NMDA-mediated excitotoxicity. Administration of drugs that recover this effect, such as simvastatin could be beneficial for the treatment of HD.

Dysregulation of gene expression is one of the mechanisms involved in the pathophysiology of Huntington's disease (HD). Here, we examined whether mutant huntingtin regulates the levels of PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), a phosphatase that specifically dephosphorylates Akt at Ser473. Our results show decreased PHLPP1 protein levels in knock-in models (Hdh(Q111/Q111) mouse striatum and STHdh(Q111/Q111) cells), in the striatum of N-terminal exon-1 mutant huntingtin transgenic mouse models (R6/1; R6/1 : BDNF + or - , R6/2 and Tet/HD94) and in the putamen of HD patients. Quantitative PCR analysis revealed a reduction in PHLPP1 mRNA levels in the striatum of R6/1 compared with wild-type mice. Coincident with reduced PHLPP1 protein levels, we observed increased phosphorylated Akt (Ser473) levels specifically in the striatum. The analysis of the conditional mouse model Tet/HD94 disclosed that after mutant huntingtin shutdown PHLPP1 levels returned to wild-type levels whereas phospho-Akt levels were partially reduced. In conclusion, our results show that mutant huntingtin downregulates PHLPP1 expression. In the striatum, these reduced levels of PHLPP1 can contribute to maintain high levels of activated Akt that may delay cell death and allow the recovery of neuronal viability after mutant huntingtin silencing.

Two different methods have been adopted for the cryopreservation of human embryonic stem cells (hESCs): vitrification and conventional slow freezing/rapid thawing. However, these methods present poor viability and high differentiation rates. Therefore, the development of an efficient cryopreservation protocol for hESCs is one of the major challenges for the application of these cells in clinical therapy and regenerative medicine. A novel method for the cryopreservation of dissociated hESCs in the presence of a selective Rho-associated kinase (ROCK) inhibitor that increases cell survival and the efficiency of colony formation of cryopreserved hESCs has been developed. Moreover, this protocol improves the existing methods presenting short recovery times and hardly any differentiation rates. Thus, an easy handling protocol that allows the cryopreservation of large amounts of hESCs is described.

The precise mechanism by which mutant huntingtin elicits its toxicity remains unknown. However, synaptic alterations and increased susceptibility to neuronal death are known contributors to Huntington's disease (HD) symptomatology. While decreased metabolism has long been associated with HD, recent findings have surprisingly demonstrated reduced neuronal apoptosis in Caenorhabditis elegans and Drosophila models of HD by drugs that diminish ATP production. Interestingly, extracellular ATP has been recently reported to elicit neuronal death through stimulation of P2X7 receptors. These are ATP-gated cation channels known to modulate neurotransmitter release from neuronal presynaptic terminals and to regulate cytokine production and release from microglia. We hypothesized that alteration in P2X7-mediated calcium permeability may contribute to HD synaptic dysfunction and increased neuronal apoptosis. Using mouse and cellular models of HD, we demonstrate increased P2X7-receptor level and altered P2X7-mediated calcium permeability in somata and terminals of HD neurons. Furthermore, cultured neurons expressing mutant huntingtin showed increased susceptibility to apoptosis triggered by P2X7-receptor stimulation. Finally, in vivo administration of the P2X7-antagonist Brilliant Blue-G (BBG) to HD mice prevented neuronal apoptosis and attenuated body weight loss and motor-coordination deficits. These in vivo data strongly suggest that altered P2X7-receptor level and function contribute to HD pathogenesis and highlight the therapeutic potential of P2X7 receptor antagonists.

BACKGROUND: Human embryonic stem cells (hESCs) have potential use in clinical therapy and regenerative medicine. One of the major challenges regarding the application of these cells is the development of an efficient cryopreservation protocol, since current methods, which include slow-freezing–rapid thawing and vitrification of colonies in suspension, present poor viability and high differentiation rates. Dissociated hESC suspensions do not survive cryopreservation because they are susceptible to apoptosis upon cell detachment and dissociation. A selective Rho-associated kinase (ROCK) inhibitor has been reported to increase the survival of dissociated hESCs and their cloning efficiency. METHODS AND RESULTS: Here, we describe a novel method for dissociated hESCs cryopreservation in the presence of the ROCK inhibitor Y-27632. The addition of this inhibitor to the freezing and post-thawing medium significantly increased the survival rate and efficiency of colony formation. Moreover, the hESC colonies obtained after the cryopreservation in the presence of the ROCK inhibitor showed a very low rate of differentiation and a reduced time of recovery. After prolonged culture of frozen–thawed dissociated hESCs, the characteristic properties of pluripotent cells were observed, including normal karyotype, morphological features, marker expression (SSEA-4, TRA-1-60, TRA-1-81 and Oct-4) and the potential to differentiate into derivatives of all three germ layers after embryoid bodies formation. CONCLUSION: This novel method for the cryopreservation of dissociated hESCs may reduce the time required to amplify frozen stocks, and facilitate not only the storage of large numbers of hESCs but also the widespread use of these cells in regenerative medicine.

We study the striatal susceptibility to NMDA receptor (NMDAR)-mediated injury of two Huntington's disease (HD) transgenic mice: R6/1 and R6/1:BDNF(+/-). We found that R6/1:BDNF(+/-) mice--which express reduced levels of BDNF--were more resistant than R6/1 mice to intrastriatal injection of quinolinate. This increased resistance is related to a differential reduction in expression of NMDAR scaffolding proteins, MAGUKs (PSD-95, PSD-93, SAP-102 and SAP-97) but not to altered levels or synaptic location of NMDAR. A robust reorganization of postsynaptic density (PSD) was detected in HD transgenic mice, shown by a switch of PSD-93 by PSD-95 in PSD. Furthermore, NMDAR signaling pathways were affected by different BDNF levels in HD mice; we found a reduction of synaptic alpha CaMKII (but not of nNOS) in R6/1:BDNF(+/-) compared to R6/1 mice. The specific regulation of MAGUKs and alpha CaMKII in striatal neurons may reflect a protective mechanism against expression of mutant huntingtin exon-1.

There is no treatment for the neurodegenerative disorder Huntington disease (HD). Cystamine is a candidate drug; however, the mechanisms by which it operates remain unclear. We show here that cystamine increases levels of the heat shock DnaJ-containing protein 1b (HSJ1b) that are low in HD patients. HSJ1b inhibits polyQ-huntingtin¿induced death of striatal neurons and neuronal dysfunction in Caenorhabditis elegans. This neuroprotective effect involves stimulation of the secretory pathway through formation of clathrin-coated vesicles containing brain-derived neurotrophic factor (BDNF). Cystamine increases BDNF secretion from the Golgi region that is blocked by reducing HSJ1b levels or by overexpressing transglutaminase. We demonstrate that cysteamine, the FDA-approved reduced form of cystamine, is neuroprotective in HD mice by increasing BDNF levels in brain. Finally, cysteamine increases serum levels of BDNF in mouse and primate models of HD. Therefore, cysteamine is a potential treatment for HD, and serum BDNF levels can be used as a biomarker for drug efficacy.

Deficits of neurotrophic support caused by reduced levels of brain-derived neurotrophic factor (BDNF) have been implicated in the selective vulnerability of striatal neurones in Huntington's disease (HD). Therapeutic strategies based on BDNF administration have been proposed to slow or prevent the disease progression. However, the effectiveness of BDNF may depend on the proper expression of its receptor TrkB. In this study, we analysed the expression of TrkB in several HD models and in postmortem HD brains. We found a specific reduction of TrkB receptors in transgenic exon-1 and full-length knock-in HD mouse models and also in the motor cortex and caudate nucleus of HD brains. Our findings also demonstrated that continuous expression of mutant huntingtin is required to down-regulate TrkB levels. This was shown by findings in an inducible HD mouse model showing rescue of TrkB by turning off mutant huntingtin expression. Interestingly, the length of the polyglutamine tract in huntingtin appears to modulate the reduction of TrkB. Finally, to analyse the effect of BDNF in TrkB we compared TrkB expression in mutant huntingtin R6/1 and double mutant (R6/1 : BDNF+/-) mice. Similar TrkB expression was found in both transgenic mice suggesting that reduced TrkB is not a direct consequence of decreased BDNF. Therefore, taken together our findings identify TrkB as an additional component that potentially might contribute to the altered neurotrophic support in HD.

Brain-derived neurotrophic factor (BDNF) polymorphism is associated with the pathophysiology of several neurodegenerative disorders, including Huntington's disease. In view of these data and the involvement of huntingtin in intracellular trafficking, we examined the intracellular transport and release of Val66Val BDNF (Val-BDNF) and Val66Met BDNF (Met-BDNF) in transfected striatal knock-in cells expressing wild-type or mutant full-length huntingtin. Colocalization studies with specific markers for endoplasmic reticulum showed no differences between the Val-BDNF and Met-BDNF and were not modified by mutant huntingtin. However, post-Golgi trafficking was altered by mutant huntingtin dependent on the BDNF form. Thus, fluorescence recovery after photobleaching (FRAP) and inverse FRAP analysis showed retention of Met-BDNF in the Golgi apparatus with respect to Val-BDNF in wild-type cells. Strikingly, mutant huntingtin diminished post-Golgi trafficking of Val-BDNF, whereas Met-BDNF was not modified. Accordingly, a reduction in the number of transport vesicles was only observed in mutant huntingtin cells transfected with Val-BDNF but not Met-BDNF. Moreover, mutant huntingtin severely affected the KCl-evoked release of Val-BDNF, although it had little effect on Met-BDNF regulated release. The constitutive release of Val-BDNF or Met-BDNF in mutant cells was only slightly reduced. Interestingly, mutant huntingtin only perturbed post-Golgi trafficking of proteins that follow the regulated secretory pathway (epidermal growth factor receptor or atrial natriuretic factor), whereas it did not change those that follow the constitutive pathway (p75NTR).We conclude that mutant huntingtin differently affects intracellular transport and release of Val-BDNF and Met-BDNF. In addition, our findings reveal a new role for huntingtin in the regulation of the post-Golgi trafficking of the regulated secretory pathway.

Huntington disease (HD) is caused by CAG repeat expansion in exon 1 of the IT15 gene, which encodes huntingtin protein. The influence of expanded CAG repeats on the age at onset is clear in the case of low and high number of repeats, but the predictive value is weaker in intermediate ranges.1 Therefore, environmental factors, sex, or interacting genes may alter the course of the disease.1 Although a genome scan report did not link the age at onset with BDNF region,2 other studies have shown that huntingtin functions as a positive regulator of BDNF transcription.3 This may explain the decrease in BDNF levels reported in patients with HD.3 Furthermore, BDNF levels modulate the age at onset in transgenic models.4 Recently, the presence of a polymorphism in the BDNF gene leading to a valine (val) to methionine (met) substitution at position 66 in the BDNF prodomain (Val66Met) has been correlated with deleterious5 or beneficial6 effects in several neuropsychiatric diseases. Here we studied the effect of BDNF on the age at onset, analyzing the relationship of the Val66Met polymorphism and CAG repeat number in patients …

We have investigated functional roles of EphA/ephrin-A signaling in the development and function of the nigrostriatal system by overexpressing a soluble, broad-range EphA receptor antagonist in the central nervous system of transgenic mice. Adult transgenic mice showed a 30–40% reduction in the total volume of the substantia nigra (SN) without detectable differences in the number of dopaminergic neurons. Using fluorogold retrograde tracing from the striatum, we detected a 40–50% reduction in the number of dopaminergic neurons that could be traced from this structure in transgenic mice, suggesting that, a lower proportion of these cells were able to reach the striatum after disruption of EphA/ephrin-A signaling. In spite of this, total dopamine content in the striatum of transgenic mice was comparable to wild type. Analysis of locomotor activity and its regulation by pharmacological treatments that stimulate dopaminergic transmission revealed an unexpected dissociation of the behavioral responses to amphetamine and cocaine. In particular, transgenic mice were relatively insensitive to amphetamine while retaining normal responsiveness to cocaine, which, to the best of our knowledge, represents the first report of a dissociation of the behavioral responses to these two psychostimulants. Together, these results reveal an unexpected role for EphA/ephrin-A signaling in the normal connectivity and function of midbrain dopaminergic neurons.

Huntington's disease is a neurodegenerative disorder characterized by the selective loss of striatal neurons and, to a lesser extent, cortical neurons. The neurodegenerative process is caused by the mutation of huntingtin gene. Recent studies have established a link between mutant huntingtin, excitotoxicity and neurotrophic factors. Neurotrophic factors prevent cell death in degenerative processes but they can also enhance growth and function of neurons that are affected in Huntington's disease. The endogenous regulation of the expression of neurotrophic factors and their receptors in the striatum and its connections can be important to protect striatal cells and maintains basal ganglia connectivity. The administration of exogenous neurotrophic factors, in animal models of Huntington's disease, has been used to characterize the trophic requirements of striatal and cortical neurons. Neurotrophins, glial cell line-derived neurotrophic factor family members and ciliary neurotrophic factor have shown a potent neuroprotective effects on different neuronal populations of the striatum. Furthermore, they are also useful to maintain the integrity of the corticostriatal pathway. Thus, these neurotrophic factors may be suitable for the development of a neuroprotective therapy for neurodegenerative disorders of the basal ganglia.

Huntington's disease is a neurodegenerative disorder characterized by a selective degeneration of striatal projection neurons, which deal with choreic movements. Neuroprotective therapy could be achieved with the knowledge of the specific trophic requirements of these neuronal populations. Thus, the induction of endogenous trophic response or the exogenous administration of neurotrophic factors may help to prevent or stop the progression of the illness. Excitotoxicity has been implicated in the etiology of Huntington's disease, because intrastriatal injection of glutamate receptor agonists reproduces some of the neuropathological features of this disorder. Activation of glutamate receptors in the striatum differentially regulates the expression of neurotrophins, glial cell line-derived neurotrophic factor (GDNF), neurturin, and their receptors in the striatum and in its connections, cortex, and substantia nigra, showing a selective trophic response against excitotoxic insults. Transplantation of cells genetically engineered to release neurotrophic factors in the striatum has been used to study the neuroprotective effects of neurotrophin and GDNF family members in the excitotoxic model of Huntington's disease. Neurotrophins (brain-derived neurotrophic factor [BDNF], neurotrophin-3, and neurotrophin-4) protected striatal projection neurons against quinolinic or kainic acid treatment. However, GDNF family members showed a more specific action. Neurturin only protected gamma-aminobutyric acid (GABA)/enkephalinergic neurons that project to the external segment of the globus pallidus, whereas GDNF exerts its effects on GABA/substance P positive neurons, which project to the substantia nigra pars compacta and the internal segment of the globus pallidus. In conclusion, the trophic requirements of each population of striatal projection neurons are due to a complex interaction between several neurotrophic factors, such as neurotrophins and GDNF family members, which can be modified, in different pathological conditions. Moreover, these neurotrophic factors may be able to provide selective protection for basal ganglia circuits, which are affected in striatonigral degenerative disorders, such as Huntington's disease or multisystem atrophy.

Human Dopamine- and cAMP-regulated phosphoprotein of molecular weight 32kDa (DARPP-32, also known as PPP1R1B) gene codes for different transcripts that are mainly translated into two DARPP-32 protein isoforms, full length (fl)-DARPP-32 and truncated (t)-DARPP. The t-DARPP lacks the first 36 residues at the N-terminal, which alters its function. In the central nervous system, fl-DARPP-32 is highly expressed in GABAergic striatal medium spiny neurons (MSNs), where it integrates dopaminergic and glutamatergic input signaling. However, no information about human DARPP-32 isoform expression during MSNs maturation is available. In this study, our aim is to determine the expression of the two DARPP-32 isoforms in human fetal and adult striatal samples. We show that DARPP-32 isoform expression is differentially regulated during human striatal development, with the t-DARPP isoform being virtually absent from whole ganglionic eminence (WGE) and highly induced in the adult striatum (in both caudate and putamen). We next compared the four most common anti-DARPP-32 antibodies used in human specimens, to study their recognition of the two isoforms in fetal and adult human striatal samples by western blot and immunohistochemistry. The four antibodies specifically identify the fl-DARPP-32 in both fetal and adult samples, while t-DARPP form was only detected in adult striatal samples. In addition, the lack of t-DARPP recognition in human adult striatum by the antibody generated against the full-length domain produces in turn different efficacy by immunohistochemical analysis. In conclusion, our results show that expression of human DARPP-32 protein isoforms depends on the striatal neurodevelopmental stage with t-DARPP being specific for the human adult striatum.

Induced pluripotent stem cells (iPSC) offer an unprecedented opportunity to model human disease in relevant cell types, but it is unclear whether they could successfully model age-related diseases such as Parkinson’s disease (PD). Here, we generated iPSC lines from seven patients with idiopathic PD (ID-PD), four patients with familial PD associated to the G2019S mutation in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene (LRRK2-PD) and four age- and sex-matched healthy individuals (Ctrl). Over long-time culture, dopaminergic neurons (DAn) differentiated from either ID-PD- or LRRK2-PD-iPSC showed morphological alterations, including reduced numbers of neurites and neurite arborization, as well as accumulation of autophagic vacuoles, which were not evident in DAn differentiated from Ctrl-iPSC. Further induction of autophagy and/or inhibition of lysosomal proteolysis greatly exacerbated the DAn morphological alterations, indicating autophagic compromise in DAn from ID-PD- and LRRK2-PD-iPSC, which we demonstrate occurs at the level of autophagosome clearance. Our study provides an iPSC-based in vitro model that captures the patients’ genetic complexity and allows investigation of the pathogenesis of both sporadic and familial PD cases in a disease-relevant cell type., AS-D was partially supported by a pre-doctoral fellowship from MEC. Additional support was provided by grants from MICINN (BFU2009- 13277, PLE2009-0144 and ACI2010-1117 to AR; RyC-2008- 02772 and BFU2010-21823 to AC; SAF2008-04360, to JA; SAF2009-07774 and PLE2009-0089, to JMC), FIS (PI10/00849 to MV; RD06/0010/0006 to JMC), NIH/NIA AG031782/ AG038072 (to AMC) and Fondazione Guido Berlucchi (to AC). The Cell Therapy Program was supported by the CMRB (Promt-0901 to JMC). The work was also part of a CIBERNED Cooperative Project (to JA, JL-B, MV, ET and AR).

Neurodegenerative disorders affecting the central nervous system, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s chorea (HD) and amyotrophic lateral sclerosis are characterized by the loss of selected neuronal populations. Another striking feature shared by these diseases is the deposition of proteinaceous inclusion bodies in the brain, which may be intracytoplasmatic or intranuclear, or even extracellular. However, the density and prevalence of aggregates are not always directly related to neurodegeneration. Although some of these diseases are the result of mutations in known proteins, with HD a clear example, the expression and location of the affected protein do not explain the selective neurodegeneration. Therefore, other intrinsic mechanisms, characteristic of each neuronal population, might be involved in the neurodegenerative process. In this review we focus on several proposed mechanisms such as excitotoxicity, mitochondrial dysfunction and altered expression of trophic factors, which could account for the pathogenesis of HD.