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Highlights • Astrocytes and microglia key fulfil homeostatic and immune functions in the CNS. • Dysfunction of these cell types is implicated in neurodegenerative diseases. • Understanding cellular autonomy and early pathogenic changes is a key goal. • New human iPSC models will inform on disease mechanisms and therapy development., Both astrocytes and microglia fulfil homeostatic and immune functions in the healthy CNS. Dysfunction of these cell types have been implicated in the pathomechanisms of several neurodegenerative diseases. Understanding the cellular autonomy and early pathological changes in these cell types may inform drug screening and therapy development. While animal models and post-mortem tissue have been invaluable in understanding disease processes, the advent of human in vitro models provides a unique insight into disease biology as a manipulable model system obtained directly from patients. Here, we discuss the different human in vitro models of astrocytes and microglia and outline the phenotypes that have been recapitulated in these systems.

SummaryCoordinated development of brain stem and spinal target neurons is pivotal for the emergence of a precisely functioning locomotor system. Signals that match the development of these far-apart regions of the central nervous system may be redeployed during spinal cord regeneration. Here we show that descending dopaminergic projections from the brain promote motor neuron generation at the expense of V2 interneurons in the developing zebrafish spinal cord by activating the D4a receptor, which acts on the hedgehog pathway. Inhibiting this essential signal during early neurogenesis leads to a long-lasting reduction of motor neuron numbers and impaired motor responses of free-swimming larvae. Importantly, during successful spinal cord regeneration in adult zebrafish, endogenous dopamine promotes generation of spinal motor neurons, and dopamine agonists augment this process. Hence, we describe a supraspinal control mechanism for the development and regeneration of specific spinal cell types that uses dopamine as a signal.

Post-transcriptional regulation plays a major role in the generation of cell type diversity. In particular, alternative splicing increases diversification of transcriptome between tissues, in different cell types within a tissue, and even in different compartments of the same cell. The complexity of alternative splicing has increased during evolution. With increasing sophistication, however, comes greater potential for malfunction of these intricate processes. Indeed, recent years have uncovered a wealth of disease-causing mutations affecting RNA-binding proteins and non-coding regions on RNAs, highlighting the importance of studying disease mechanisms that act at the level of RNA processing. For instance, mutations in TARDBP and FUS, or a repeat expansion in the intronic region of the C9ORF72 gene, can all cause amyotrophic lateral sclerosis. We discuss how interspecies differences highlight the necessity for human model systems to complement existing non-human approaches to study neurodegenerative disorders. We conclude by discussing the improvements that could further increase the promise of human pluripotent stem for cell-based disease modeling. This article is part of a Special Issue entitled “RNA-Binding Proteins”.

BackgroundMore than half of patients with multiple sclerosis have progressive disease characterised by accumulating disability. The absence of treatments for progressive multiple sclerosis represents a major unmet clinical need. On the basis of evidence that mesenchymal stem cells have a beneficial effect in acute and chronic animal models of multiple sclerosis, we aimed to assess the safety and efficacy of these cells as a potential neuroprotective treatment for secondary progressive multiple sclerosis. MethodsPatients with secondary progressive multiple sclerosis involving the visual pathways (expanded disability status score 5·5–6·5) were recruited from the East Anglia and north London regions of the UK. Participants received intravenous infusion of autologous bone-marrow-derived mesenchymal stem cells in this open-label study. Our primary objective was to assess feasibility and safety; we compared adverse events from up to 20 months before treatment until up to 10 months after the infusion. As a secondary objective, we chose efficacy outcomes to assess the anterior visual pathway as a model of wider disease. Masked endpoint analyses was used for electrophysiological and selected imaging outcomes. We used piecewise linear mixed models to assess the change in gradients over time at the point of intervention. This trial is registered with ClinicalTrials.gov, number NCT00395200. FindingsWe isolated, expanded, characterised, and administered mesenchymal stem cells in ten patients. The mean dose was 1·6×106 cells per kg bodyweight (range 1·1–2·0). One patient developed a transient rash shortly after treatment; two patients had self-limiting bacterial infections 3–4 weeks after treatment. We did not identify any serious adverse events. We noted improvement after treatment in visual acuity (difference in monthly rates of change −0·02 logMAR units, 95% CI −0·03 to −0·01; p=0·003) and visual evoked response latency (−1·33 ms, −2·44 to −0·21; p=0·020), with an increase in optic nerve area (difference in monthly rates of change 0·13 mm2, 0·04 to 0·22; p=0·006). We did not identify any significant effects on colour vision, visual fields, macular volume, retinal nerve fibre layer thickness, or optic nerve magnetisation transfer ratio. InterpretationAutologous mesenchymal stem cells were safely given to patients with secondary progressive multiple sclerosis in our study. The evidence of structural, functional, and physiological improvement after treatment in some visual endpoints is suggestive of neuroprotection.

Parkinson's disease (PD) is a common neurodegenerative disease characterised initially loss of dopaminergic neurons in the substantia nigra (SN) and later widespread nondopaminergic neuronal loss including in the cortex. Genetic mutations can cause familial PD, but the sequence of molecular events that trigger neuronal vulnerability is poorly understood. Of the familial forms of PD, point mutations of gene encoding alpha-synuclein (SNCA), the major component of the predominant pathological hallmark called Lewy bodies/neurites, result in dominant PD. To characterize the cellular pathophysiology, we differentiated patient-specific induced pluripotent stem cells (iPSCs) from PD patient fibroblasts carrying alpha-synuclein point mutation, A53T, into functional cortical and midbrain neurons. We demonstrated that endogenous levels of SNCA-A53T reduce cell survival and the source of vulnerability is the excess reactive oxygen species (ROS) evidenced by increased superoxide production and lipid peroxidation levels compared to control. By utilizing the power of patient specific iPSC derived neurons, this study provides an insight into the primary pathological events leading to neuronal cell death and may furthermore be an important direct tool for therapeutic development.

Background A hexanucleotide expansion in C9orf72 is a common cause of the fatal neurodegenerative disorder amyotrophic lateral sclerosis. We have found evidence in a Drosophila model that neurotoxicity is mediated by dipeptide repeat (DPR) proteins generated by repeat-associated non-ATG translation. Here we aimed to evaluate in models of amyotrophic lateral sclerosis caused by the C9orf72 mutation ( C9orf72 -ALS) whether DPR proteins cause nucleolar dysfunction and whether novel small molecules that bind C9orf72 -repeat RNA reduce DPR formation and neurotoxicity. Methods We assessed nucleolar function in in-vivo Drosophila models. Nucleolar size was measured with immunofluorescence and confocal microscopy, using automated image analysis. Human induced pluripotent stem cells (iPSCs) from patients with C9orf72 -ALS and from healthy controls were taken through neural induction and patterning to derive spinal motor neuron populations. Disease phenotypes were measured with fluorescence in-situ hybridisation for the typical RNA foci seen in C9orf72 -ALS patients. Small molecules binding to C9orf72 -repeat RNA were fed to C9orf72 Drosophila and applied to the human iPSC-derived spinal motor neurons to evaluate rescue of disease phenotypes. Findings DPR proteins colocalised with nucleoli in C9orf72 - Drosophila brain tissue, with effects on nucleolar morphology. C9orf72 - Drosophila had significantly reduced egg-to-adult viability (p Drosophila was investigated. Interpretation Emerging evidence suggests that nucleolar dysfunction is a key mechanism in C9orf72 -ALS. It is crucial that this finding is validated in relevant disease models to rapidly translate findings into promising therapeutic targets. The high prevalence of C9orf72 -ALS makes use of targeted therapies a compelling strategy. These experiments might provide novel mechanistic insights into a common form of amyotrophic lateral sclerosis and deliver preclinical data on an exciting therapeutic approach. Funding RB is a Leonard Wolfson Clinical Research Training Fellow and is funded by a Wellcome Trust Clinical Research Training Fellowship (107196/Z/14/Z).

Background Cooling is the single most effective treatment for acute neuronal injury. Understanding the molecular mechanisms that mediate cooling-induced neuroprotection might yield novel therapeutic targets for neurodegenerative disease. Many of these disorders involve modulation of tau, a protein that is enriched in neurons and becomes hyperphosphorylated in hypothermic, injury-resistant rodent brains as well as in Alzheimer's disease. We sought to establish a new model for exploring human tau physiology and its role in neuroprotection in the context of therapeutic hypothermia. Methods Functional cortical neurons (hCNs) were differentiated from three independent human pluripotent stem-cell lines and validated for regional identity and tau status. Matched cultures were incubated at 37°C or clinically targeted temperatures for therapeutic hypothermia (32°C) and suspended animation (28°C). Effects of cooling on tau status and neuronal injury elicited by common neurotoxic stressors were established. Injury experiments were repeated while manipulating tau phosphorylation. Findings hCN differentiation featured transitions in tau status, recapitulating transcriptional and post-translational human in-vivo cortical tau development. Key aspects of this development were reversed by cooling. Notably, cooling induced tau hyperphosphorylation via rapid inhibition of the major tau phosphatase, protein phosphatase 2A (PP2A). Multiplexed injury analysis confirmed that hypothermia robustly protected hCNs from oxidative (100 μM hydrogen peroxide) and excitotoxic (30 μM glutamate) stress (at 28°C, injury was reduced by 78% and 56%, respectively; p Interpretation To our knowledge, this is the first study of human neuronal tau physiology under hypothermic conditions. Although definitive experiments are needed, our findings support previous work linking phospho-tau to neuroprotection. Furthermore, cold-induced tau hyperphosphorylation is a potential trigger for proteostatic priming, a recently discovered mechanism of hypothermic preconditioning in hCNs. Exploiting these cryobiological effects might lead to new treatments for acute and chronic neuronal injury. Funding Wellcome Trust, Anne Rowling Regenerative Neurology Clinic, Euan MacDonald Centre for Motor Neurone Disease Research.