Generation of three induced pluripotent stem cell lines from individuals with Aicardi-Goutières syndrome caused by a c.3019G>A (p.G1007R) autosomal dominant pathogenic variant in ADAR1.

Mutations in Adenosine deaminase acting on RNA 1 (ADAR1) gene encoding RNA editing enzyme ADAR1 results in the neuroinflammatory leukodystrophy Aicardi Goutières Syndrome (AGS). AGS is an early onset leukoencephalopathy with an exacerbated interferon response leading to neurological regression with intellectual disability, spasticity, and motor deficits. We have generated three induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) of individuals with ADAR1G1007R mutation. The generated iPSCs were investigated to confirm a normal karyotype, pluripotency, and trilineage differentiation potential. The reprogrammed iPSCs will allow us to model AGS, dissect the cellular mechanisms and testing different treatment targets.

Generation of three induced Pluripotent Stem Cell lines from individuals with Hypomyelination with Atrophy of Basal Ganglia and Cerebellum caused by a c.745G>A (p.D249N) autosomal dominant mutation in TUBB4A.

Mutations in tubulin alpha 4a (TUBB4A) result in a spectrum of leukodystrophies, including Hypomyelination with atrophy of basal ganglia and cerebellum (H-ABC), resulting from a recurring mutation p.Asp249Asn (TUBB4AD249N). H-ABC presents with dystonia, motor and cognitive impairment and pathological features of hypomyelination and loss of cerebellar and striatal neurons. We have generated three induced pluripotent stem cell (iPSC) lines from fibroblast and peripheral blood mononuclear cells (PBMCs) of individuals with TUBB4AD249N mutation. The iPSCs were assessed to confirm a normal karyotype, pluripotency, and trilineage differentiation potential. The iPSCs will allow for disease modeling, understanding mechanisms and testing of therapeutic targets.

Cx43 hemichannels contribute to astrocyte-mediated toxicity in sporadic and familial ALS.

Connexin 43 (Cx43) gap junctions and hemichannels mediate astrocyte intercellular communication in the central nervous system under normal conditions and contribute to astrocyte-mediated neurotoxicity in amyotrophic lateral sclerosis (ALS). Here, we show that astrocyte-specific knockout of Cx43 in a mouse model of ALS slows disease progression both spatially and temporally, provides motor neuron (MN) protection, and improves survival. In addition, Cx43 expression is up-regulated in human postmortem tissue and cerebrospinal fluid from ALS patients. Using human induced pluripotent stem cell­derived astrocytes (hiPSC-A) from both familial and sporadic ALS, we establish that Cx43 is up-regulated and that Cx43-hemichannels are enriched at the astrocyte membrane. We also demonstrate that the pharmacological blockade of Cx43-hemichannels in ALS astrocytes using GAP 19, a mimetic peptide blocker, and tonabersat, a clinically tested small molecule, provides neuroprotection of hiPSC-MN and reduces ALS astrocyte-mediated neuronal hyperexcitability. Extending the in vitro application of tonabersat with chronic administration to SOD1G93A mice results in MN protection with a reduction in reactive astrocytosis and microgliosis. Taking these data together, our studies identify Cx43 hemichannels as conduits of astrocyte-mediated disease progression and a pharmacological target for disease-modifying ALS therapies.

Further Delineation of the Clinical and Pathologic Features of HIKESHI-Related Hypomyelinating Leukodystrophy.

BACKGROUND: A recurrent homozygous missense variant, c.160G>C;p.(Val54Leu) in HIKESHI, was found to cause a hypomyelinating leukodystrophy with high frequency in the Ashkenazi Jewish population. We provide extended phenotypic classification of this disorder based on clinical history of a further seven affected individuals, assess carrier frequency in the Ashkenazi Jewish population, and provide a neuropathological study. METHODS: Clinical information, neuroimaging, and biosamples were collected. Brain autopsy was performed for one case. RESULTS: Individuals with HIKESHI-related disease share common clinical features: early axial hypotonia evolving to dystonia or with progressive spasticity, hyperreflexia and clonus, feeding difficulties with poor growth, and nystagmus. Severe morbidity or death during febrile illness occurred in five of the nine affected individuals. Magnetic resonance images of seven patients were analyzed and demonstrated diffuse hypomyelination and thin corpus callosum. Genotyping data of more than 125,000 Ashkenazi Jewish individuals revealed a carrier frequency of 1 in 216. Gross pathology examination in one case revealed abnormal white matter. Microscopically, there was a near-total absence of myelin with a relative preservation of axons. The cerebral white matter showed several reactive astrocytes and microglia. CONCLUSIONS: We provide pathologic evidence for a primary disorder of the myelin in HIKESHI-related leukodystrophy. These findings are consistent with the hypomyelination seen in brain magnetic resonance imaging and with the clinical features of early-onset spastic/dystonic quadriplegia and nystagmus. The high carrier rate of the recurrent variant seen in the Ashkenazi Jewish population requires increased attention to screening and diagnosis of this condition, particularly in this population.

Glial cells in the driver seat of leukodystrophy pathogenesis.

Glia cells are often viewed as support cells in the central nervous system, but recent discoveries highlight their importance in physiological functions and in neurological diseases. Central to this are leukodystrophies, a group of progressive, neurogenetic disease affecting white matter pathology. In this review, we take a closer look at multiple leukodystrophies, classified based on the primary glial cell type that is affected. While white matter diseases involve oligodendrocyte and myelin loss, we discuss how astrocytes and microglia are affected and impinge on oligodendrocyte, myelin and axonal pathology. We provide an overview of the leukodystrophies covering their hallmark features, clinical phenotypes, diverse molecular pathways, and potential therapeutics for clinical trials. Glial cells are gaining momentum as cellular therapeutic targets for treatment of demyelinating diseases such as leukodystrophies, currently with no treatment options. Here, we bring the much needed attention to role of glia in leukodystrophies, an integral step towards furthering disease comprehension, understanding mechanisms and developing future therapeutics.

TUBB4A mutations result in both glial and neuronal degeneration in an H-ABC leukodystrophy mouse model.

Mutations in TUBB4A result in a spectrum of leukodystrophy including Hypomyelination with Atrophy of Basal Ganglia and Cerebellum (H-ABC), a rare hypomyelinating leukodystrophy, often associated with a recurring variant p.Asp249Asn (D249N). We have developed a novel knock-in mouse model harboring heterozygous (Tubb4aD249N/+) and the homozygous (Tubb4aD249N/D249N) mutation that recapitulate the progressive motor dysfunction with tremor, dystonia and ataxia seen in H-ABC. Tubb4aD249N/D249N mice have myelination deficits along with dramatic decrease in mature oligodendrocytes and their progenitor cells. Additionally, a significant loss occurs in the cerebellar granular neurons and striatal neurons in Tubb4aD249N/D249N mice. In vitro studies show decreased survival and dysfunction in microtubule dynamics in neurons from Tubb4aD249N/D249N mice. Thus Tubb4aD249N/D249N mice demonstrate the complex cellular physiology of H-ABC, likely due to independent effects on oligodendrocytes, striatal neurons, and cerebellar granule cells in the context of altered microtubule dynamics, with profound neurodevelopmental deficits.

Inside human and other animal cells, filaments known as microtubules help support the shape of the cell and move proteins to where they need to be. Defects in microtubules may lead to disease. For example, genetic mutations affecting a microtubule component called TUBB4A cause a rare brain disease in humans known as H-ABC. Individuals with H-ABC display many symptoms including abnormal walking, speech defects, impaired swallowing, and several cognitive defects. Abnormalities in several areas of the brain, including the cerebellum and striatum contribute to these defects. . In these structures, the neurons that carry messages around the brain and their supporting cells, known as oligodendrocytes, die, which causes these parts of the brain to gradually waste away. At this time, there are no therapies available to treat H-ABC. Furthermore, research into the disease has been hampered by the lack of a suitable "model" in mice or other laboratory animals. To address this issue, Sase, Almad et al. generated mice carrying a mutation in a gene which codes for the mouse equivalent of the human protein TUBB4A. Experiments showed that the mutant mice had similar physical symptoms to humans with H-ABC, including an abnormal walking gait, poor coordination and involuntary movements such as twitching and reduced reflexes. H-ABC mice had smaller cerebellums than normal mice, which was consistent with the wasting away of the cerebellum observed in individuals with H-ABC. The mice also lost neurons in the striatum and cerebellum, and oligodendrocytes in the brain and spinal cord. Furthermore, the mutant TUBB4A protein affected the behavior and formation of microtubules in H-ABC mice. The findings of Sase, Almad et al. provide the first mouse model that shares many features of H-ABC disease in humans. This model provides a useful tool to study the disease and develop potential new therapies.

Perfluorocarbon Labeling of Human Glial-Restricted Progenitors for 19 F Magnetic Resonance Imaging.

One of the fundamental limitations in assessing potential efficacy in Central Nervous System (CNS) transplantation of stem cells is the capacity for monitoring cell survival and migration noninvasively and longitudinally. Human glial-restricted progenitor (hGRP) cells (Q-Cells) have been investigated for their utility in providing neuroprotection following transplantation into models of amyotrophic lateral sclerosis (ALS) and have been granted a Food and Drug Administration (FDA) Investigational New Drug (IND) for intraspinal transplantation in ALS patients. Furthermore, clinical development of these cells for therapeutic use will rely on the ability to track the cells using noninvasive imaging methodologies as well as the verification that the transplanted GRPs have disease-relevant activity. As a first step in development, we investigated the use of a perfluorocarbon (PFC) dual-modal (19 F magnetic resonance imaging [MRI] and fluorescence) tracer agent to label Q-Cells in culture and following spinal cord transplantation. PFCs have a number of potential benefits that make them appealing for clinical use. They are quantitative, noninvasive, biologically inert, and highly specific. In this study, we developed optimized PFC labeling protocols for Q-Cells and demonstrate that PFCs do not significantly alter the glial identity of Q-Cells. We also show that PFCs do not interfere with the capacity for differentiation into astrocytes either in vitro or following transplantation into the ventral horn of the mouse spinal cord, and can be visualized in vivo by hot spot 19 F MRI. These studies provide a foundation for further preclinical development of PFCs within the context of evaluating Q-Cell transplantation in the brain and spinal cord of future ALS patients using 19 F MRI. Stem Cells Translational Medicine 2019;8:355-365.

Astrocytes, an active player in Aicardi-Goutières syndrome.

Aicardi-Goutières syndrome (AGS) is an early-onset, autoimmune and genetically heterogeneous disorder with severe neurologic injury. Molecular studies have established that autosomal recessive mutations in one of the following genes are causative: TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR1 and IFIH1/MDA5. The phenotypic presentation and pathophysiology of AGS is associated with over-production of the cytokine Interferon-alpha (IFN-α) and its downstream signaling, characterized as type I interferonopathy. Astrocytes are one of the major source of IFN in the central nervous system (CNS) and it is proposed that they could be key players in AGS pathology. Astrocytes are the most ubiquitous glial cell in the CNS and perform a number of crucial and complex functions ranging from formation of blood-brain barrier, maintaining ionic homeostasis, metabolic support to synapse formation and elimination in healthy CNS. Involvement of astrocytic dysfunction in neurological diseases-Alexander's disease, Epilepsy, Alzheimer's and amyotrophic lateral sclerosis (ALS)-has been well-established. It is now known that compromised astrocytic function can contribute to CNS abnormalities and severe neurodegeneration, nevertheless, its contribution in AGS is unclear. The current review discusses known molecular and cellular pathways for AGS mutations and how it stimulates IFN-α signaling. We shed light on how astrocytes might be key players in the phenotypic presentations of AGS and emphasize the cell-autonomous and non-cell-autonomous role of astrocytes. Understanding the contribution of astrocytes will help reveal mechanisms underlying interferonopathy and develop targeted astrocyte specific therapeutic treatments in AGS.

A stocked toolbox for understanding the role of astrocytes in disease.

Our understanding of astrocytes and their role in neurological diseases has increased considerably over the past two decades as the diverse roles of these cells have become recognized. Our evolving understanding of these cells suggests that they are more than support cells for neurons and that they play important roles in CNS homeostasis under normal conditions, in neuroprotection and in disease exacerbation. These multiple functions make them excellent candidates for targeted therapies to treat neurological disorders. New technological advances, including in vivo imaging, optogenetics and chemogenetics, have allowed us to examine astrocytic functions in ways that have uncovered new insights into the dynamic roles of these cells. Furthermore, the use of induced pluripotent stem cell-derived astrocytes from patients with a host of neurological disorders can help to tease out the contributions of astrocytes to human disease. In this Review, we explore some of the technological advances developed over the past decade that have aided our understanding of astrocyte function. We also highlight neurological disorders in which astrocyte function or dysfunction is believed to have a role in disease pathogenesis or propagation and discuss how the technological advances have been and could be used to study each of these diseases.

Serial in vivo imaging of transplanted allogeneic neural stem cell survival in a mouse model of amyotrophic lateral sclerosis.

Neural stem cells (NSCs) are being investigated as a possible treatment for amyotrophic lateral sclerosis (ALS) through intraspinal transplantation, but no longitudinal imaging studies exist that describe the survival of engrafted cells over time. Allogeneic firefly luciferase-expressing murine NSCs (Luc+-NSCs) were transplanted bilaterally (100,000 cells/2µl) into the cervical spinal cord (C5) parenchyma of pre-symptomatic (63day-old) SOD1G93A ALS mice (n=14) and wild-type age-matched littermates (n=14). Six control SOD1G93A ALS mice were injected with saline. Mice were immunosuppressed using a combination of tacrolimus+sirolimus (1mg/kg each, i.p.) daily. Compared to saline-injected SOD1G93A ALS control mice, a transient improvement (p<0.05) in motor performance (rotarod test) was observed after NSC transplantation only at the early disease stage (weeks 2 and 3 post-transplantation). Compared to day one post-transplantation, there was a significant decline in bioluminescent imaging (BLI) signal in SOD1G93A ALS mice at the time of disease onset (71.7±17.9% at 4weeks post-transplantation, p<0.05), with a complete loss of BLI signal at endpoint (120day-old mice). In contrast, BLI signal intensity was observed in wild-type littermates throughout the entire study period, with only a 41.4±8.7% decline at the endpoint. In SOD1G93A ALS mice, poor cell survival was accompanied by accumulation of mature macrophages and the presence of astrogliosis and microgliosis. We conclude that the disease progression adversely affects the survival of engrafted murine Luc+-NSCs in SOD1G93A ALS mice as a result of the hostile ALS spinal cord microenvironment, further emphasizing the challenges that face successful cell therapy of ALS.

Connexin 43 in astrocytes contributes to motor neuron toxicity in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons in the CNS. Astrocytes play a critical role in disease progression of ALS. Astrocytes are interconnected through a family of gap junction proteins known as connexins (Cx). Cx43 is a major astrocyte connexin conducting crucial homeostatic functions in the CNS. Under pathological conditions, connexin expression and functions are altered. Here we report that an abnormal increase in Cx43 expression serves as one of the mechanisms for astrocyte-mediated toxicity in ALS. We observed a progressive increase in Cx43 expression in the SOD1(G93A) mouse model of ALS during the disease course. Notably, this increase in Cx43 was also detected in the motor cortex and spinal cord of ALS patients. Astrocytes isolated from SOD1(G93A) mice as well as human induced pluripotent stem cell (iPSC)-derived astrocytes showed an increase in Cx43 protein, which was found to be an endogenous phenomenon independent of neuronal co-culture. Increased Cx43 expression led to important functional consequences when tested in SOD1(G93A) astrocytes when compared to control astrocytes over-expressing wild-type SOD1 (SOD1(WT) ). We observed SOD1(G93A) astrocytes exhibited enhanced gap junction coupling, increased hemichannel-mediated activity, and elevated intracellular calcium levels. Finally, we tested the impact of increased expression of Cx43 on MN survival and observed that use of both a pan Cx43 blocker and Cx43 hemichannel blocker conferred neuroprotection to MNs cultured with SOD1(G93A) astrocytes. These novel findings show a previously unrecognized role of Cx43 in ALS-related motor neuron loss. GLIA 2016;64:1154-1169.

Serum exosomes in pregnancy-associated immune modulation and neuroprotection during CNS autoimmunity.

In multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), relapses are markedly reduced during pregnancy. Exosomes are lipid-bound vesicles and are more abundant in the serum during pregnancy. Using murine EAE, we demonstrate that serum exosomes suppress T cell activation, promote the maturation of oligodendrocyte precursor cells (OPC), and pregnancy exosomes facilitate OPC migration into active CNS lesions. However, exosomes derived from both pregnant and non-pregnant mice reduced the severity of established EAE. Thus, during pregnancy, serum exosomes modulate the immune and central nervous systems and contribute to pregnancy-associated suppression of EAE.

Glia: an emerging target for neurological disease therapy.

Therapeutic strategies using stem cells for treating neurological diseases are receiving more attention as the scientific community appreciates cell-autonomous contributions to several diseases of the central nervous system. The transplantation of stem cells from various sources is now being employed for both neuronal and glial replacement. This review provides an assessment of glial contributions to some of the central nervous system diseases and the advancements in cellular replacement approaches. The rationale for glial replacement in individual diseases and the potential hurdles for cell-replacement strategies are also emphasized. The significant progress in the field of stem cell biology with the advent of tools such as induced pluripotent stem cells and imaging techniques holds promise for the clinical application of cell therapeutics.

The PPAR alpha agonist gemfibrozil is an ineffective treatment for spinal cord injured mice.

Peroxisome Proliferator Activated Receptor (PPAR)-α is a key regulator of lipid metabolism and recent studies reveal it also regulates inflammation in several different disease models. Gemfibrozil, an agonist of PPAR-α, is a FDA approved drug for hyperlipidemia and has been shown to inhibit clinical signs in a rodent model of multiple sclerosis. Since many studies have shown improved outcome from spinal cord injury (SCI) by anti-inflammatory and neuroprotective agents, we tested the efficacy of oral gemfibrozil given before or after SCI for promoting tissue preservation and behavioral recovery after spinal contusion injury in mice. Unfortunately, the results were contrary to our hypothesis; in our first attempt, gemfibrozil treatment exacerbated locomotor deficits and increased tissue pathology after SCI. In subsequent experiments, the behavioral effects were not replicated but histological outcomes again were worse. We also tested the efficacy of a different PPAR-α agonist, fenofibrate, which also modulates immune responses and is beneficial in several neurodegenerative disease models. Fenofibrate treatment did not improve recovery, although there was a slight trend for a modest increase in histological tissue sparing. Based on our results, we conclude that PPAR-α agonists yield either no effect or worsen recovery from spinal cord injury, at least at the doses and the time points of drug delivery tested here. Further, patients sustaining spinal cord injury while taking gemfibrozil might be prone to exacerbated tissue damage.

Oligodendrocyte fate after spinal cord injury.

Oligodendrocytes (OLs) are particularly susceptible to the toxicity of the acute lesion environment after spinal cord injury (SCI). They undergo both necrosis and apoptosis acutely, with apoptosis continuing at chronic time points. Loss of OLs causes demyelination and impairs axon function and survival. In parallel, a rapid and protracted OL progenitor cell proliferative response occurs, especially at the lesion borders. Proliferating and migrating OL progenitor cells differentiate into myelinating OLs, which remyelinate demyelinated axons starting at 2 weeks post-injury. The progression of OL lineage cells into mature OLs in the adult after injury recapitulates development to some degree, owing to the plethora of factors within the injury milieu. Although robust, this endogenous oligogenic response is insufficient against OL loss and demyelination. First, in this review we analyze the major spatial-temporal mechanisms of OL loss, replacement, and myelination, with the purpose of highlighting potential areas of intervention after SCI. We then discuss studies on OL protection and replacement. Growth factors have been used both to boost the endogenous progenitor response, and in conjunction with progenitor transplantation to facilitate survival and OL fate. Considerable progress has been made with embryonic stem cell-derived cells and adult neural progenitor cells. For therapies targeting oligogenesis to be successful, endogenous responses and the effects of the acute and chronic lesion environment on OL lineage cells must be understood in detail, and in relation, the optimal therapeutic window for such strategies must also be determined.

Chronic expression of PPAR-delta by oligodendrocyte lineage cells in the injured rat spinal cord.

The transcription factor peroxisome proliferator-activated receptor (PPAR)-delta promotes oligodendrocyte differentiation and myelin formation in vitro and is prevalent throughout the brain and spinal cord. Its expression after injury, however, has not been examined. Thus, we used a spinal contusion model to examine the spatiotemporal expression of PPAR-delta in naïve and injured spinal cords from adult rats. As previously reported, PPAR-delta was expressed by neurons and oligodendrocytes in uninjured spinal cords; PPAR-delta was also detected in NG2 cells (potential oligodendrocyte progenitors) within the white matter and gray matter. After spinal cord injury (SCI), PPAR-delta mRNA and protein were present early and increased over time. Overall PPAR-delta+ cell numbers declined at 1 day post injury (dpi), likely reflecting neuron loss, and then rose through 14 dpi. A large proportion of NG2 cells expressed PPAR-delta after SCI, especially along lesion borders. PPAR-delta+ NG2 cell numbers were significantly higher than naive by 7 dpi and remained elevated through at least 28 dpi. PPAR-delta+ oligodendrocyte numbers declined at 1 dpi and then increased over time such that >20% of oligodendrocytes expressed PPAR-delta after SCI compared with approximately 10% in uninjured tissue. The most prominent increase in PPAR-delta+ oligodendrocytes was along lesion borders where at least a portion of newly generated oligodendrocytes (bromodeoxyuridine+) were PPAR-delta+. Consistent with its role in cellular differentiation, the early rise in PPAR-delta+ NG2 cells followed by an increase in new PPAR-delta+ oligodendrocytes suggests that this transcription factor may be involved in the robust oligodendrogenesis detected previously along SCI lesion borders.

Protein kinase C modulates the phase-delaying effects of light in the mammalian circadian clock.

The mammalian circadian pacemaker located in the suprachiasmatic nuclei (SCN) drives a vast array of biochemical and physiological processes with 24-h periodicity. The phasing of SCN pacemaker activity is tightly regulated by photic input from the retina. Recent work has implicated protein kinase C (PKC) as a regulator of photic input, although stimulus-induced PKC activity has not been examined. Here we used a combination of biochemical, immunohistochemical and behavioral techniques to examine both the regulation and role of PKC in light-induced clock entrainment in mice. We report that photic stimulation during the subjective night, but not during the subjective day, stimulates PKC activity within the SCN. To assess the role of PKC in clock entrainment, we employed an in-vivo infusion approach to deliver the PKC inhibitor bisindolylmaleimide I to the SCN. The disruption of PKC activity significantly enhanced the phase-shifting effects of light, indicating that PKC functions as a negative regulator of light entrainment. Importantly, bisindolylmaleimide I infusion in the absence of light treatment did not phase shift the clock, demonstrating that transient disruption of basal PKC activity does not affect inherent pacemaker activity. The capacity of light to stimulate immediate early gene expression in the SCN was not substantively altered by PKC inhibition, suggesting that PKC does not couple light to rapid transcriptional activation. Rather, a combination of in-vivo and cell culture assays indicates that PKC functions as an inhibitor of PERIOD1 degradation. Thus, PKC may influence clock entrainment via a post-translational mechanism that influences clock protein stability.