Oligodendrocyte progenitor cell maturation is dependent on dual function of MCT8 in the transport of thyroid hormone across brain barriers and the plasma membrane.

Inactivating mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) causes a rare and debilitating form of X-linked psychomotor disability known as Allan Herndon Dudley syndrome (AHDS). One of the most prominent pathophysiological symptoms of MCT8-deficiency is hypomyelination. Here, patient-derived induced pluripotent stem cells (iPSCs) were used to study the role of MCT8 and TH on the maturation of oligodendrocytes. Interestingly, neither MCT8 mutations nor reduced TH affected the in vitro differentiation of control or MCT8-deficient iPSCs into oligodendrocytes. To assess whether patient-derived iPSC-derived oligodendrocyte progenitor cells (iOPCs) could provide myelinating oligodendrocytes in vivo, cells were transplanted into the shiverer mouse corpus callosum where they survived, migrated, and matured into myelinating oligodendrocytes, though the myelination efficiency was reduced compared with control cells. When MCT8-deficient and healthy control iOPCs were transplanted into a novel hypothyroid immunodeficient triple knockout mouse (tKO, mct8-/- ; oatp1c1-/- ; rag2-/- ), they failed to provide behavioral recovery and did not mature into oligodendrocytes in the hypothyroid corpus callosum, demonstrating the critical role of TH transport across brain barriers in oligodendrocyte maturation. We conclude that MCT8 plays a cell autonomous role in oligodendrocyte maturation and that functional TH transport into the central nervous system will be required for developing an effective treatment for MCT8-deficient patients.

HD iPSC-derived neural progenitors accumulate in culture and are susceptible to BDNF withdrawal due to glutamate toxicity.

Huntington's disease (HD) is a fatal neurodegenerative disease, caused by expansion of polyglutamine repeats in the Huntingtin gene, with longer expansions leading to earlier ages of onset. The HD iPSC Consortium has recently reported a new in vitro model of HD based on the generation of induced pluripotent stem cells (iPSCs) from HD patients and controls. The current study has furthered the disease in a dish model of HD by generating new non-integrating HD and control iPSC lines. Both HD and control iPSC lines can be efficiently differentiated into neurons/glia; however, the HD-derived cells maintained a significantly greater number of nestin-expressing neural progenitor cells compared with control cells. This cell population showed enhanced vulnerability to brain-derived neurotrophic factor (BDNF) withdrawal in the juvenile-onset HD (JHD) lines, which appeared to be CAG repeat-dependent and mediated by the loss of signaling from the TrkB receptor. It was postulated that this increased death following BDNF withdrawal may be due to glutamate toxicity, as the N-methyl-d-aspartate (NMDA) receptor subunit NR2B was up-regulated in the cultures. Indeed, blocking glutamate signaling, not just through the NMDA but also mGlu and AMPA/Kainate receptors, completely reversed the cell death phenotype. This study suggests that the pathogenesis of JHD may involve in part a population of 'persistent' neural progenitors that are selectively vulnerable to BDNF withdrawal. Similar results were seen in adult hippocampal-derived neural progenitors isolated from the BACHD model mouse. Together, these results provide important insight into HD mechanisms at early developmental time points, which may suggest novel approaches to HD therapeutics.

Induced pluripotent stem cells from a spinal muscular atrophy patient.

Spinal muscular atrophy is one of the most common inherited forms of neurological disease leading to infant mortality. Patients have selective loss of lower motor neurons resulting in muscle weakness, paralysis and often death. Although patient fibroblasts have been used extensively to study spinal muscular atrophy, motor neurons have a unique anatomy and physiology which may underlie their vulnerability to the disease process. Here we report the generation of induced pluripotent stem cells from skin fibroblast samples taken from a child with spinal muscular atrophy. These cells expanded robustly in culture, maintained the disease genotype and generated motor neurons that showed selective deficits compared to those derived from the child's unaffected mother. This is the first study to show that human induced pluripotent stem cells can be used to model the specific pathology seen in a genetically inherited disease. As such, it represents a promising resource to study disease mechanisms, screen new drug compounds and develop new therapies.

CircHTT(2,3,4,5,6) - co-evolving with the HTT CAG-repeat tract - modulates Huntington's disease phenotypes.

Circular RNA (circRNA) molecules have critical functions during brain development and in brain-related disorders. Here, we identified and validated a circRNA, circHTT(2,3,4,5,6), stemming from the Huntington's disease (HD) gene locus that is most abundant in the central nervous system (CNS). We uncovered its evolutionary conservation in diverse mammalian species, and a correlation between circHTT(2,3,4,5,6) levels and the length of the CAG-repeat tract in exon-1 of HTT in human and mouse HD model systems. The mouse orthologue, circHtt(2,3,4,5,6), is expressed during embryogenesis, increases during nervous system development, and is aberrantly upregulated in the presence of the expanded CAG tract. While an IRES-like motif was predicted in circH TT (2,3,4,5,6), the circRNA does not appear to be translated in adult mouse brain tissue. Nonetheless, a modest, but consistent fraction of circHtt(2,3,4,5,6) associates with the 40S ribosomal subunit, suggesting a possible role in the regulation of protein translation. Finally, circHtt(2,3,4,5,6) overexpression experiments in HD-relevant STHdh striatal cells revealed its ability to modulate CAG expansion-driven cellular defects in cell-to-substrate adhesion, thus uncovering an unconventional modifier of HD pathology.

Optimizing maturity and dose of iPSC-derived dopamine progenitor cell therapy for Parkinson's disease.

In pursuit of treating Parkinson's disease with cell replacement therapy, differentiated induced pluripotent stem cells (iPSC) are an ideal source of midbrain dopaminergic (mDA) cells. We previously established a protocol for differentiating iPSC-derived post-mitotic mDA neurons capable of reversing 6-hydroxydopamine-induced hemiparkinsonism in rats. In the present study, we transitioned the iPSC starting material and defined an adapted differentiation protocol for further translation into a clinical cell transplantation therapy. We examined the effects of cellular maturity on survival and efficacy of the transplants by engrafting mDA progenitors (cryopreserved at 17 days of differentiation, D17), immature neurons (D24), and post-mitotic neurons (D37) into immunocompromised hemiparkinsonian rats. We found that D17 progenitors were markedly superior to immature D24 or mature D37 neurons in terms of survival, fiber outgrowth and effects on motor deficits. Intranigral engraftment to the ventral midbrain demonstrated that D17 cells had a greater capacity than D24 cells to innervate over long distance to forebrain structures, including the striatum. When D17 cells were assessed across a wide dose range (7,500-450,000 injected cells per striatum), there was a clear dose response with regards to numbers of surviving neurons, innervation, and functional recovery. Importantly, although these grafts were derived from iPSCs, we did not observe teratoma formation or significant outgrowth of other cells in any animal. These data support the concept that human iPSC-derived D17 mDA progenitors are suitable for clinical development with the aim of transplantation trials in patients with Parkinson's disease.

Delivery of recombinant follistatin lessens disease severity in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is the most common genetic cause of infant mortality. SMA is caused by loss of functional survival motor neuron 1 (SMN1), resulting in death of spinal motor neurons. Current therapeutic research focuses on modulating the expression of a partially functioning copy gene, SMN2, which is retained in SMA patients. However, a treatment strategy that improves the SMA phenotype by slowing or reversing the skeletal muscle atrophy may also be beneficial. Myostatin, a member of the TGF-beta super-family, is a potent negative regulator of skeletal muscle mass. Follistatin is a natural antagonist of myostatin, and over-expression of follistatin in mouse muscle leads to profound increases in skeletal muscle mass. To determine whether enhanced muscle mass impacts SMA, we administered recombinant follistatin to an SMA mouse model. Treated animals exhibited increased mass in several muscle groups, elevation in the number and cross-sectional area of ventral horn cells, gross motor function improvement and mean lifespan extension by 30%, by preventing some of the early deaths, when compared with control animals. SMN protein levels in spinal cord and muscle were unchanged in follistatin-treated SMA mice, suggesting that follistatin exerts its effect in an SMN-independent manner. Reversing muscle atrophy associated with SMA may represent an unexploited therapeutic target for the treatment of SMA.

Delivery of a read-through inducing compound, TC007, lessens the severity of a spinal muscular atrophy animal model.

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality and is caused by the loss of a functional SMN1 gene. In humans, there exists a nearly-identical copy gene known as SMN2 that encodes an identical protein as SMN1, but differs by a silent C to T transition within exon 7. This single nucleotide difference produces an alternatively spliced isoform, SMNDelta7, which encodes a rapidly degraded protein. The absence of the short peptide encoded by SMN exon 7 is critical in the disease development process; however, heterologous sequences can partially compensate for the SMN exon 7 peptide in several cellular assays. Consistent with this, aminoglycosides, compounds that can suppress efficient recognition of stop codons, resulted in significantly increased levels of SMN protein in SMA patient fibroblasts. We now examine the potential therapeutic capabilities of a novel aminoglycoside, TC007. In an intermediate SMA model (Smn-/-; SMN2+/+; SMNDelta7), when delivered directly to the central nervous system (CNS), TC007 induces SMN in both the brain and spinal cord, significantly increases lifespan ( approximately 30%) and increases ventral horn cell number, consistent with its ability to increase SMN levels in induced pluripotent stem cell-derived human SMA motor neuron cultures. Collectively, these experiments are the first in vivo examination of therapeutics for SMA designed to induce read-through of the SMNDelta7 stop codon to show increased benefit by direct administration to the CNS.

The difficulty to model Huntington's disease in vitro using striatal medium spiny neurons differentiated from human induced pluripotent stem cells.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanded polyglutamine repeat in the huntingtin gene. The neuropathology of HD is characterized by the decline of a specific neuronal population within the brain, the striatal medium spiny neurons (MSNs). The origins of this extreme vulnerability remain unknown. Human induced pluripotent stem cell (hiPS cell)-derived MSNs represent a powerful tool to study this genetic disease. However, the differentiation protocols published so far show a high heterogeneity of neuronal populations in vitro. Here, we compared two previously published protocols to obtain hiPS cell-derived striatal neurons from both healthy donors and HD patients. Patch-clamp experiments, immunostaining and RT-qPCR were performed to characterize the neurons in culture. While the neurons were mature enough to fire action potentials, a majority failed to express markers typical for MSNs. Voltage-clamp experiments on voltage-gated sodium (Nav) channels revealed a large variability between the two differentiation protocols. Action potential analysis did not reveal changes induced by the HD mutation. This study attempts to demonstrate the current challenges in reproducing data of previously published differentiation protocols and in generating hiPS cell-derived striatal MSNs to model a genetic neurodegenerative disorder in vitro.

Therapeutics that directly increase SMN expression to treat spinal muscular atrophy.

Spinal muscular atrophy (SMA) is the second most common autosomal recessive disease and is a leading cause of infantile death. This disease has a carrier frequency of 1:35, affecting 1/6,000 live births and is the result of a homozygous loss of the survival of motor neuron 1 gene (SMN1). Humans carry a nearly identical copy gene, SMN2, that codes for very low levels of the full-length protein, ∼10% when compared to SMN1. This is due to one silent nucleotide transition at the 5' end of exon 7 that disrupts a critical splicing regulatory domain. The underlying protein coding region, however, is unaffected by this and other nucleotide differences between SMN1 and SMN2. SMN2 has, therefore, been envisioned as an outstanding target for therapeutic strategies that 1) increases SMN2 expression, 2) alters the pre-messenger RNA splicing of exon 7 or 3) stabilizes the SMN2-derived protein products. In this review, we summarize numerous therapeutic approaches including nucleic acid-based and drug-oriented therapies that have progressed toward treating SMA.

The Interaction of Aging and Cellular Stress Contributes to Pathogenesis in Mouse and Human Huntington Disease Neurons.

Huntington disease (HD) is a fatal, inherited neurodegenerative disorder caused by a mutation in the huntingtin (HTT) gene. While mutant HTT is present ubiquitously throughout life, HD onset typically occurs in mid-life. Oxidative damage accumulates in the aging brain and is a feature of HD. We sought to interrogate the roles and interaction of age and oxidative stress in HD using primary Hu97/18 mouse neurons, neurons differentiated from HD patient induced pluripotent stem cells (iPSCs), and the brains of HD mice. We find that primary neurons must be matured in culture for canonical stress responses to occur. Furthermore, when aging is accelerated in mature HD neurons, mutant HTT accumulates and sensitivity to oxidative stress is selectively enhanced. Furthermore, we observe HD-specific phenotypes in neurons and mouse brains that have undergone accelerated aging, including a selective increase in DNA damage. These findings suggest a role for aging in HD pathogenesis and an interaction between the biological age of HD neurons and sensitivity to exogenous stress.

Subcutaneous administration of TC007 reduces disease severity in an animal model of SMA.

BACKGROUND: Spinal Muscular Atrophy (SMA) is the leading genetic cause of infantile death. It is caused by the loss of functional Survival Motor Neuron 1 (SMN1). There is a nearly identical copy gene, SMN2, but it is unable to rescue from disease due to an alternative splicing event that excises a necessary exon (exon 7) from the majority of SMN2-derived transcripts. While SMNDelta7 protein has severely reduced functionality, the exon 7 sequences may not be specifically required for all activities. Therefore, aminoglycoside antibiotics previously shown to suppress stop codon recognition and promote translation read-through have been examined to increase the length of the SMNDelta7 C-terminus. RESULTS: Here we demonstrate that subcutaneous-administration of a read-through inducing compound (TC007) to an intermediate SMA model (Smn-/-; SMN2+/+; SMNDelta7) had beneficial effects on muscle fiber size and gross motor function. CONCLUSION: Delivery of the read-through inducing compound TC007 reduces the disease-associated phenotype in SMA mice, however, does not significantly extend survival.

Characterization of Neurodevelopmental Abnormalities in iPSC-Derived Striatal Cultures from Patients with Huntington's Disease.

BACKGROUND: Huntington's disease (HD) is an inherited neurodegenerative disease and is characterized by atrophy of certain regions of the brain in a progressive manner. HD patients experience behavioral changes and uncontrolled movements which can be primarily attributed to the atrophy of striatal neurons. Previous publications describe the models of the HD striatum using induced pluripotent stem cells (iPSCs) derived from HD patients with a juvenile onset (JHD). In this model, the JHD iPSC-derived striatal cultures had altered neurodevelopment and contained a high number of nestin expressing progenitor cells at 42 days of differentiation. OBJECTIVE: To further characterize the altered neurodevelopmental phenotype and evaluate potential phenotypic reversal. METHODS: Differentiation of human iPSCs towards striatal fate and characterization by means of immunocytochemistry and stereological quantification. RESULTS: Here this study demonstrates a distinct delay in the differentiation of the JHD neural progenitor population. However, reduction of the JHD aberrant progenitor populations can be accomplished either by targeting the canonical Notch signaling pathway or by treatment with HTT antisense oligonucleotides (ASOs). CONCLUSIONS: In summary, this data is postulated to reflect a potential overall developmental delay in JHD.

Huntington's Disease Patient-Derived Astrocytes Display Electrophysiological Impairments and Reduced Neuronal Support.

In Huntington's disease (HD), while the ubiquitously expressed mutant Huntingtin (mtHTT) protein primarily compromises striatal and cortical neurons, glia also undergo disease-contributing alterations. Existing HD models using human induced pluripotent stem cells (iPSCs) have not extensively characterized the role of mtHTT in patient-derived astrocytes. Here physiologically mature astrocytes are generated from HD patient iPSCs. These human astrocytes exhibit hallmark HD phenotypes that occur in mouse models, including impaired inward rectifying K+ currents, lengthened spontaneous Ca2+ waves and reduced cell membrane capacitance. HD astrocytes in co-culture provided reduced support for the maturation of iPSC-derived neurons. In addition, neurons exposed to chronic glutamate stimulation are not protected by HD astrocytes. This iPSC-based HD model demonstrates the critical effects of mtHTT on human astrocytes, which not only broadens the understanding of disease susceptibility beyond cortical and striatal neurons but also increases potential drug targets.

Detection of human survival motor neuron (SMN) protein in mice containing the SMN2 transgene: applicability to preclinical therapy development for spinal muscular atrophy.

Spinal muscular atrophy (SMA), the leading genetic cause of infant death results from loss of spinal motor neurons causing atrophy of skeletal muscle. SMA is caused by loss of the Survival Motor Neuron 1 (SMN1) gene, however, an identically coding gene called SMN2 is retained, but is alternatively spliced to produce approximately 90% truncated protein. Most SMA translational and preclinical drug development has relied on the use of SMA mice to determine changes in SMN protein levels. However, the SMA mouse models are relatively severe and analysis of SMN-inducing compounds is confounded by the early mortality of these animals. An antibody that could detect SMN protein on a Smn background could circumvent this limitation and allow unaffected, heterozygous animals to be examined. Here we describe the generation and characterization of a monoclonal anti-SMN antibody, 4F11, which specifically recognizes human SMN protein. 4F11 detects SMN (human) but not native Smn (mouse) protein in SMN2 transgenic mice and in SMA cell lines. We demonstrate the feasibility of using 4F11 to detect changes in SMN2-derived SMN protein in SMA patient fibroblasts and in healthy SMN2 transgenic mice. This antibody is, therefore, an excellent tool for examining SMN2-inducing therapeutics in patient cells as well as in transgenic mice.

A SMNDelta7 read-through product confers functionality to the SMNDelta7 protein.

Spinal muscular atrophy (SMA) affects about 1 in every 6000 children born and is the leading genetic cause of infant death. SMA is a recessive disorder caused by the mutation or deletion of Survival Motor Neuron-1 (SMN1). SMN2, a nearly identical copy gene, has the potential to encode the same protein as SMN1 and is retained in all SMA patients. The majority of SMN2-derived transcripts are alternatively spliced and therefore encode a truncated isoform lacking exon 7 (SMNDelta7), which is a defective protein because it is unstable, has a reduced ability to self-associate and is unable to efficiently function in SMN cellular activities. However, we have shown that the SMN C-terminus functions non-specifically, since heterologous sequences can compensate for the exon 7 sequence. Several classes of compounds identified in SMN-inducing high throughput screens have been proposed to function through a read-through mechanism; however, a functional analysis of the SMNDelta7 read-through product has not been performed. In this report, the SMNDelta7 read-through product is characterized and compared to the SMNDelta7 protein. In a series of in vitro and cell based assays, SMNDelta7 read-through product is shown to increase protein stability, promote neurite outgrowths in SMN deficient neurons, and significantly elevate SMN-dependent UsnRNP assembly in extracts from SMA patient fibroblasts. Collectively, these results demonstrate that SMNDelta7 read-through product is more active than the SMNDelta7 protein and suggest that SMA therapeutics that specifically induce SMNDelta7 read-through may provide an alternative platform for drug discovery.

Cellular Models: HD Patient-Derived Pluripotent Stem Cells.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by expanded polyglutamine (polyQ)-encoding repeats in the Huntingtin (HTT) gene. Traditionally, HD cellular models consisted of either patient cells not affected by disease or rodent neurons expressing expanded polyQ repeats in HTT. As these models can be limited in their disease manifestation or proper genetic context, respectively, human HD pluripotent stem cells (PSCs) are currently under investigation as a way to model disease in patient-derived neurons and other neural cell types. This chapter reviews embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) models of disease, including published differentiation paradigms for neurons and their associated phenotypes, as well as current challenges to the field such as validation of the PSCs and PSC-derived cells. Highlighted are potential future technical advances to HD PSC modeling, including transdifferentiation, complex in vitro multiorgan/system reconstruction, and personalized medicine. Using a human HD patient model of the central nervous system, hopefully one day researchers can tease out the consequences of mutant HTT (mHTT) expression on specific cell types within the brain in order to identify and test novel therapies for disease.

Human Huntington's Disease iPSC-Derived Cortical Neurons Display Altered Transcriptomics, Morphology, and Maturation.

Huntington's disease (HD) is a neurodegenerative disease caused by an expanded CAG repeat in the Huntingtin (HTT) gene. Induced pluripotent stem cell (iPSC) models of HD provide an opportunity to study the mechanisms underlying disease pathology in disease-relevant patient tissues. Murine studies have demonstrated that HTT is intricately involved in corticogenesis. However, the effect of mutant Hungtintin (mtHTT) in human corticogenesis has not yet been thoroughly explored. This examination is critical, due to inherent differences in cortical development and timing between humans and mice. We therefore differentiated HD and non-diseased iPSCs into functional cortical neurons. While HD patient iPSCs can successfully differentiate toward a cortical fate in culture, the resulting neurons display altered transcriptomics, morphological and functional phenotypes indicative of altered corticogenesis in HD.

Inducible Expression of GDNF in Transplanted iPSC-Derived Neural Progenitor Cells.

Trophic factor delivery to the brain using stem cell-derived neural progenitors is a powerful way to bypass the blood-brain barrier. Protection of diseased neurons using this technology is a promising therapy for neurodegenerative diseases. Glial cell line-derived neurotrophic factor (GDNF) has provided benefits to Parkinsonian patients and is being used in a clinical trial for amyotrophic lateral sclerosis. However, chronic trophic factor delivery prohibits dose adjustment or cessation if side effects develop. To address this, we engineered a doxycycline-regulated vector, allowing inducible and reversible expression of a therapeutic molecule. Human induced pluripotent stem cell (iPSC)-derived neural progenitors were stably transfected with the vector and transplanted into the adult mouse brain. Doxycycline can penetrate the graft, with addition and withdrawal providing inducible and reversible GDNF expression in vivo, over multiple cycles. Our findings provide proof of concept for combining gene and stem cell therapy for effective modulation of ectopic protein expression in transplanted cells.

Modeling Huntington׳s disease with patient-derived neurons.

Huntington׳s Disease (HD) is a fatal neurodegenerative disorder caused by expanded polyglutamine repeats in the Huntingtin (HTT) gene. While the gene was identified over two decades ago, it remains poorly understood why mutant HTT (mtHTT) is initially toxic to striatal medium spiny neurons (MSNs). Models of HD using non-neuronal human patient cells and rodents exhibit some characteristic HD phenotypes. While these current models have contributed to the field, they are limited in disease manifestation and may vary in their response to treatments. As such, human HD patient MSNs for disease modeling could greatly expand the current understanding of HD and facilitate the search for a successful treatment. It is now possible to use pluripotent stem cells, which can generate any tissue type in the body, to study and potentially treat HD. This review covers disease modeling in vitro and, via chimeric animal generation, in vivo using human HD patient MSNs differentiated from embryonic stem cells or induced pluripotent stem cells. This includes an overview of the differentiation of pluripotent cells into MSNs, the established phenotypes found in cell-based models and transplantation studies using these cells. This review not only outlines the advancements in the rapidly progressing field of HD modeling using neurons derived from human pluripotent cells, but also it highlights several remaining controversial issues such as the 'ideal' series of pluripotent lines, the optimal cell types to use and the study of a primarily adult-onset disease in a developmental model. This article is part of a Special Issue entitled SI: Exploiting human neurons.

Optimization of trans-Splicing for Huntington's Disease RNA Therapy.

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by a polyglutamine (polyQ) expansion in exon 1 of the Huntingtin (HTT) gene. We have previously demonstrated that spliceosome-mediated trans-splicing is a viable molecular strategy to specifically reduce and repair mutant HTT (mtHTT). Here, the targeted tethering efficacy of the pre-mRNA trans-splicing modules (PTM) in HTT was optimized. Various PTMs that targeted the 3' end of HTT intron 1 or the intron 1 branch point were shown trans-splice into an HTT mini-gene, as well as the endogenous HTT pre-mRNA. PTMs that specifically target the endogenous intron 1 branch point increased the trans-splicing efficacy from 1-5 to 10-15%. Furthermore, lentiviral expression of PTMs in a human HD patient iPSC-derived neural culture significantly reversed two previously established polyQ-length dependent phenotypes. These results suggest that pre-mRNA repair of mtHTT could hold therapeutic benefit and it demonstrates an alternative platform to correct the mRNA product produced by the mtHTT allele in the context of HD.