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.

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.

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.

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.

Survival of iPSC-derived grafts within the striatum of immunodeficient mice: Importance of developmental stage of both transplant and host recipient.

Degeneration of the striatum can occur in multiple disorders with devastating consequences for the patients. Infantile infections with streptococcus, measles, or herpes can cause striatal necrosis associated with dystonia or dyskinesia; and in patients with Huntington's disease the striatum undergoes massive degeneration, leading to behavioral, psychological and movement issues, ultimately resulting in death. Currently, only supportive therapies are available for striatal degeneration. Clinical trials have shown some efficacy using transplantation of fetal-derived primary striatal progenitors. Large banks of fetal progenitors that give rise to medium spiny neurons (MSNs), the primary neuron of the striatum, are needed to make transplantation therapy a reality. However, fetal tissue is of limited supply, has ethical concerns, and is at risk of graft immunorejection. An alternative potential source of MSNs is induced pluripotent stem cells (iPSCs), adult somatic tissues reprogrammed back to a stem cell fate. Multiple publications have demonstrated the ability to differentiate striatal MSNs from iPSCs. Previous publications have demonstrated that the efficacy of fetal progenitor transplants is critically dependent upon the age of the donor embryo/fetus as well as the age of the transplant recipient. With the advent of iPSC technology, a question that remains unanswered concerns the graft's "age," which is crucial since transplanting pluripotent cells has an inherent risk of over proliferation and teratoma formation. Therefore, in order to also determine the effect of transplant recipient age on the graft, iPSCs were differentiated to three stages along a striatal differentiation paradigm and transplanted into the striatum of both neonatal and adult immunodeficient mice. This study demonstrated that increased murine transplant-recipient age (adult vs neonate) resulted in decreased graft survival and volume/rostro-caudal spread after six weeks in vivo, regardless of "age" of the cells transplanted. Importantly, this study implicates that the in vivo setting may provide a better neurogenic niche for iPSC-based modeling as compared to the in vitro setting. Together, these results recapitulate findings from fetal striatal progenitor transplantation studies and further demonstrate the influence of the host environment on cellular survival and maturation.