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We have previously established induced pluripotent stem cell (iPSC) models of Huntington's disease (HD), demonstrating CAG-repeat-expansion-dependent cell biological changes and toxicity. However, the current differentiation protocols are cumbersome and time consuming, making preparation of large quantities of cells for biochemical or screening assays difficult. Here, we report the generation of immortalized striatal precursor neurons (ISPNs) with normal (33) and expanded (180) CAG repeats from HD iPSCs, differentiated to a phenotype resembling medium spiny neurons (MSN), as a proof of principle for a more tractable patient-derived cell model. For immortalization, we used co-expression of the enzymatic component of telomerase hTERT and conditional expression of c-Myc. ISPNs can be propagated as stable adherent cell lines, and rapidly differentiated into highly homogeneous MSN-like cultures within 2 weeks, as demonstrated by immunocytochemical criteria. Differentiated ISPNs recapitulate major HD-related phenotypes of the parental iPSC model, including brain-derived neurotrophic factor (BDNF)-withdrawal-induced cell death that can be rescued by small molecules previously validated in the parental iPSC model. Proteome and RNA-seq analyses demonstrate separation of HD versus control samples by principal component analysis. We identified several networks, pathways, and upstream regulators, also found altered in HD iPSCs, other HD models, and HD patient samples. HD ISPN lines may be useful for studying HD-related cellular pathogenesis, and for use as a platform for HD target identification and screening experimental therapeutics. The described approach for generation of ISPNs from differentiated patient-derived iPSCs could be applied to a larger allelic series of HD cell lines, and to comparable modeling of other genetic disorders., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Altered cellular metabolism is believed to be an important contributor to pathogenesis of the neurodegenerative disorder Huntington's disease (HD). Research has primarily focused on mitochondrial toxicity, which can cause death of the vulnerable striatal neurons, but other aspects of metabolism have also been implicated. Most previous studies have been carried out using postmortem human brain or non-human cells. Here, we studied bioenergetics in an induced pluripotent stem cell-based model of the disease. We found decreased adenosine triphosphate (ATP) levels in HD cells compared to controls across differentiation stages and protocols. Proteomics data and multiomics network analysis revealed normal or increased levels of mitochondrial messages and proteins, but lowered expression of glycolytic enzymes. Metabolic experiments showed decreased spare glycolytic capacity in HD neurons, while maximal and spare respiratory capacities driven by oxidative phosphorylation were largely unchanged. ATP levels in HD neurons could be rescued with addition of pyruvate or late glycolytic metabolites, but not earlier glycolytic metabolites, suggesting a role for glycolytic deficits as part of the metabolic disturbance in HD neurons. Pyruvate or other related metabolic supplements could have therapeutic benefit in HD.

Neural cultures derived from Huntington's disease (HD) patient-derived induced pluripotent stem cells were used for 'omics' analyses to identify mechanisms underlying neurodegeneration. RNA-seq analysis identified genes in glutamate and GABA signaling, axonal guidance and calcium influx whose expression was decreased in HD cultures. One-third of gene changes were in pathways regulating neuronal development and maturation. When mapped to stages of mouse striatal development, the profiles aligned with earlier embryonic stages of neuronal differentiation. We observed a strong correlation between HD-related histone marks, gene expression and unique peak profiles associated with dysregulated genes, suggesting a coordinated epigenetic program. Treatment with isoxazole-9, which targets key dysregulated pathways, led to amelioration of expanded polyglutamine repeat-associated phenotypes in neural cells and of cognitive impairment and synaptic pathology in HD model R6/2 mice. These data suggest that mutant huntingtin impairs neurodevelopmental pathways that could disrupt synaptic homeostasis and increase vulnerability to the pathologic consequence of expanded polyglutamine repeats over time.

Huntington's disease (HD) is caused by an expanded CAG repeat in the Huntingtin (HTT) gene. The mechanism(s) by which mutant HTT (mHTT) causes disease is unclear. Nucleocytoplasmic transport, the trafficking of macromolecules between the nucleus and cytoplasm, is tightly regulated by nuclear pore complexes (NPCs) made up of nucleoporins (NUPs). Previous studies offered clues that mHTT may disrupt nucleocytoplasmic transport and a mutation of an NUP can cause HD-like pathology. Therefore, we evaluated the NPC and nucleocytoplasmic transport in multiple models of HD, including mouse and fly models, neurons transfected with mHTT, HD iPSC-derived neurons, and human HD brain regions. These studies revealed severe mislocalization and aggregation of NUPs and defective nucleocytoplasmic transport. HD repeat-associated non-ATG (RAN) translation proteins also disrupted nucleocytoplasmic transport. Additionally, overexpression of NUPs and treatment with drugs that prevent aberrant NUP biology also mitigated this transport defect and neurotoxicity, providing future novel therapy targets.

Huntington's disease (HD) is a neurodegenerative disease caused by a CAG repeat expansion in the Huntingtin gene (HTT), translated into a Huntingtin protein with a polyglutamine expansion. There is preferential loss of medium spiny neurons within the striatum and cortical pyramidal neurons. Pridopidine is a small molecule showing therapeutic potential in HD preclinical and clinical studies. Pridopidine has nanomolar affinity to the sigma-1 receptor (sigma-1R), which is located predominantly at the endoplasmic reticulum (ER) and mitochondrial associated ER membrane, and activates neuroprotective pathways. Here we evaluate the neuroprotective effects of pridopidine against mutant Huntingtin toxicity in mouse and human derived in vitro cell models. We also investigate the involvement of the sigma-1 receptor in the mechanism of pridopidine. Pridopidine protects mutant Huntingtin transfected mouse primary striatal and cortical neurons, with an EC50 in the mid nanomolar range, as well as HD patient-derived induced pluripotent stem cells (iPSCs). This protection by pridopidine is blocked by NE-100, a purported sigma-1 receptor antagonist, and not blocked by ANA-12, a reported TrkB receptor antagonist. 3PPP, a documented sigma-1 receptor agonist, shows similar neuroprotective effects. Genetic knock out of the sigma-1 receptor dramatically decreases protection from pridopidine and 3PPP, but not protection via brain derived neurotrophic factor (BDNF). The neuroprotection afforded by pridopidine in our HD cell models is robust and sigma-1 receptor dependent. These studies support the further development of pridopidine, and other sigma-1 receptor agonists as neuroprotective agents for HD and perhaps for other disorders. • Pridopidine protects against mutant Huntingtin toxicity in mouse primary neurons. • Protection is blocked by sigma-1R antagonist, but not by TrkB receptor antagonist. • Pridopidine shows similar protection of HD patient iPSCs. • Sigma-1R knockout specifically blocks pridopidine neuroprotection. [ABSTRACT FROM AUTHOR]

Background: The potential benefit of cysteamine for Huntington's disease has been demonstrated in HD animal models. Cysteamine and its derivate cystamine were shown to reduce neuropathology and prolong lifespan. Human studies have demonstrated safety, and suggestive results regarding efficacy. Despite all the studies available in vivo, there are only few in vitro studies, and the mechanism of action of cysteamine remains unclear. Objective: The objective of this study is to assess the capacity of cysteamine for neuroprotection against mutant Huntingtin in vitro using cellular models of HD, and to provide initial data regarding mechanism of action. Methods: We tested the neuroprotective properties of cysteamine in vitro in our primary neuron and iPSC models of HD. Results: Cysteamine showed a strong neuroprotective effect (EC50 = 7.1 nM) against mutant Htt-(aa-1-586 82Q) toxicity, in a nuclear condensation cell toxicity assay. Cysteamine also rescued mitochondrial changes induced by mutant Htt. Modulation of the levels of cysteine or glutathione failed to protect neurons, suggesting that cysteamine neuroprotection is not mediated through cysteine metabolism. Taurine and Hypotaurine, which are metabolites of cysteamine can protect neurons against Htt toxicity, but the inhibition of the enzyme converting cysteamine to hypotaurine does not block either protective activity, suggesting independent protective pathways. Cysteamine has been suggested to activate BDNF secretion; however, cysteamine protection was not blocked by BDNF pathway antagonists. Conclusions: Cysteamine was strongly neuroprotective with relatively high potency. We demonstrated that the main neuroprotective pathways that have been proposed to be the mechanism of protection by cysteamine can all be blocked and still not prevent the neuroprotective effect. The results suggest the involvement of other yet-to-be-determined neuroprotective pathways. [ABSTRACT FROM AUTHOR]