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

AbstractHuntington’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.

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Dysregulation of Neuronal Calcium Signaling via Store-Operated Channels in Huntington's Disease

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.611735 ◽

2020 ◽

Vol 8 ◽

Author(s):

Magdalena Czeredys

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Transient Receptor Potential ◽

Neurodegenerative Disorder ◽

Induced Pluripotent Stem Cell ◽

Receptor Potential ◽

Medium Spiny Neurons ◽

Trpc Channels ◽

Polyglutamine Expansion ◽

Spiny Neurons

Huntington's disease (HD) is a progressive neurodegenerative disorder that is characterized by motor, cognitive, and psychiatric problems. It is caused by a polyglutamine expansion in the huntingtin protein that leads to striatal degeneration via the transcriptional dysregulation of several genes, including genes that are involved in the calcium (Ca2+) signalosome. Recent research has shown that one of the major Ca2+ signaling pathways, store-operated Ca2+ entry (SOCE), is significantly elevated in HD. SOCE refers to Ca2+ flow into cells in response to the depletion of endoplasmic reticulum Ca2+ stores. The dysregulation of Ca2+ homeostasis is postulated to be a cause of HD progression because the SOCE pathway is indirectly and abnormally activated by mutant huntingtin (HTT) in γ-aminobutyric acid (GABA)ergic medium spiny neurons (MSNs) from the striatum in HD models before the first symptoms of the disease appear. The present review summarizes recent studies that revealed a relationship between HD pathology and elevations of SOCE in different models of HD, including YAC128 mice (a transgenic model of HD), cellular HD models, and induced pluripotent stem cell (iPSC)-based GABAergic medium spiny neurons (MSNs) that are obtained from adult HD patient fibroblasts. SOCE in MSNs was shown to be mediated by currents through at least two different channel groups, Ca2+ release-activated Ca2+ current (ICRAC) and store-operated Ca2+ current (ISOC), which are composed of stromal interaction molecule (STIM) proteins and Orai or transient receptor potential channel (TRPC) channels. Their role under physiological and pathological conditions in HD are discussed. The role of Huntingtin-associated protein 1 isoform A in elevations of SOCE in HD MSNs and potential compounds that may stabilize elevations of SOCE in HD are also summarized. Evidence is presented that shows that the dysregulation of molecular components of SOCE or pathways upstream of SOCE in HD MSN neurons is a hallmark of HD, and these changes could lead to HD pathology, making them potential therapeutic targets.

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Bioenergetic deficits in Huntington’s disease iPSC-derived neural cells and rescue with glycolytic metabolites

Human Molecular Genetics ◽

10.1093/hmg/ddy430 ◽

2019 ◽

Vol 29 (11) ◽

pp. 1757-1771 ◽

Cited By ~ 4

Author(s):

◽

Amanda J Kedaigle ◽

Ernest Fraenkel ◽

Ranjit S Atwal ◽

Min Wu ◽

...

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Neurodegenerative Disorder ◽

Induced Pluripotent Stem Cell ◽

Therapeutic Benefit ◽

Neural Cells ◽

Striatal Neurons ◽

Proteomics Data ◽

Postmortem Human Brain ◽

Atp Levels

Abstract 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.

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A Human Induced Pluripotent Stem Cell-Derived Isogenic Model of Huntington’s Disease Based on Neuronal Cells Has Several Relevant Phenotypic Abnormalities

Journal of Personalized Medicine ◽

10.3390/jpm10040215 ◽

2020 ◽

Vol 10 (4) ◽

pp. 215

Author(s):

Tuyana Malankhanova ◽

Lyubov Suldina ◽

Elena Grigor’eva ◽

Sergey Medvedev ◽

Julia Minina ◽

...

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Neurodegenerative Disorder ◽

Microscopic Analysis ◽

Induced Pluripotent Stem Cell ◽

Cell System ◽

Neural Cells ◽

Electron Microscopic ◽

Healthy Human ◽

Induced Pluripotent

Huntington’s disease (HD) is a severe neurodegenerative disorder caused by a CAG triplet expansion in the first exon of the HTT gene. Here we report the introduction of an HD mutation into the genome of healthy human embryonic fibroblasts through CRISPR/Cas9-mediated homologous recombination. We verified the specificity of the created HTT-editing system and confirmed the absence of undesirable genomic modifications at off-target sites. We showed that both mutant and control isogenic induced pluripotent stem cells (iPSCs) derived by reprogramming of the fibroblast clones can be differentiated into striatal medium spiny neurons. We next demonstrated phenotypic abnormalities in the mutant iPSC-derived neural cells, including impaired neural rosette formation and increased sensitivity to growth factor withdrawal. Moreover, using electron microscopic analysis, we detected a series of ultrastructural defects in the mutant neurons, which did not contain huntingtin aggregates, suggesting that these defects appear early in HD development. Thus, our study describes creation of a new isogenic iPSC-based cell system that models HD and recapitulates HD-specific disturbances in the mutant cells, including some ultrastructural features implemented for the first time.

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Dysfunction of cAMP-PKA-calcium signaling axis in striatal medium spiny neurons: a role in schizophrenia and Huntington’s disease pathogenesis

10.1101/2021.10.08.463614 ◽

2021 ◽

Author(s):

Marija Fjodorova ◽

Zoe Noakes ◽

Daniel C. De La Fuente ◽

Adam C. Errington ◽

Meng Li

Keyword(s):

Huntington's Disease ◽

Calcium Signaling ◽

Huntington’S Disease ◽

Cortical Neurons ◽

Medium Spiny Neurons ◽

Striatal Neurons ◽

Cell Type ◽

Direct Role ◽

Spiny Neurons ◽

Signaling Axis

SummaryBackgroundStriatal medium spiny neurons (MSNs) are preferentially lost in Huntington’s disease. Genomic studies also implicate a direct role for MSNs in schizophrenia, a psychiatric disorder known to involve cortical neuron dysfunction. It remains unknown whether the two diseases share similar MSN pathogenesis or if neuronal deficits can be attributed to cell type-dependent biological pathways. Transcription factor BCL11B, which is expressed by all MSNs and deep layer cortical neurons, was recently proposed to drive selective neurodegeneration in Huntington’s disease and identified as a candidate risk gene in schizophrenia.MethodsUsing human stem cell-derived neurons lacking BCL11B as a model, we investigated cellular pathology in MSNs and cortical neurons in the context of these disorders. Integrative analyses between differentially expressed transcripts and published GWAS datasets identified cell type-specific disease-related phenotypes.ResultsWe uncover a role for BCL11B in calcium homeostasis in both neuronal types, while deficits in mitochondrial function and protein kinase A (PKA)-dependent calcium transients are detected only in MSNs. Moreover, BCL11B-deficient MSNs display abnormal responses to glutamate and fail to integrate dopaminergic and glutamatergic stimulation, a key feature of striatal neurons in vivo. Gene enrichment analysis reveals overrepresentation of disorder risk genes among BCL11B-regulated pathways, primarily relating to cAMP-PKA-calcium signaling axis and synaptic signaling.ConclusionsOur study indicates that Huntington’s disease and schizophrenia are likely to share neuronal pathogenesis where dysregulation of intracellular calcium levels is found in both striatal and cortical neurons. In contrast, reduction in PKA signaling and abnormal dopamine/glutamate receptor signaling is largely specific to MSNs.

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Oxidative Stress Research on Huntington's Disease Neurons Using Genetically Encoded Biosensors

KnE Life Sciences ◽

10.18502/kls.v7i1.10152 ◽

2022 ◽

Author(s):

Tuyana Malankhanova ◽

Dayaana Vasileva ◽

Elena Grigor'eva ◽

Sergey Medvedev ◽

Suren Zakian ◽

...

Keyword(s):

Oxidative Stress ◽

Stem Cells ◽

Huntington's Disease ◽

Induced Pluripotent Stem Cells ◽

Huntington’S Disease ◽

Pluripotent Stem Cells ◽

Cell Model ◽

Medium Spiny Neurons ◽

Spiny Neurons ◽

Induced Pluripotent

Many neurodegenerative diseases, including Huntington’s disease (HD), are associated with oxidative stress in the neurons of the brain. Genetically encoded biosensorsare useful for studying these processesin vitro. Human cell cultures expressing the biosensors can serve as a cell model for developing and testing effective agents that reduce oxidative stress. In this work, transgenes encoding biosensors of glutathione oxidative potential(Grx1-roGFP2) with cytoplasmic and mitochondrial localization were introduced into human induced pluripotent stem cells of a healthy donor and an HD patient using CRISPR/Cas9-mediated genome editing. The cells were subsequently differentiated into medium spiny neurons of the striatum. The expression of the biosensors was detected in the iPSCs, neuronal precursors and mature neurons.The obtained cells could be used to study the redox potential of glutathione in HD neurons and to screen for new drug compounds aimed at reducing oxidative stress. Keywords: genetically encoded biosensors, Huntington’s disease, induced pluripotent stem cells, medium spiny neurons, oxidative stress, glutathione, Grx1-roGFP2

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Cell Reprogramming to Model Huntington’s Disease: A Comprehensive Review

Cells ◽

10.3390/cells10071565 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1565

Author(s):

Ruth Monk ◽

Bronwen Connor

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Neurodegenerative Disorder ◽

Cag Repeat ◽

Cell Reprogramming ◽

Medium Spiny Neurons ◽

High Throughput Drug Screening ◽

Mutant Huntingtin Protein ◽

Neurological Conditions

Huntington’s disease (HD) is a neurodegenerative disorder characterized by the progressive decline of motor, cognitive, and psychiatric functions. HD results from an autosomal dominant mutation that causes a trinucleotide CAG repeat expansion and the production of mutant Huntingtin protein (mHTT). This results in the initial selective and progressive loss of medium spiny neurons (MSNs) in the striatum before progressing to involve the whole brain. There are currently no effective treatments to prevent or delay the progression of HD as knowledge into the mechanisms driving the selective degeneration of MSNs has been hindered by a lack of access to live neurons from individuals with HD. The invention of cell reprogramming provides a revolutionary technique for the study, and potential treatment, of neurological conditions. Cell reprogramming technologies allow for the generation of live disease-affected neurons from patients with neurological conditions, becoming a primary technique for modelling these conditions in vitro. The ability to generate HD-affected neurons has widespread applications for investigating the pathogenesis of HD, the identification of new therapeutic targets, and for high-throughput drug screening. Cell reprogramming also offers a potential autologous source of cells for HD cell replacement therapy. This review provides a comprehensive analysis of the use of cell reprogramming to model HD and a discussion on recent advancements in cell reprogramming technologies that will benefit the HD field.

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Striatal Neurodegeneration that Mimics Huntington’s Disease Modifies GABA-induced Currents

Brain Sciences ◽

10.3390/brainsci8120217 ◽

2018 ◽

Vol 8 (12) ◽

pp. 217

Author(s):

Jorge Flores-Hernández ◽

Jeanette Garzón-Vázquez ◽

Gustavo Hernández-Carballo ◽

Elizabeth Nieto-Mendoza ◽

Evelyn Ruíz-Luna ◽

...

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Gaba Receptors ◽

Gaba Receptor ◽

Receptor Subtype ◽

Medium Spiny Neurons ◽

Nitropropionic Acid ◽

Spiny Neurons ◽

Excitotoxic Damage ◽

Induced Currents

Huntington’s Disease (HD) is a degenerative disease which produces cognitive and motor disturbances. Treatment with GABAergic agonists improves the behavior and activity of mitochondrial complexes in rodents treated with 3-nitropropionic acid to mimic HD symptomatology. Apparently, GABA receptors activity may protect striatal medium spiny neurons (MSNs) from excitotoxic damage. This study evaluates whether mitochondrial inhibition with 3-NP that mimics the early stages of HD, modifies the kinetics and pharmacology of GABA receptors in patch clamp recorded dissociated MSNs cells. The results show that MSNs from mice treated with 3-NP exhibited differences in GABA-induced dose-response currents and pharmacological responses that suggests the presence of GABAC receptors in MSNs. Furthermore, there was a reduction in the effect of the GABAC antagonist that demonstrates a lessening of this GABA receptor subtype activity as a result of mitochondria inhibition.

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Electrophysiological and Morphological Changes in Striatal Spiny Neurons in R6/2 Huntington's Disease Transgenic Mice

Journal of Neurophysiology ◽

10.1152/jn.2001.86.6.2667 ◽

2001 ◽

Vol 86 (6) ◽

pp. 2667-2677 ◽

Cited By ~ 209

Author(s):

Gloria J. Klapstein ◽

Robin S. Fisher ◽

Hadi Zanjani ◽

Carlos Cepeda ◽

Eve S. Jokel ◽

...

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Action Potentials ◽

Morphological Changes ◽

Brain Slices ◽

Cag Repeats ◽

Membrane Properties ◽

Striatal Neurons ◽

Spiny Neurons ◽

Active Membrane

We examined passive and active membrane properties and synaptic responses of medium-sized spiny striatal neurons in brain slices from presymptomatic (∼40 days of age) and symptomatic (∼90 days of age) R6/2 transgenics, a mouse model of Huntington's disease (HD) and their age-matched wild-type (WT) controls. This transgenic expresses exon 1 of the human HD gene with ∼150 CAG repeats and displays a progressive behavioral phenotype associated with numerous neuronal alterations. Intracellular recordings were obtained using standard techniques from R6/2 and age-matched WT mice. Few electrophysiological changes occurred in striatal neurons from presymptomatic R6/2 mice. The changes in this age group were increased neuronal input resistance and lower stimulus intensity to evoke action potentials (rheobase). Symptomatic R6/2 mice exhibited numerous electrophysiological alterations, including depolarized resting membrane potentials, increased input resistances, decreased membrane time constants, and alterations in action potentials. Increased stimulus intensities were required to evoke excitatory postsynaptic potentials (EPSPs) in neurons from symptomatic R6/2 transgenics. These EPSPs had slower rise times and did not decay back to baseline by 45 ms, suggesting a more prominent component mediated by activation of N-methyl-d-aspartate receptors. Neurons from both pre- and symptomatic R6/2 mice exhibited reduced paired-pulse facilitation. Data from biocytin-filled or Golgi-impregnated neurons demonstrated decreased dendritic spine densities, smaller diameters of dendritic shafts, and smaller dendritic fields in symptomatic R6/2 mice. Taken together, these findings indicate that passive and active membrane and synaptic properties of medium-sized spiny neurons are altered in the R6/2 transgenic. These physiological and morphological alterations will affect communication in the basal ganglia circuitry. Furthermore, they suggest areas to target for pharmacotherapies to alleviate and reduce the symptoms of HD.

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BACHD Mice Recapitulate the Striatal Parvalbuminergic Interneuron Loss Found in Huntington’s Disease

Frontiers in Neuroanatomy ◽

10.3389/fnana.2021.673177 ◽

2021 ◽

Vol 15 ◽

Author(s):

Vyshnavi Rallapalle ◽

Annesha C. King ◽

Michelle Gray

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Neuronal Population ◽

Medium Spiny Neurons ◽

Cholinergic Interneurons ◽

Spiny Neurons ◽

Parvalbumin Interneurons ◽

Area And Perimeter ◽

Old Mice ◽

Disease Grade

Huntington’s disease (HD) is a dominantly inherited, adult-onset neurodegenerative disease characterized by motor, psychiatric, and cognitive abnormalities. Neurodegeneration is prominently observed in the striatum where GABAergic medium spiny neurons (MSN) are the most affected neuronal population. Interestingly, recent reports of pathological changes in HD patient striatal tissue have identified a significant reduction in the number of parvalbumin-expressing interneurons which becomes more robust in tissues of higher disease grade. Analysis of other interneuron populations, including somatostatin, calretinin, and cholinergic, did not reveal significant neurodegeneration. Electrophysiological experiments in BACHD mice have identified significant changes in the properties of parvalbumin and somatostatin expressing interneurons in the striatum. Furthermore, their interactions with MSNs are altered as the mHTT expressing mouse models age with increased input onto MSNs from striatal somatostatin and parvalbumin-expressing neurons. In order to determine whether BACHD mice recapitulate the alterations in striatal interneuron number as observed in HD patients, we analyzed the number of striatal parvalbumin, somatostatin, calretinin, and choline acetyltransferase positive cells in symptomatic 12–14 month-old mice by immunofluorescent labeling. We observed a significant decrease in the number of parvalbumin-expressing interneurons as well as a decrease in the area and perimeter of these cells. No significant changes were observed for somatostatin, calretinin, or cholinergic interneuron numbers while a significant decrease was observed for the area of cholinergic interneurons. Thus, the BACHD mice recapitulate the degenerative phenotype observed in the parvalbumin interneurons in HD patient striata without affecting the number of other interneuron populations in the striatum.

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