Disease related changes in ATAC-seq of iPSC-derived motor neuron lines from ALS patients and controls.

Amyotrophic Lateral Sclerosis (ALS), like many other neurodegenerative diseases, is highly heritable, but with only a small fraction of cases explained by monogenic disease alleles. To better understand sporadic ALS, we report epigenomic profiles, as measured by ATAC-seq, of motor neuron cultures derived from a diverse group of 380 ALS patients and 80 healthy controls. We find that chromatin accessibility is heavily influenced by sex, the iPSC cell type of origin, ancestry, and the inherent variance arising from sequencing. Once these covariates are corrected for, we are able to identify ALS-specific signals in the data. Additionally, we find that the ATAC-seq data is able to predict ALS disease progression rates with similar accuracy to methods based on biomarkers and clinical status. These results suggest that iPSC-derived motor neurons recapitulate important disease-relevant epigenomic changes.

SOX9 switch links regeneration to fibrosis at the single-cell level in mammalian kidneys.

The steps governing healing with or without fibrosis within the same microenvironment are unclear. After acute kidney injury (AKI), injured proximal tubular epithelial cells activate SOX9 for self-restoration. Using a multimodal approach for a head-to-head comparison of injury-induced SOX9 lineages, we identified a dynamic SOX9 switch in repairing epithelia. Lineages that regenerated epithelia silenced SOX9 and healed without fibrosis (SOX9on-off). By contrast, lineages with unrestored apicobasal polarity maintained SOX9 activity in sustained efforts to regenerate, which were identified as a SOX9on-on Cadherin6pos cell state. These reprogrammed cells generated substantial single-cell WNT activity to provoke a fibroproliferative response in adjacent fibroblasts, driving AKI to chronic kidney disease. Transplanted human kidneys displayed similar SOX9/CDH6/WNT2B responses. Thus, we have uncovered a sensor of epithelial repair status, the activity of which determines regeneration with or without fibrosis.

Multi-lineage heart-chip models drug cardiotoxicity and enhances maturation of human stem cell-derived cardiovascular cells.

Cardiovascular toxicity causes adverse drug reactions and may lead to drug removal from the pharmaceutical market. Cancer therapies can induce life-threatening cardiovascular side effects such as arrhythmias, muscle cell death, or vascular dysfunction. New technologies have enabled cardiotoxic compounds to be identified earlier in drug development. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) and vascular endothelial cells (ECs) can screen for drug-induced alterations in cardiovascular cell function and survival. However, most existing hiPSC models for cardiovascular drug toxicity utilize two-dimensional, immature cells grown in static culture. Improved in vitro models to mechanistically interrogate cardiotoxicity would utilize more adult-like, mature hiPSC-derived cells in an integrated system whereby toxic drugs and protective agents can flow between hiPSC-ECs that represent systemic vasculature and hiPSC-CMs that represent heart muscle (myocardium). Such models would be useful for testing the multi-lineage cardiotoxicities of chemotherapeutic drugs such as VEGFR2/PDGFR-inhibiting tyrosine kinase inhibitors (VPTKIs). Here, we develop a multi-lineage, fully-integrated, cardiovascular organ-chip that can enhance hiPSC-EC and hiPSC-CM functional and genetic maturity, model endothelial barrier permeability, and demonstrate long-term functional stability. This microfluidic organ-chip harbors hiPSC-CMs and hiPSC-ECs on separate channels that can be subjected to active fluid flow and rhythmic biomechanical stretch. We demonstrate the utility of this cardiovascular organ-chip as a predictive platform for evaluating multi-lineage VPTKI toxicity. This study may lead to the development of new modalities for the evaluation and prevention of cancer therapy-induced cardiotoxicity.

Human Neural Progenitors Expressing GDNF Enhance Retinal Protection in a Rodent Model of Retinal Degeneration.

Stem cell therapy for retinal degenerative diseases has been extensively tested in preclinical and clinical studies. However, preclinical studies performed in animal models at the early stage of disease do not optimally translate to patients that present to the clinic at a later stage of disease. As the retina degenerates, inflammation and oxidative stress increase and trophic factor support declines. Testing stem cell therapies in animal models at a clinically relevant stage is critical for translation to the clinic. Human neural progenitor cells (hNPC) and hNPC engineered to stably express GDNF (hNPCGDNF) were subretinally injected into the Royal College of Surgeon (RCS) rats, a well-established model for retinal degeneration, at early and later stages of the disease. hNPCGDNF treatment at the early stage of retinal degeneration provided enhanced visual function compared to hNPC alone. Treatment with both cell types resulted in preserved retinal morphology compared to controls. hNPCGDNF treatment led to significantly broader photoreceptor protection than hNPC treatment at both early and later times of intervention. The phagocytic role of hNPC appears to support RPE cell functions and the secreted GDNF offers neuroprotection and enables the extended survival of photoreceptor cells in transplanted animal eyes. Donor cells in the RCS rat retina survived with only limited proliferation, and hNPCGDNF produced GDNF in vivo. Cell treatment led to significant changes in various pathways related to cell survival, antioxidative stress, phagocytosis, and autophagy. A combined stem cell and trophic factor therapy holds great promise for treating retinal degenerative diseases including retinitis pigmentosa and age-related macular degeneration.

Roadmap for C9ORF72 in Frontotemporal Dementia and Amyotrophic Lateral Sclerosis: Report on the C9ORF72 FTD/ALS Summit.

A summit held March 2023 in Scottsdale, Arizona (USA) focused on the intronic hexanucleotide expansion in the C9ORF72 gene and its relevance in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS; C9ORF72-FTD/ALS). The goal of this summit was to connect basic scientists, clinical researchers, drug developers, and individuals affected by C9ORF72-FTD/ALS to evaluate how collaborative efforts across the FTD-ALS disease spectrum might break down existing disease silos. Presentations and discussions covered recent discoveries in C9ORF72-FTD/ALS disease mechanisms, availability of disease biomarkers and recent advances in therapeutic development, and clinical trial design for prevention and treatment for individuals affected by C9ORF72-FTD/ALS and asymptomatic pathological expansion carriers. The C9ORF72-associated hexanucleotide repeat expansion is an important locus for both ALS and FTD. C9ORF72-FTD/ALS may be characterized by loss of function of the C9ORF72 protein and toxic gain of functions caused by both dipeptide repeat (DPR) proteins and hexanucleotide repeat RNA. C9ORF72-FTD/ALS therapeutic strategies discussed at the summit included the use of antisense oligonucleotides, adeno-associated virus (AAV)-mediated gene silencing and gene delivery, and engineered small molecules targeting RNA structures associated with the C9ORF72 expansion. Neurofilament light chain, DPR proteins, and transactive response (TAR) DNA-binding protein 43 (TDP-43)-associated molecular changes were presented as biomarker candidates. Similarly, brain imaging modalities (i.e., magnetic resonance imaging [MRI] and positron emission tomography [PET]) measuring structural, functional, and metabolic changes were discussed as important tools to monitor individuals affected with C9ORF72-FTD/ALS, at both pre-symptomatic and symptomatic disease stages. Finally, summit attendees evaluated current clinical trial designs available for FTD or ALS patients and concluded that therapeutics relevant to FTD/ALS patients, such as those specifically targeting C9ORF72, may need to be tested with composite endpoints covering clinical symptoms of both FTD and ALS. The latter will require novel clinical trial designs to be inclusive of all patient subgroups spanning the FTD/ALS spectrum.

The C9ORF72 Summit was held in March 2023 in Scottsdale, Arizona (USA). Some people who have the disease frontotemporal dementia or the disease amyotrophic lateral sclerosis have a change in one of their genes; the name of the gene is C9ORF72. People who carry this genetic difference usually inherited it from a parent. Researchers are improving their understanding of how the change in the C9ORF72 gene affects people, and efforts are being made to use this knowledge to develop treatments for amyotrophic lateral sclerosis and frontotemporal dementia. In addition to studying the cellular and molecular mechanisms of how the C9ORF72 mutation leads to cellular dysfunction and frontotemporal dementia and amyotrophic lateral sclerosis clinical symptoms, a large effort of the research community is aimed at developing measurements, called biomarkers, that could enhance therapy development efforts in multiple ways. Examples include monitoring of disease activity, identifying those at risk of developing amyotrophic lateral sclerosis or frontotemporal dementia, predicting which people might benefit from a particular treatment, and showing that a drug has had a biological effect. Markers that identify healthy people who are at risk of developing amyotrophic lateral sclerosis or frontotemporal dementia could be used to test treatments that would start before a person shows any symptoms and hopefully would delay or even prevent their onset.

Organ-Chips Enhance the Maturation of Human iPSC-Derived Dopamine Neurons.

While cells in the human body function in an environment where the blood supply constantly delivers nutrients and removes waste, cells in conventional tissue culture well platforms are grown with a static pool of media above them and often lack maturity, limiting their utility to study cell biology in health and disease. In contrast, organ-chip microfluidic systems allow the growth of cells under constant flow, more akin to the in vivo situation. Here, we differentiated human induced pluripotent stem cells into dopamine neurons and assessed cellular properties in conventional multi-well cultures and organ-chips. We show that organ-chip cultures, compared to multi-well cultures, provide an overall greater proportion and homogeneity of dopaminergic neurons as well as increased levels of maturation markers. These organ-chips are an ideal platform to study mature dopamine neurons to better understand their biology in health and ultimately in neurological disorders.

GMP-grade human neural progenitors delivered subretinally protect vision in rat model of retinal degeneration and survive in minipigs.

BACKGROUND: Stem cell products are increasingly entering early stage clinical trials for treating retinal degeneration. The field is learning from experience about comparability of cells proposed for preclinical and clinical use. Without this, preclinical data supporting translation to a clinical study might not adequately reflect the performance of subsequent clinical-grade cells in patients. METHODS: Research-grade human neural progenitor cells (hNPC) and clinical-grade hNPC (termed CNS10-NPC) were injected into the subretinal space of the Royal College of Surgeons (RCS) rat, a rodent model of retinal degeneration such as retinitis pigmentosa. An investigational new drug (IND)-enabling study with CNS10-NPC was performed in the same rodent model. Finally, surgical methodology for subretinal cell delivery in the clinic was optimized in a large animal model with Yucatan minipigs. RESULTS: Both research-grade hNPC and clinical-grade hNPC can survive and provide functional and morphological protection in a dose-dependent fashion in RCS rats and the optimal cell dose was defined and used in IND-enabling studies. Grafted CNS10-NPC migrated from the injection site without differentiation into retinal cell phenotypes. Additionally, CNS10-NPC showed long-term survival, safety and efficacy in a good laboratory practice (GLP) toxicity and tumorigenicity study, with no observed cell overgrowth even at the maximum deliverable dose. Finally, using a large animal model with the Yucatan minipig, which has an eye size comparable to the human, we optimized the surgical methodology for subretinal cell delivery in the clinic. CONCLUSIONS: These extensive studies supported an approved IND and the translation of CNS10-NPC to an ongoing Phase 1/2a clinical trial (NCT04284293) for the treatment of retinitis pigmentosa.

Translational roadmap for regenerative therapies of eye disease.

The translation of regenerative therapies to neuronal eye diseases requires a roadmap specific to the nature of the target diseases, patient population, methodologies for assessing outcome, and other factors. This commentary focuses on critical issues for translating regenerative eye therapies relevant to retinal neurons to human clinical trials.

Reversal of dual epigenetic repression of non-canonical Wnt-5a normalises diabetic corneal epithelial wound healing and stem cells.

AIMS/HYPOTHESIS: Diabetes is associated with epigenetic modifications including DNA methylation and miRNA changes. Diabetic complications in the cornea can cause persistent epithelial defects and impaired wound healing due to limbal epithelial stem cell (LESC) dysfunction. In this study, we aimed to uncover epigenetic alterations in diabetic vs non-diabetic human limbal epithelial cells (LEC) enriched in LESC and identify new diabetic markers that can be targeted for therapy to normalise corneal epithelial wound healing and stem cell expression. METHODS: Human LEC were isolated, or organ-cultured corneas were obtained, from autopsy eyes from non-diabetic (59.87±20.89 years) and diabetic (71.93±9.29 years) donors. The groups were not statistically different in age. DNA was extracted from LEC for methylation analysis using Illumina Infinium 850K MethylationEPIC BeadChip and protein was extracted for Wnt phospho array analysis. Wound healing was studied using a scratch assay in LEC or 1-heptanol wounds in organ-cultured corneas. Organ-cultured corneas and LEC were transfected with WNT5A siRNA, miR-203a mimic or miR-203a inhibitor or were treated with recombinant Wnt-5a (200 ng/ml), DNA methylation inhibitor zebularine (1-20 µmol/l) or biodegradable nanobioconjugates (NBCs) based on polymalic acid scaffold containing antisense oligonucleotide (AON) to miR-203a or a control scrambled AON (15-20 µmol/l). RESULTS: There was significant differential DNA methylation between diabetic and non-diabetic LEC. WNT5A promoter was hypermethylated in diabetic LEC accompanied with markedly decreased Wnt-5a protein. Treatment of diabetic LEC and organ-cultured corneas with exogenous Wnt-5a accelerated wound healing by 1.4-fold (p<0.05) and 37% (p<0.05), respectively, and increased LESC and diabetic marker expression. Wnt-5a treatment in diabetic LEC increased the phosphorylation of members of the Ca2+-dependent non-canonical pathway (phospholipase Cγ1 and protein kinase Cß; by 1.15-fold [p<0.05] and 1.36-fold [p<0.05], respectively). In diabetic LEC, zebularine treatment increased the levels of Wnt-5a by 1.37-fold (p<0.01)and stimulated wound healing in a dose-dependent manner with a 1.6-fold (p<0.01) increase by 24 h. Moreover, zebularine also improved wound healing by 30% (p<0.01) in diabetic organ-cultured corneas and increased LESC and diabetic marker expression. Transfection of these cells with WNT5A siRNA abrogated wound healing stimulation by zebularine, suggesting that its effect was primarily due to inhibition of WNT5A hypermethylation. Treatment of diabetic LEC and organ-cultured corneas with NBC enhanced wound healing by 1.4-fold (p<0.01) and 23.3% (p<0.05), respectively, with increased expression of LESC and diabetic markers. CONCLUSIONS/INTERPRETATION: We provide the first account of epigenetic changes in diabetic corneas including dual inhibition of WNT5A by DNA methylation and miRNA action. Overall, Wnt-5a is a new corneal epithelial wound healing stimulator that can be targeted to improve wound healing and stem cells in the diabetic cornea. DATA AVAILABILITY: The DNA methylation dataset is available from the public GEO repository under accession no. GSE229328 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229328 ).

Human iPSC-derived neural progenitor cells secreting GDNF provide protection in rodent models of ALS and retinal degeneration.

Human induced pluripotent stem cells (iPSCs) are a renewable cell source that can be differentiated into neural progenitor cells (iNPCs) and transduced with glial cell line-derived neurotrophic factor (iNPC-GDNFs). The goal of the current study is to characterize iNPC-GDNFs and test their therapeutic potential and safety. Single-nuclei RNA-seq show iNPC-GDNFs express NPC markers. iNPC-GDNFs delivered into the subretinal space of the Royal College of Surgeons rodent model of retinal degeneration preserve photoreceptors and visual function. Additionally, iNPC-GDNF transplants in the spinal cord of SOD1G93A amyotrophic lateral sclerosis (ALS) rats preserve motor neurons. Finally, iNPC-GDNF transplants in the spinal cord of athymic nude rats survive and produce GDNF for 9 months, with no signs of tumor formation or continual cell proliferation. iNPC-GDNFs survive long-term, are safe, and provide neuroprotection in models of both retinal degeneration and ALS, indicating their potential as a combined cell and gene therapy for various neurodegenerative diseases.

Large-scale differentiation of iPSC-derived motor neurons from ALS and control subjects.

Using induced pluripotent stem cells (iPSCs) to understand the mechanisms of neurological disease holds great promise; however, there is a lack of well-curated lines from a large array of participants. Answer ALS has generated over 1,000 iPSC lines from control and amyotrophic lateral sclerosis (ALS) patients along with clinical and whole-genome sequencing data. The current report summarizes cell marker and gene expression in motor neuron cultures derived from 92 healthy control and 341 ALS participants using a 32-day differentiation protocol. This is the largest set of iPSCs to be differentiated into motor neurons, and characterization suggests that cell composition and sex are significant sources of variability that need to be carefully controlled for in future studies. These data are reported as a resource for the scientific community that will utilize Answer ALS data for disease modeling using a wider array of omics being made available for these samples.

NeuroLINCS Proteomics: Defining human-derived iPSC proteomes and protein signatures of pluripotency.

The National Institute of Health (NIH) Library of integrated network-based cellular signatures (LINCS) program is premised on the generation of a publicly available data resource of cell-based biochemical responses or "signatures" to genetic or environmental perturbations. NeuroLINCS uses human inducible pluripotent stem cells (hiPSCs), derived from patients and healthy controls, and differentiated into motor neuron cell cultures. This multi-laboratory effort strives to establish i) robust multi-omic workflows for hiPSC and differentiated neuronal cultures, ii) public annotated data sets and iii) relevant and targetable biological pathways of spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Here, we focus on the proteomics and the quality of the developed workflow of hiPSC lines from 6 individuals, though epigenomics and transcriptomics data are also publicly available. Known and commonly used markers representing 73 proteins were reproducibly quantified with consistent expression levels across all hiPSC lines. Data quality assessments, data levels and metadata of all 6 genetically diverse human iPSCs analysed by DIA-MS are parsable and available as a high-quality resource to the public.

Tissue clearing of human iPSC-derived organ-chips enables high resolution imaging and analysis.

Engineered microfluidic organ-chips enable increased cellular diversity and function of human stem cell-derived tissues grown in vitro. These three dimensional (3D) cultures, however, are met with unique challenges in visualization and quantification of cellular proteins. Due to the dense 3D nature of cultured nervous tissue, classical methods of immunocytochemistry are complicated by sub-optimal light and antibody penetrance as well as image acquisition parameters. In addition, complex polydimethylsiloxane scaffolding surrounding the tissue of interest can prohibit high resolution microscopy and spatial analysis. Hyperhydration tissue clearing methods have been developed to mitigate similar challenges of in vivo tissue imaging. Here, we describe an adaptation of this approach to efficiently clear human pluripotent stem cell-derived neural tissues grown on organ-chips. We also describe critical imaging considerations when designing signal intensity-based approaches to complex 3D architectures inherent in organ-chips. To determine morphological and anatomical features of cells grown in organ-chips, we have developed a reliable protocol for chip sectioning and high-resolution microscopic acquisition and analysis.

Transplantation of human neural progenitor cells secreting GDNF into the spinal cord of patients with ALS: a phase 1/2a trial.

Amyotrophic lateral sclerosis (ALS) involves progressive motor neuron loss, leading to paralysis and death typically within 3-5 years of diagnosis. Dysfunctional astrocytes may contribute to disease and glial cell line-derived neurotrophic factor (GDNF) can be protective. Here we show that human neural progenitor cells transduced with GDNF (CNS10-NPC-GDNF) differentiated to astrocytes protected spinal motor neurons and were safe in animal models. CNS10-NPC-GDNF were transplanted unilaterally into the lumbar spinal cord of 18 ALS participants in a phase 1/2a study (NCT02943850). The primary endpoint of safety at 1 year was met, with no negative effect of the transplant on motor function in the treated leg compared with the untreated leg. Tissue analysis of 13 participants who died of disease progression showed graft survival and GDNF production. Benign neuromas near delivery sites were common incidental findings at post-mortem. This study shows that one administration of engineered neural progenitors can provide new support cells and GDNF delivery to the ALS patient spinal cord for up to 42 months post-transplantation.

Regenerative and restorative medicine for eye disease.

Causes of blindness differ across the globe; in higher-income countries, most blindness results from the degeneration of specific classes of cells in the retina, including retinal pigment epithelium (RPE), photoreceptors, and retinal ganglion cells. Advances over the past decade in retinal regenerative medicine have allowed each of these cell types to be produced ex vivo from progenitor stem cells. Here, we review progress in applying these technologies to cell replacement - with the goal of vision restoration in degenerative disease. We discuss the landscape of human clinical trials for RPE transplantation and advanced preclinical studies for other cell types. We also review progress toward in situ repair of retinal degeneration using endogenous progenitor cells. Finally, we provide a high-level overview of progress toward prosthetic ocular vision restoration, including advanced photovoltaic devices, opsin-based gene therapy, and small-molecule photoswitches. Progress in each of these domains is at or near the human clinical-trial stage, bringing the audacious goal of vision restoration within sight.

The proSAAS Chaperone Provides Neuroprotection and Attenuates Transsynaptic α-Synuclein Spread in Rodent Models of Parkinson's Disease.

BACKGROUND: Parkinson's disease involves aberrant aggregation of the synaptic protein alpha-synuclein (aSyn) in the nigrostriatal tract. We have previously shown that proSAAS, a small neuronal chaperone, blocks aSyn-induced dopaminergic cytotoxicity in primary nigral cultures. OBJECTIVE: To determine if proSAAS overexpression is neuroprotective in animal models of Parkinson's disease. METHODS: proSAAS- or GFP-encoding lentivirus was injected together with human aSyn-expressing AAV unilaterally into the substantia nigra of rats and motor asymmetry assessed using a battery of motor performance tests. Dopamine neuron survival was assessed by nigral stereology and striatal tyrosine hydroxylase (TH) densitometry. To examine transsynaptic spread of aSyn, aSyn AAV was injected into the vagus of mice in the presence of AAVs encoding either GFP or proSAAS; the spread of aSyn-positive neurites into rostral nuclei was quantified following immunohistochemistry. RESULTS: Coinjection of proSAAS-encoding lentivirus profoundly reduced the motor asymmetry caused by unilateral nigral AAV-mediated human aSyn overexpression. This was accompanied by significant amelioration of the human aSyn-induced loss of both nigral TH-positive cells and striatal TH-positive terminals, demonstrating clear proSAAS-mediated protection of the nigrostriatal tract. ProSAAS overexpression reduced human aSyn protein levels in nigra and striatum and reduced the loss of TH protein in both regions. Following vagal administration of human aSyn-encoding AAV, the number of human aSyn-positive neurites in the pons and caudal midbrain was considerably reduced in mice coinjected with proSAAS-, but not GFP-encoding AAV, supporting proSAAS-mediated blockade of transsynaptic aSyn transmission. CONCLUSION: The proSAAS chaperone may represent a promising target for therapeutic development in Parkinson's disease.

AAV9-MCT8 Delivery at Juvenile Stage Ameliorates Neurological and Behavioral Deficits in a Mouse Model of MCT8-Deficiency.

Background: Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor disability disorder that also manifests characteristic abnormal thyroid hormone (TH) levels. AHDS is caused by inactivating mutations in monocarboxylate transporter 8 (MCT8), a specific TH plasma membrane transporter widely expressed in the central nervous system (CNS). MCT8 mutations cause impaired transport of TH across brain barriers, leading to insufficient neural TH supply. There is currently no successful therapy for the neurological symptoms. Earlier work has shown that intravenous (IV), but not intracerebroventricular adeno-associated virus serotype 9 (AAV9) -based gene therapy given to newborn Mct8 knockout (Mct8-/y) male mice increased triiodothyronine (T3) brain content and partially rescued TH-dependent gene expression, suggesting a promising approach to treat this neurological disorder. Methods: The potential of IV delivery of AAV9 carrying human MCT8 was tested in the well-established Mct8-/y/Organic anion-transporting polypeptide 1c1 (Oatp1c1)-/ - double knockout (dKO) mouse model of AHDS, which, unlike Mct8-/y mice, displays both neurological and TH phenotype. Further, as the condition is usually diagnosed during childhood, treatment was given intravenously to P30 mice and psychomotor tests were carried out blindly at P120-P140 after which tissues were collected and analyzed. Results: Systemic IV delivery of AAV9-MCT8 at a juvenile stage led to improved locomotor and cognitive functions at P120-P140, which was accompanied by a near normalization of T3 content and an increased response of positively regulated TH-dependent gene expression in different brain regions examined (thalamus, hippocampus, and parietal cortex). The effects on serum TH concentrations and peripheral tissues were less pronounced, showing only improvement in the serum T3/reverse T3 (rT3) ratio and in liver deiodinase 1 expression. Conclusion: IV administration of AAV9, carrying the human MCT8, to juvenile dKO mice manifesting AHDS has long-term beneficial effects, predominantly on the CNS. This preclinical study indicates that this gene therapy has the potential to ameliorate the devastating neurological symptoms in patients with AHDS.

Sodium Phenylbutyrate Rescues Thyroid Hormone Transport in Brain Endothelial-Like Cells.

Background: Monocarboxylate transporter 8 (MCT8) deficiency is a rare genetic disease leading to a severe developmental delay due to a lack of thyroid hormones (THs) during critical stages of human brain development. Some MCT8-deficient patients are not as severely affected as others. Previously, we hypothesized that these patients' mutations do not affect the functionality but destabilize the MCT8 protein, leading to a diminished number of functional MCT8 molecules at the cell surface. Methods: We have already demonstrated that the chemical chaperone sodium phenylbutyrate (NaPB) rescues the function of these mutants by stabilizing their protein expression in an overexpressing cell system. Here, we expanded our previous work and used iPSC (induced pluripotent stem cell)-derived brain microvascular endothelial-like cells (iBMECs) as a physiologically relevant cell model of human origin to test for NaPB responsiveness. The effects on mutant MCT8 expression and function were tested by Western blotting and radioactive uptake assays. Results: We found that NaPB rescues decreased mutant MCT8 expression and restores transport function in iBMECs carrying patient's mutation MCT8-P321L. Further, we identified MCT10 as an alternative TH transporter in iBMECs that contributes to triiodothyronine uptake, the biological active TH. Our results indicate an upregulation of MCT10 after NaPB treatment. In addition, we detected an increase in thyroxine (T4) uptake after NaPB treatment that was not mediated by rescued MCT8 but an unidentified T4 transporter. Conclusions: We demonstrate that NaPB is suitable to stabilize a pathogenic missense mutation in a human-derived cell model. Further, it activates TH transport independent of MCT8. Both options fuel future studies to investigate repurposing the Food and Drug Administration-approved drug NaPB in selected cases of MCT8 deficiency.