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BackgroundThe expansion of GGC repeats (typically exceeding 60 repeats) in the 5’ untranslated region (UTR) of the NOTCH2NLC gene (N2C) is linked to N2C-related repeat expansion disorders (NREDs), such as neuronal intranuclear inclusion disease (NIID), frontotemporal dementia (FTD), essential tremor (ET), and Parkinson’s disease (PD). These disorders share common clinical manifestations, including parkinsonism, dementia, seizures, and muscle weakness. Intermediate repeat sizes ranging from 40 to 60 GGC repeats, particularly those with AGC-encoded serine insertions, have been reported to be associated with PD; however, the functional implications of these intermediate repeats with serine insertion remain unexplored.MethodsHere, we utilized cellular models harbouring different sizes of N2C variant 2 (N2C2) GGC repeat expansion and CRISPR-Cas9 engineered transgenic mouse models carrying N2C2 GGC intermediate repeats with and without serine insertion to elucidate the underlying pathophysiology associated with N2C intermediate repeat with serine insertion in NREDs.ResultsOur findings revealed that the N2C2 GGC intermediate repeat with serine insertion (32G13S) led to mitochondrial dysfunction and cell death in vitro. The neurotoxicity was influenced by the length of the repeat and was exacerbated by the presence of the serine insertion. In 12-month-old transgenic mice, 32G13S intensified intranuclear aggregation and exhibited early PD-like characteristics, including the formation of ?-synuclein fibers in the midbrain and the loss of tyrosine hydroxylase (TH)-positive neurons in both the cortex and striatum. Additionally, 32G13S induced neuronal hyperexcitability and caused locomotor behavioural impairments. Transcriptomic analysis of the mouse cortex indicated dysregulation in calcium signaling and MAPK signaling pathways, both of which are critical for mitochondrial function. Notably, genes associated with myelin sheath components, including MBP and MOG, were dysregulated in the 32G13S mouse. Further investigations using immunostaining and transmission electron microscopy revealed that the N2C intermediate repeat with serine induced mitochondrial dysfunction-related hypermyelination in the cortex.ConclusionsOur in vitro and in vivo investigations provide the first evidence that the N2C-GGC intermediate repeat with serine promotes intranuclear aggregation of N2C, leading to mitochondrial dysfunction-associated hypermyelination and neuronal hyperexcitability. These changes contribute to motor deficits in early PD-like neurodegeneration in NREDs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13024-024-00780-2.

During a heart attack, ischemia causes losses of billions of cells; this is especially concerning given the minimal regenerative capability of cardiomyocytes (CMs). Heart remuscularization utilizing stem cells has improved cardiac outcomes despite little cell engraftment, thereby shifting focus to cell-free therapies. Consequently, we chose induced pluripotent stem cells (iPSCs) given their pluripotent nature, efficacy in previous studies, and easy obtainability from minimally invasive techniques. Nonetheless, using iPSC secretome-based therapies for treating injured CMs in a clinical setting is ill-understood. We hypothesized that the iPSC secretome, regardless of donor health, would improve cardiovascular outcomes in the CM model of ischemia–reperfusion (IR) injury. Episomal-generated iPSCs from healthy and dilated cardiomyopathy (DCM) donors, passaged 6–10 times, underwent 24 h incubation in serum-free media. Protein content of the secretome was analyzed by mass spectroscopy and used to treat AC16 immortalized CMs during 5 h reperfusion following 24 h of hypoxia. IPSC-derived secretome content, independent of donor health status, had elevated expression of proteins involved in cell survival pathways. In IR conditions, iPSC-derived secretome increased cell survival as measured by metabolic activity (p < 0.05), cell viability (p < 0.001), and maladaptive cellular remodelling (p = 0.052). Healthy donor-derived secretome contained increased expression of proteins related to calcium contractility compared to DCM donors. Congruently, only healthy donor-derived secretomes improved CM intracellular calcium concentrations (p < 0.01). Heretofore, secretome studies mainly investigated differences relating to cell type rather than donor health. Our work suggests that healthy donors provide more efficacious iPSC-derived secretome compared to DCM donors in the context of IR injury in human CMs. These findings illustrate that the regenerative potential of the iPSC secretome varies due to donor-specific differences.

The overproduction of reactive oxygen species (ROS) and mitochondrial dysregulation are regarded as key mechanisms in the progression of cardiac remodelling in cardiometabolic diseases including heart failure. Conventional treatments are often ineffective as they do not specifically target the underlying pathological mechanisms. Mitoquinone mesylate (MitoQ), a mitochondrial-targeted antioxidant has been reported to be protective against vascular dysfunction in hypertension, diabetic kidney disease and alcohol-induced liver damage. However, the cardioprotective potential of MitoQ to limit oxidative stress-induced mitochondrial remodelling in cardiomyocytes has not been fully resolved. We sought to investigate the effect of MitoQ and its mitochondrial-targeting moiety dodecyl-triphenylphosphonium (dTPP) on hydrogen peroxide-induced overproduction of ROS, mitochondrial dysregulation and cell death in H9C2 rat cardiomyoblasts (H9C2-rCM) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Cardiomyocytes were exposed to acute or chronic treatment (5–60 min or 48 h) of vehicle control (0.0001 % Ultrapure Milli-Q water), hydrogen peroxide (100 ?M) ± MitoQ (1 ?M) or dTPP (1 ?M) control. Hydrogen peroxide-induced overproduction of ROS, extracellular superoxide, mitochondrial ROS, mitochondrial hyperpolarisation and cell death were significantly blunted by MitoQ, but not dTPP, suggesting that the coenzyme Q10 moiety of MitoQ is protective under these conditions. Interestingly, both MitoQ and dTPP exhibited a pro-mitochondrial fusion effect by preserving mitochondrial network and reducing mitochondrial fragmentation in oxidative stress conditions. Overall, our findings confirm the cytoprotective potential of MitoQ to limit oxidative stress-induced adverse mitochondrial remodelling and dysregulation that is clinically observed in cardiometabolic-induced cardiac dysfunction in the failing heart. Graphical abstractBioRender Scientific Image and Illustration Software were used to generate the graphical abstract.Unlabelled Image Highlights•Oxidative stress is a key driver of mitochondrial dysregulation and cell death in cardiomyocytes.•MitoQ exhibits cytoprotection against elevated ROS production in human cardiomyocytes.•Mitochondrial structure and regulation were preserved in human cardiomyocytes with MitoQ treatment.

BackgroundThioredoxin-interacting protein (TXNIP) plays a role in regulating endoplasmic reticulum (ER) and oxidative stress, which disrupt glucose homeostasis in diabetes. However, the impact of TXNIP deficiency on the differentiation and functionality of human stem cell-derived somatic metabolic cells remains unclear.MethodsWe used CRISPR-Cas12a genome editing to generate TXNIP-deficient (TXNIP?/?) H1 human embryonic stem cells (H1-hESCs). These cells were differentiated into hepatocyte-like cells (HLCs) and stem-cell-derived insulin-producing islets (SC-islets). The maturation and functionality TXNIP?/? and TXNIP+/+ SC-islets were assessed by implantation under the kidney capsule of male or female NOD-SCID mice.ResultsTXNIP deficiency significantly increased H1-hESC proliferation without affecting pluripotency, viability, or differentiation potential into HLCs and SC-islets. Bulk RNA-sequencing of thapsigargin-treated TXNIP?/? and TXNIP+/+ hESCs revealed differential expression of stress-responsive genes, with enriched apoptosis-related pathways in TXNIP+/+ cells, but minimal transcriptional changes specific to TXNIP deficiency. In HLCs, TXNIP deletion reduced albumin secretion and insulin signalling, as indicated by decreased AKT phosphorylation, while showing no differences in glycolytic activity or lipid metabolism markers. Under thapsigargin-induced ER stress, TXNIP?/? HLCs exhibited transiently reduced eIF2? phosphorylation and lower BiP expression, suggesting compromised adaptive responses to prolonged stress. SC-islets derived from TXNIP?/? hESCs showed comparable viability, endocrine cell composition, and cytokine responses to TXNIP+/+ islets. Following IFN? or IFN? treatment, STAT1 phosphorylation was increased in TXNIP?/? SC-islets, indicating that IFN signalling remained intact despite TXNIP deficiency. Upon implantation into NOD-SCID mice, both TXNIP?/? and TXNIP+/+ SC-islets produced human C-peptide and responded to glucose stimulation. However, TXNIP?/? SC-islets did not demonstrate enhanced glycaemic control or glucose-stimulated insulin secretion compared to controls.ConclusionsOur study demonstrates that TXNIP deficiency does not improve the differentiation or functionality of HLCs and SC-islets. We present the generation and characterisation of TXNIP?/? and TXNIP+/+ H1-hESCs, HLCs, and SC-islets as valuable models for future studies on the role of TXNIP in metabolic cell biology.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04314-5.

MicroRNAs (miRNAs) are small non-coding RNAs that play crucial roles in post-transcriptional gene regulation. Poly(A) RNA polymerase D5 (PAPD5) catalyzes the addition of adenosine to the 3? end of miRNAs. In this study, we demonstrate that the Yin Yang 1 protein, a transcriptional repressor of PAPD5, is recruited to both RNA foci and protein aggregates, resulting in an upregulation of PAPD5 expression in Huntington’s disease (HD). Additionally, we identify a subset of PAPD5-regulated miRNAs with increased adenylation and reduced expression in our disease model. We focus on miR-7-5p and find that its reduction causes the activation of the TAB2-mediated TAK1–MKK4–JNK pro-apoptotic pathway. This pathway is also activated in induced pluripotent stem cell-derived striatal neurons and post-mortem striatal tissues isolated from HD patients. In addition, we discover that a small molecule PAPD5 inhibitor, BCH001, can mitigate cell death and neurodegeneration in our disease models. This study highlights the importance of PAPD5-mediated miRNA dysfunction in HD pathogenesis and suggests a potential therapeutic direction for the disease. PAPD5 is responsible for adenylation of microRNAs. Here, the authors show that elevated level of PAPD5 enhances the adenylation and reduced expression of miR-7-5p. As a result, expression of TAB2, a target of miR-7-5p, is induced triggering neuronal apoptosis in Huntington’s disease.