Mitochondria-Endoplasmic Reticulum Contact Sites Dynamics and Calcium Homeostasis Are Differentially Disrupted in PINK1-PD or PRKN-PD Neurons.

BACKGROUND: It is generally believed that the pathogenesis of PINK1/parkin-related Parkinson's disease (PD) is due to a disturbance in mitochondrial quality control. However, recent studies have found that PINK1 and Parkin play a significant role in mitochondrial calcium homeostasis and are involved in the regulation of mitochondria-endoplasmic reticulum contact sites (MERCSs). OBJECTIVE: The aim of our study was to perform an in-depth analysis of the role of MERCSs and impaired calcium homeostasis in PINK1/Parkin-linked PD. METHODS: In our study, we used induced pluripotent stem cell-derived dopaminergic neurons from patients with PD with loss-of-function mutations in PINK1 or PRKN. We employed a split-GFP-based contact site sensor in combination with the calcium-sensitive dye Rhod-2 AM and applied Airyscan live-cell super-resolution microscopy to determine how MERCSs are involved in the regulation of mitochondrial calcium homeostasis. RESULTS: Our results showed that thapsigargin-induced calcium stress leads to an increase of the abundance of narrow MERCSs in wild-type neurons. Intriguingly, calcium levels at the MERCSs remained stable, whereas the increased net calcium influx resulted in elevated mitochondrial calcium levels. However, PINK1-PD or PRKN-PD neurons showed an increased abundance of MERCSs at baseline, accompanied by an inability to further increase MERCSs upon thapsigargin-induced calcium stress. Consequently, calcium distribution at MERCSs and within mitochondria was disrupted. CONCLUSIONS: Our results demonstrated how the endoplasmic reticulum and mitochondria work together to cope with calcium stress in wild-type neurons. In addition, our results suggests that PRKN deficiency affects the dynamics and composition of MERCSs differently from PINK1 deficiency, resulting in differentially affected calcium homeostasis. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Assessment of Gene Variant Amenability for Pharmacological Chaperone Therapy with 1-Deoxygalactonojirimycin in Fabry Disease.

Fabry disease is one of the most common lysosomal storage disorders caused by mutations in the gene encoding lysosomal α-galactosidase A (α-Gal A) and resultant accumulation of glycosphingolipids. The sugar mimetic 1-deoxygalactonojirimycin (DGJ), an orally available pharmacological chaperone, was clinically approved as an alternative to intravenous enzyme replacement therapy. The decision as to whether a patient should be treated with DGJ depends on the genetic variant within the α-galactosidase A encoding gene (GLA). A good laboratory practice (GLP)-validated cell culture-based assay to investigate the biochemical responsiveness of the variants is currently the only source available to obtain pivotal information about susceptibility to treatment. Herein, variants were defined amenable when an absolute increase in enzyme activity of ≥3% of wild type enzyme activity and a relative increase in enzyme activity of ≥1.2-fold was achieved following DGJ treatment. Efficacy testing was carried out for over 1000 identified GLA variants in cell culture. Recent data suggest that about one-third of the variants comply with the amenability criteria. A recent study highlighted the impact of inter-assay variability on DGJ amenability, thereby reducing the power of the assay to predict eligible patients. This prompted us to compare our own α-galactosidase A enzyme activity data in a very similar in-house developed assay with those from the GLP assay. In an essentially retrospective approach, we reviewed 148 GLA gene variants from our former studies for which enzyme data from the GLP study were available and added novel data for 30 variants. We also present data for 18 GLA gene variants for which no data from the GLP assay are currently available. We found that both differences in experimental biochemical data and the criteria for the classification of amenability cause inter-assay discrepancy. We conclude that low baseline activity, borderline biochemical responsiveness, and inter-assay discrepancy are alarm signals for misclassifying a variant that must not be ignored. Furthermore, there is no solid basis for setting a minimum response threshold on which a clinical indication with DGJ can be justified.

Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test.

Niemann-Pick Type C (NP-C) is a rare disorder of lipid metabolism caused by mutations within the NPC1 and NPC2 genes. NP-C is a neurovisceral disease leading to a heterogeneous, multisystemic spectrum of symptoms in those affected. Until now, there is no investigative tool to demonstrate the significance of single variants within the NPC genes. Hence, the aim of the study was to establish a test that allows for an objective assessment of the pathological potential of NPC1 gene variants. Chinese hamster ovary cells defective in the NPC1 gene accumulate cholesterol in lysosomal storage organelles. The cells were transfected with NPC1-GFP plasmid vectors carrying distinct sequence variants. Filipin staining was used to test for complementation of the phenotype. The known variant p.Ile1061Thr showed a significantly impaired cholesterol clearance after 12 and 24 h compared to the wild type. Among the investigated variants, p.Ser954Leu and p.Glu1273Lys showed decelerated cholesterol clearance as well. The remaining variants p.Gln60His, p.Val494Met, and p.Ile787Val showed a cholesterol clearance indistinguishable from wild type. Further, p.Ile1061Thr acquired an enhanced clearance ability upon 25-hydroxycholesterol treatment. We conclude that the variants that caused an abnormal clearance phenotype are highly likely to be of clinical relevance. Moreover, we present a system that can be utilized to screen for new drugs.

Corrosion Products from Metallic Implants Induce ROS and Cell Death in Human Motoneurons In Vitro.

Due to advances in surgical procedures and the biocompatibility of materials used in total joint replacement, more and younger patients are undergoing these procedures. Although state-of-the-art joint replacements can last 20 years or longer, wear and corrosion is still a major risk for implant failure, and patients with these implants are exposed for longer to these corrosive products. It is therefore important to investigate the potential effects on the whole organism. Released nanoparticles and ions derived from commonly used metal implants consist, among others, of cobalt, nickel, and chromium. The effect of these metallic products in the process of osteolysis and aseptic implant loosening has already been studied; however, the systemic effect on other cell types, including neurons, remains elusive. To this end, we used human iPSC-derived motoneurons to investigate the effects of metal ions on human neurons. We treated human motoneurons with ion concentrations regularly found in patients, stained them with MitoSOX and propidium iodide, and analyzed them with fluorescence-assisted cell sorting (FACS). We found that upon treatment human motoneurons suffered from the formation of ROS and subsequently died. These effects were most prominent in motoneurons treated with 500 µM of cobalt or nickel, in which we observed significant cell death, whereas chromium showed fewer ROS and no apparent impairment of motoneurons. Our results show that the wear and corrosive products of metal implants at concentrations readily available in peri-implant tissues induced ROS and subsequently cell death in an iPSC-derived motoneuron cell model. We therefore conclude that monitoring of neuronal impairment is important in patients undergoing total joint replacement.

A molecular genetics view on Mucopolysaccharidosis Type II.

Mucopolysaccharidosis Type II (MPS II) is an X-linked recessive genetic disorder that primarily affects male patients. With an incidence of 1 in 100,000 male live births, the disease is one of the orphan diseases. MPS II symptoms are caused by mutations in the lysosomal iduronate-2-sulfatase (IDS) gene. The mutations cause a loss of enzymatic performance and result in the accumulation of glycosaminoglycans (GAGs), heparan sulfate and dermatan sulfate, which are no longer degradable. This inadvertent accumulation causes damage in multiple organs and leads either to a severe neurological course or to an attenuated course of the disease, although the exact relationship between mutation, extent of GAG accumulation and disease progression is not yet fully understood. This review is intended to present current diagnostic procedures and therapeutic interventions. In times when the genetic profile of patients plays an increasingly important role in the assessment of therapeutic success and future drug design, we chose to further elucidate the impact of genetic diversity within the IDS gene on disease phenotype and potential implications in current diagnosis, prognosis and therapy. We report recent advances in the structural biological elucidation of I2S enzyme that that promises to improve our future understanding of the molecular damage of the hundreds of IDS gene variants and will aid damage prediction of novel mutations in the future.

Direct Interaction of ATP7B and LC3B Proteins Suggests a Cooperative Role of Copper Transportation and Autophagy.

Macroautophagy/autophagy plays an important role in cellular copper clearance. The means by which the copper metabolism and autophagy pathways interact mechanistically is vastly unexplored. Dysfunctional ATP7B, a copper-transporting ATPase, is involved in the development of monogenic Wilson disease, a disorder characterized by disturbed copper transport. Using in silico prediction, we found that ATP7B contains a number of potential binding sites for LC3, a central protein in the autophagy pathway, the so-called LC3 interaction regions (LIRs). The conserved LIR3, located at the C-terminal end of ATP7B, was found to directly interact with LC3B in vitro. Replacing the two conserved hydrophobic residues W1452 and L1455 of LIR3 significantly reduced interaction. Furthermore, autophagy was induced in normal human hepatocellular carcinoma cells (HepG2) leading to enhanced colocalization of ATP7B and LC3B on the autophagosome membranes. By contrast, HepG2 cells deficient of ATP7B (HepG2 ATP7B-/-) showed autophagy deficiency at elevated copper condition. This phenotype was complemented by heterologous ATP7B expression. These findings suggest a cooperative role of ATP7B and LC3B in autophagy-mediated copper clearance.

Generation of two induced pluripotent stem cell lines from a female adult homozygous for the Wilson disease associated ATP7B variant p.H1069Q (AKOSi008-A) and a healthy control (AKOSi009-A).

Wilson disease (WD) is a rare, monogenic disorder caused by mutations in the gene ATP7B. A loss of function of the expressed protein leads to excessive hepatic and cerebral copper storage. In this study, we present the generation of two induced pluripotent stem cell (iPSC) lines derived from fibroblasts of a clinically asymptomatic, chelator treated female WD patient carrying the common missense mutation p.H1069Q and an age-matched female healthy control subject. The generated iPSC lines expressed pluripotency markers, showed differentiation potential and retained their parental genotype. Therefore, these cells provide a valuable resource to understand the pathophysiology of WD and can be used as model systems for drug testing.

Generation of an iPSC line (AKOSi006-A) from fibroblasts of an NPC1 patient, carrying the homozygous mutation p.I1061T (c.3182 T > C) and a control iPSC line (AKOSi007-A) using a non-integrating Sendai virus system.

Niemann-Pick disease type C1 (NPC1) is a rare inherited lipid storage disorder caused by mutations in the NPC1 gene. Mutations lead to impaired lipid trafficking and subsequently to accumulation of cholesterol and sphingolipids. NPC1-patients present variable multisystemic symptoms, including neurological deficits. Here, we describe the generation of human iPSC lines obtained from fibroblasts of a male individual, carrying the homozygous mutation p.I1061T, and an unrelated and healthy male individual. A non-integrating Sendai virus system, containing KLF4, OCT3/4, SOX2 and C-MYC, was used for reprogramming. These cell lines provide a valuable resource for studying the pathophysiology of multisystemic NPC1-disease.

Generation of induced pluripotent stem cell lines AKOSi002-A and AKOSi003-A from symptomatic female adults with Wilson disease.

Wilson disease (WD) is an inherited, autosomal recessive disorder of copper metabolism caused by mutations in the ATP7B gene. Pathogenic single nucleotide variants (SNVs) lead to functional impairment of the copper transporting ATPase ATP7B, resulting in copper accumulation and toxicity in the liver and brain. We describe the generation of two induced pluripotent stem cell (iPSC) lines derived from fibroblasts of two female WD patients. Patient 1 is compound heterozygous for p.E1064A and p.H1069Q. Patient 2 is homozygous for p.M769V. These iPSCs represent a WD model for pathophysiological studies and pharmacological screening.

Generation of an iPSC line (AKOSi004-A) from fibroblasts of a female adult NPC1 patient, carrying the compound heterozygous mutation p.Val1023Serfs*15/p.Gly992Arg and of an iPSC line (AKOSi005-A) from a female adult control individual.

Niemann-Pick disease Type C (NPC) is a rare progressive neurodegenerative disorder with an incidence of 1:120,000 caused by mutations in the NPC1 or NPC2 gene leading to a massive cholesterol accumulation. Here, we describe the generation of induced pluripotent stem cells (iPSCs) of an affected female adult individual carrying the NPC1 mutation p.Val1023Serfs*15/p.Gly992Arg and an iPSC line from an unrelated healthy female adult control individual. Human iPSCs were derived from fibroblasts using retroviruses carrying the four reprogramming factors OCT4, SOX2, KLF4 and C-MYC. These lines provide a valuable resource for studying the pathophysiology of NPC and for pharmacological intervention.

Role of endoplasmic reticulum stress and protein misfolding in disorders of the liver and pancreas.

The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins. The fraction of protein passing through the ER represents a large proportion of the total protein in the cell. Protein folding, glycosylation, sorting and transport are essential tasks of the ER and a compromised ER folding network has been recognized to be a key component in the disease pathogenicity of common neurodegenerative, metabolic and malignant diseases. On the other hand, the ER protein folding machinery also holds significant potential for therapeutic interventions. Many causes can lead to ER stress. A disturbed calcium homeostasis, the generation of reactive oxygen species (ROS) and a persistent overload of misfolded proteins within the ER can drive the course of adisease. In this review the role of ER-stress in diseases of the liver and pancreas will be examined using pancreatitis and Wilson´s disease as examples. Potential therapeutic targets in ER-stress pathways will also be discussed.

Mechanisms of stem cell based cardiac repair-gap junctional signaling promotes the cardiac lineage specification of mesenchymal stem cells.

Different subtypes of bone marrow-derived stem cells are characterized by varying functionality and activity after transplantation into the infarcted heart. Improvement of stem cell therapeutics requires deep knowledge about the mechanisms that mediate the benefits of stem cell treatment. Here, we demonstrated that co-transplantation of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) led to enhanced synergistic effects on cardiac remodeling. While HSCs were associated with blood vessel formation, MSCs were found to possess transdifferentiation capacity. This cardiomyogenic plasticity of MSCs was strongly promoted by a gap junction-dependent crosstalk between myocytes and stem cells. The inhibition of cell-cell coupling significantly reduced the expression of the cardiac specific transcription factors NKX2.5 and GATA4. Interestingly, we observed that small non-coding RNAs are exchanged between MSCs and cardiomyocytes in a GJ-dependent manner that might contribute to the transdifferentiation process of MSCs within a cardiac environment. Our results suggest that the predominant mechanism of HSCs contribution to cardiac regeneration is based on their ability to regulate angiogenesis. In contrast, transplanted MSCs have the capability for intercellular communication with surrounding cardiomyocytes, which triggers the intrinsic program of cardiogenic lineage specification of MSCs by providing cardiomyocyte-derived cues.