scholarly journals Lysosomal perturbations in dopaminergic neurons derived from induced pluripotent stem cells with PARK2 mutation

2019 ◽  
Author(s):  
Justyna Okarmus ◽  
Helle Bogetofte ◽  
Sissel Ida Schmidt ◽  
Matias Ryding ◽  
Silvia Garcia Lopez ◽  
...  
Keyword(s):  
Dopaminergic Neurons ◽  
Induced Pluripotent Stem Cell ◽  
Autophagic Flux ◽  
Compensatory Mechanism ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Tem Analysis ◽  
Induced Pluripotent ◽  
And Function ◽  
The Impact

AbstractMutations in the PARK2 gene encoding parkin, an E3 ubiquitin ligase, are associated with autosomal recessive early-onset Parkinson’s disease (PD). While parkin has been implicated in the regulation of mitophagy and proteasomal degradation, the precise mechanism leading to neurodegeneration in both sporadic and familial PD upon parkin loss-of-function mutations remains unknown. Cultures of isogenic induced pluripotent stem cell (iPSC) lines with and without PARK2 knockout (KO) enable mechanistic studies of the effect of parkin deficiency in human dopaminergic neurons. In the present study, we used such cells to investigate the impact of PARK2 KO on the lysosomal compartment combining different approaches, such as mass spectrometry-based proteomics, electron microscopy (TEM) analysis and functional assays. We discovered a clear link between parkin deficiency and lysosomal alterations. PARK2 KO neurons exhibited a perturbed lysosomal morphology, displaying significantly enlarged and electron-lucent lysosomes as well as an increased total lysosomal content, which was exacerbated by mitochondrial stress. In addition, we found perturbed autophagic flux and decreased lysosomal enzyme activity suggesting an impairment of the autophagy-lysosomal pathway in parkin-deficient cells. Interestingly, activity of the GBA-encoded enzyme, β-glucocerebrosidase, was significantly increased suggesting the existence of a compensatory mechanism. In conclusion, our data provide a unique characterization of the morphology, content, and function of lysosomes in PARK2 KO neurons, thus revealing a new important connection between mitochondrial dysfunction and lysosomal dysregulation in PD pathogenesis.

scholarly journals Targeting the Microtubule EB1-CLASP2 Complex Modulates Na V 1.5 at Intercalated Discs

2021 ◽  
Author(s):  
Gerard A Marchal ◽  
Mariam Jouni ◽  
David Y Chiang ◽  
Marta Pérez-Hernández Duran ◽  
Svitlana Podliesna ◽  
...  
Keyword(s):  
Sodium Current ◽  
Induced Pluripotent Stem Cell ◽  
Loss Of Function ◽  
Whole Cell ◽  
Intercalated Discs ◽  
Cardiac Sodium Channel ◽  
Optical Reconstruction ◽  
Induced Pluripotent ◽  
And Function

Rationale: Loss-of-function of the cardiac sodium channel Na V 1.5 causes conduction slowing and arrhythmias. Na V 1.5 is differentially distributed within subcellular domains of cardiomyocytes, with sodium current (I Na ) being enriched at the intercalated discs (ID). Various pathophysiological conditions associated with lethal arrhythmias display ID-specific I Na reduction, but the mechanisms underlying microdomain-specific targeting of Na V 1.5 remain largely unknown. Objective: To investigate the role of the microtubule (MT) plus-end tracking proteins end binding protein 1 (EB1) and CLIP-associated protein 2 (CLASP2) in mediating Na V 1.5 trafficking and subcellular distribution in cardiomyocytes. Methods and Results: EB1 overexpression in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) resulted in enhanced whole-cell I Na , increased action potential (AP) upstroke velocity (V max ), and enhanced Na V 1.5 localization at the plasma membrane as detected by multi-color stochastic optical reconstruction microscopy (STORM). Fluorescence recovery after photobleaching (FRAP) experiments in HEK293A cells demonstrated that EB1 overexpression promoted Na V 1.5 forward trafficking. Knockout of MAPRE1 in hiPSC-CMs led to reduced whole-cell I Na , decreased V max and AP duration (APD) prolongation. Similarly, acute knockout of the MAPRE1 homolog in zebrafish (mapre1b) resulted in decreased ventricular conduction velocity and V max as well as increased APD. STORM imaging and macropatch I Na measurements showed that subacute treatment (2-3 hours) with SB216763 (SB2), a GSK3β inhibitor known to modulate CLASP2-EB1 interaction, reduced GSK3β localization and increased Na V 1.5 and I Na preferentially at the ID region of wild type murine ventricular cardiomyocytes. By contrast, SB2 did not affect whole cell I Na or Na V 1.5 localization in cardiomyocytes from Clasp2-deficient mice, uncovering the crucial role of CLASP2 in SB2-mediated modulation of NaV1.5 at the ID. Conclusions: Our findings demonstrate the modulatory effect of the MT plus-end tracking protein EB1 on Na V 1.5 trafficking and function, and identify the EB1-CLASP2 complex as a target for preferential modulation of I Na within the ID region of cardiomyocytes.

Abstract 224: LAMP-2 Deficiency Promotes Mitochondrial Damage in Danon Disease

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Sherin Hashem ◽  
Emily Gault ◽  
Anne Murphy ◽  
Neil Chi ◽  
Eric Adler
Keyword(s):  
Contractile Function ◽  
Induced Pluripotent Stem Cell ◽  
Mitochondrial Damage ◽  
Autophagic Flux ◽  
Gene Encoding ◽  
Mitochondrial Oxidative Stress ◽  
Danon Disease ◽  
Severe Impairment ◽  
Significant Impairment ◽  
Induced Pluripotent

Danon disease is a familial cardiomyopathy associated with impaired autophagic flux due to mutations in the gene encoding lysosomal associated membrane protein type 2 (LAMP-2). The majority of patients die from progressive heart failure in their twenties, yet to date the mechanisms by which LAMP-2 deficiency leads to cardiomyocyte dysfunction and death are not fully understood. To study the underlying molecular pathogenesis, we created induced pluripotent stem cell-derived cardiomyocytes (hiPSC- CMs) from patients with different LAMP-2 mutations, and reported an increase in apoptosis and mitochondrial oxidative stress. In Danon hiPSC-CMs, undigested mitochondria within double-membrane autophagosomes were frequently observed suggesting impaired mitophagy. Danon hiPSC-CMs displayed increased translocation of PARKIN and p62 on mitochondria, suggesting increased mitochondrial damage. When mitochondrial bioenergetic profile was assessed, Danon hiPSC-CMs showed a severe impairment in mitochondrial respiratory capacity even when offered various oxidizable substrates, suggesting global mitochondrial damage. In addition, Danon hiPSC-CMs demonstrated excessive mitochondrial fragmentation and low mitochondrial membrane potential. Lamp-2 knockout (KO) mice recapitulated key pathological features observed in Danon patients and their hiPSC-CMs. Lamp-2 KO mouse hearts exhibited abnormal mitochondria, increased mitochondrial damage, and impaired mitophagy. Interestingly, altered mitochondrial respiration was observed in Lamp-2 KO mouse hearts before the onset of significant impairment in contractile function. In summary, we have modeled Danon disease using hiPSC-CMs from patients with LAMP-2 mutations as well as Lamp-2 KO mice, allowing us to gain mechanistic insight into the pathogenesis of this disease. We demonstrate that LAMP-2 deficiency leads to impairment in mitophagic flux, accumulation of damaged mitochondria, and deterioration of mitochondrial function. Understanding the mechanisms underlying cardiomyocyte dysfunction will have important implications for the treatment of Danon disease as well as a various cardiovascular disorders associated with impaired autophagy.

scholarly journals Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity

AMB Express ◽  
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Neeraja Punde ◽  
Jennifer Kooken ◽  
Dagmar Leary ◽  
Patricia M. Legler ◽  
Evelina Angov
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
Codon Usage ◽  
Dna Sequences ◽  
Structural Integrity ◽  
Host Cells ◽  
Loss Of Function ◽  
Species Specific ◽  
And Function ◽  
The Impact

Abstract Codon usage frequency influences protein structure and function. The frequency with which codons are used potentially impacts primary, secondary and tertiary protein structure. Poor expression, loss of function, insolubility, or truncation can result from species-specific differences in codon usage. “Codon harmonization” more closely aligns native codon usage frequencies with those of the expression host particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Heterologous expression of Plasmodium falciparum genes in Escherichia coli has been a challenge due to their AT-rich codon bias and the highly repetitive DNA sequences. Here, codon harmonization was applied to the malarial antigen, CelTOS (Cell-traversal protein for ookinetes and sporozoites). CelTOS is a highly conserved P. falciparum protein involved in cellular traversal through mosquito and vertebrate host cells. It reversibly refolds after thermal denaturation making it a desirable malarial vaccine candidate. Protein expressed in E. coli from a codon harmonized sequence of P. falciparum CelTOS (CH-PfCelTOS) was compared with protein expressed from the native codon sequence (N-PfCelTOS) to assess the impact of codon usage on protein expression levels, solubility, yield, stability, structural integrity, recognition with CelTOS-specific mAbs and immunogenicity in mice. While the translated proteins were expected to be identical, the translated products produced from the codon-harmonized sequence differed in helical content and showed a smaller distribution of polypeptides in mass spectra indicating lower heterogeneity of the codon harmonized version and fewer amino acid misincorporations. Substitutions of hydrophobic-to-hydrophobic amino acid were observed more commonly than any other. CH-PfCelTOS induced significantly higher antibody levels compared with N-PfCelTOS; however, no significant differences in either IFN-γ or IL-4 cellular responses were detected between the two antigens.

scholarly journals Sodium channel current loss of function in induced pluripotent stem cell-derived cardiomyocytes from a Brugada syndrome patient

2018 ◽  
Vol 114 ◽  
pp. 10-19 ◽  
Author(s):  
Elisabet Selga ◽  
Franziska Sendfeld ◽  
Rebecca Martinez-Moreno ◽  
Claire N. Medine ◽  
Olga Tura-Ceide ◽  
...  
Keyword(s):  
Stem Cell ◽  
Sodium Channel ◽  
Brugada Syndrome ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Syndrome Patient ◽  
Loss Of Function ◽  
Current Loss ◽  
Channel Current ◽  
Induced Pluripotent

scholarly journals Rho Kinase Inhibition Is Essential During In Vitro Neurogenesis and Promotes Phenotypic Rescue of Human Induced Pluripotent Stem Cell-Derived Neurons With Oligophrenin-1 Loss of Function

2016 ◽  
Vol 5 (7) ◽  
pp. 860-869 ◽  
Author(s):  
Claudia Compagnucci ◽  
Sabina Barresi ◽  
Stefania Petrini ◽  
Pierre Billuart ◽  
Giorgia Piccini ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Rho Kinase ◽  
Induced Pluripotent Stem Cell ◽  
Loss Of Function ◽  
Kinase Inhibition ◽  
Induced Pluripotent ◽  
Phenotypic Rescue

scholarly journals Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells Provide Insights on Parenteral Nutrition Associated Cholestasis in the Immature Liver

2021 ◽  
Author(s):  
T. Hang Nghiem-Rao ◽  
Courtney Pfeifer ◽  
Michelle Asuncion ◽  
Joshua Nord ◽  
Daniel Schill ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Bile Acid ◽  
Parenteral Nutrition ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Computational Techniques ◽  
Adult Liver ◽  
Induced Pluripotent ◽  
The Impact

Abstract Parenteral nutrition-associated cholestasis (PNAC) significantly limits the safety of intravenous parenteral nutrition (PN). Critically ill infants are highly vulnerable to PNAC-related morbidity and mortality, however the impact of hepatic immaturity on PNAC is poorly understood. We examined developmental differences between fetal/infant and adult livers, and used human induced pluripotent stem cell-derived hepatocyte-like cells (iHLC) to gain insights into the contribution of development to altered sterol metabolism and PNAC. We used RNA-sequencing and computational techniques to compare gene expression patterns in human fetal/infant livers, adult liver, and iHLC. We identified distinct gene expression profiles between the human feta/infant livers compared to adult liver, and close resemblance of iHLC to human developing livers. Compared to adult, both developing livers and iHLC had significant downregulation of xenobiotic, bile acid, and fatty acid metabolism; and lower expression of the sterol metabolizing gene ABCG8. When challenged with stigmasterol, a plant sterol found in intravenous soy lipids, lipid accumulation was significantly higher in iHLC compared to adult-derived HepG2 cells. Our findings provide insights into altered bile acid and lipid metabolizing processes in the immature human liver, and support the use of iHLC as a relevant model system of developing liver to study lipid metabolism and PNAC.

Abstract 379: Effects of Microgravity on Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Structure and Function

2018 ◽  
Vol 123 (Suppl_1) ◽  
Author(s):  
Alexa Wnorowski ◽  
Arun Sharma ◽  
Haodong Chen ◽  
Haodi Wu ◽  
Ning-Yi Shao ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Structure And Function ◽  
Induced Pluripotent ◽  
And Function

scholarly journals Influence of Ferroportin Disease Mutations on Manganese Accumulation and Toxicity (P24-060-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Eun-kyung Choi ◽  
Young-Ah Seo
Keyword(s):  
Genetic Disorder ◽  
Functional Results ◽  
Site Directed Mutagenesis ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Excess Iron ◽  
Funding Sources ◽  
Disease Mutations ◽  
The Impact

Abstract Objectives Hemochromatosis is a frequent genetic disorder characterized by the accumulation of excess iron across tissues. Mutations in the FPN1 gene, encoding a cell-surface iron exporter ferroportin (Fpn), are responsible for hemochromatosis type 4, also known as ferroportin disease. Recently, Fpn has been implicated in the regulation of manganese (Mn), another essential nutrient required for numerous cellular enzymes. However, the roles of Fpn in Mn regulation remain ill defined, and the impact of disease mutations on cellular Mn levels is unknown. Thus, this study aimed to define the role of Fpn in Mn regulation and determine the functional consequences of ferroportin disease mutations in cellular Mn levels. Methods Thus far, over 50 mutations in Fpn have been identified in hemochromatosis type 4/ferroportin disease. To test whether these mutations alter cellular Mn metabolism, we constructed an expression vector encoding human Fpn with a C-terminal HA epitope tag and introduced nine clinically relevant mutations by site-directed mutagenesis. Based on previously reported in vitro functional results, we selected five ferroportin disease mutations from each of the two groups: five loss-of-function (LOF) mutations (G80S, R88G, D157G, D157Y, and V162Δ) and four gain-of-function (GOF) mutations (N144H, N144T, C326S, and and S338R). Results Here, we provide evidence that Fpn can export Mn from cells into extracellular space. Fpn appears to play protective roles in Mn-induced cellular toxicity and oxidative stress. Finally, disease mutations interfere with Fpn's role in controlling Mn levels as well as the stability of Fpn. Conclusions These results define the function of Fpn as an exporter of both iron and Mn and highlight the potential involvement of Mn dysregulation in ferroportin disease. Funding Sources National Institutes of Health (NIH) to Y.A.S. (K99/R00 ES024340).

scholarly journals Inhalational delivery of induced pluripotent stem cell secretome improves postpneumonectomy lung structure and function

2020 ◽  
Vol 129 (5) ◽  
pp. 1051-1061
Author(s):  
D. Merrill Dane ◽  
Khoa Cao ◽  
Yu-An Zhang ◽  
Kemp H. Kernstine ◽  
Amiq Gazdhar ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Induced Pluripotent Stem Cell ◽  
Repeated Administration ◽  
Control Treatment ◽  
Surgical Removal ◽  
Adaptive Responses ◽  
Lung Structure ◽  
Secretory Products ◽  
Induced Pluripotent ◽  
And Function

To examine whether the secreted products of human induced pluripotent stem cells (iPSCs) facilitate innate adaptive responses following loss of lung tissue, adult dogs underwent surgical removal of one lung, then received repeated administration of iPSC secretory products via inhalational delivery compared with control treatment. Inhalation of iPSC secretory products enhanced capillary formation and beneficial structural remodeling in the remaining lung, leading to improved lung function.

iPS models of Parkin and PINK1

2015 ◽  
Vol 43 (2) ◽  
pp. 302-307 ◽  
Author(s):  
Aleksandar Rakovic ◽  
Philip Seibler ◽  
Christine Klein
Keyword(s):  
Dopaminergic Neurons ◽  
Induced Pluripotent Stem Cell ◽  
Therapeutic Agents ◽  
Axonal Outgrowth ◽  
Disease Mechanisms ◽  
The Central Nervous System ◽  
Degenerative Disorder ◽  
Induced Pluripotent ◽  
Druggable Targets ◽  
The Brain

Parkinson disease (PD) is a degenerative disorder of the central nervous system resulting from depletion of dopaminergic neurons and currently remains incurable despite enormous international research efforts. The development of induced pluripotent stem cell (iPSC) technology opened up the unique possibility of studying disease mechanisms in human tissue that was otherwise not accessible, such as the brain. Of particular interest are the monogenetic forms of PD as they closely resemble the more common ‘idiopathic’ PD and, through the mutated protein, provide a clear research target in iPSC-derived neurons. Recessively inherited Parkin and PTEN-induced putative kinase 1 (PINK1) mutations have been investigated in this context and the present review describes the first insights gained from studies in iPSC-derived dopaminergic neurons, which comprise abnormalities in mitochondrial and dopamine homoeostasis, microtubular stability and axonal outgrowth. These new models of PD have a high translational potential that includes the identification of druggable targets, testing of known and novel therapeutic agents in the disease-relevant tissue using well-defined read-outs and potential regenerative approaches.

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