scholarly journals Inhibition of VCP preserves retinal structure and function in autosomal dominant retinal degeneration

2020 ◽  
Author(s):  
Blanca Arango-Gonzalez ◽  
Merve Sen ◽  
Rosellina Guarascio ◽  
Kalliopi Ziaka ◽  
Eva M. del Amo ◽  
...  
Keyword(s):  
Autosomal Dominant ◽  
Transgenic Rat ◽  
Aaa Atpase ◽  
Rod Cells ◽  
High Production ◽  
Retinal Structure ◽  
Photoreceptor Neurons ◽  
And Function

ABSTRACTDue to continuously high production rates of rhodopsin (RHO) and high metabolic activity, photoreceptor neurons are especially vulnerable to defects in proteostasis. A proline to histidine substitution at position 23 (P23H) leads to production of structurally misfolded RHO, causing the most common form of autosomal dominant Retinitis Pigmentosa (adRP) in North America. The AAA-ATPase valosin-containing protein (VCP) extracts misfolded proteins from the ER membrane for cytosolic degradation. Here, we provide the first evidence that inhibition of VCP activity rescues degenerating P23H rod cells and improves their functional properties in P23H transgenic rat and P23H knock-in mouse retinae, both in vitro and in vivo. This improvement correlates with the restoration of the physiological RHO localization to rod outer segments (OS) and properly-assembled OS disks. As a single intravitreal injection suffices to deliver a long-lasting benefit in vivo, we suggest VCP inhibition as a potential therapeutic strategy for adRP patients carrying mutations in the RHO gene.

scholarly journals Dihydroceramide desaturase directs the switch between exosome production and autophagy for neuronal maintenance

2020 ◽  
Author(s):  
Chen-Yi Wu ◽  
Jhih-Gang Jhang ◽  
Chih-Wei Lin ◽  
Han-Chen Ho ◽  
Chih-Chiang Chan ◽  
...  
Keyword(s):  
Nervous System ◽  
Desaturase Gene ◽  
Dihydroceramide Desaturase ◽  
Autophagic Degradation ◽  
Photoreceptor Neurons ◽  
And Function ◽  
Neuronal Maintenance ◽  
Autophagy Inhibition

ABSTRACTExosomes play important roles in the nervous system. Mutations in the human dihydroceramide desaturase gene, DEGS1, are recently linked to severe neurological disorders, but the cause remains unknown. Here, we show that Ifc is required for the morphology and function of Drosophila photoreceptor neurons and not in the surrounding glia, but the degeneration of ifc-KO eyes can be rescued by glial expression of ifc, possibly mediated by exosomes. We develop an in vivo assay using Drosophila eye imaginal discs and show that the level and activity of Ifc correlates with the detection of exosome-like vesicles. While ifc overexpression and autophagy inhibition both enhances exosome production, combining the two had no additive effect. Moreover, ifc-KO reduces the density of the exosome precursor intraluminal vesicles (ILVs) in vivo, and DEGS1 promotes ILV formation in vitro. In conclusion, dihydroceramide desaturase promotes exosome formation and prevents its autophagic degradation in the nervous system.

scholarly journals Grafts Derived from an α-Synuclein Triplication Patient Mediate Functional Recovery but Develop Disease-Associated Pathology in the 6-OHDA Model of Parkinson’s Disease

2020 ◽  
pp. 1-14
Author(s):  
Shelby Shrigley ◽  
Fredrik Nilsson ◽  
Bengt Mattsson ◽  
Alessandro Fiorenzano ◽  
Janitha Mudannayake ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Lines ◽  
Functional Recovery ◽  
Embryonic Stem ◽  
Hesc Line ◽  
Alternative Source ◽  
And Function

Background: Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson’s disease (PD) and they provide the option of using the patient’s own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD. Objective: To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control. Methods: Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo. Results: Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line. Conclusion: This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.

scholarly journals Extracellular vesicles as mediators and markers of acute organ injury: current concepts

2021 ◽  
Author(s):  
Birte Weber ◽  
Niklas Franz ◽  
Ingo Marzi ◽  
Dirk Henrich ◽  
Liudmila Leppik
Keyword(s):  
Extracellular Vesicles ◽  
Inflammatory Diseases ◽  
Organ Injury ◽  
Isolation And Characterization ◽  
Communication Processes ◽  
Incidence And Mortality ◽  
New Biomarkers ◽  
And Function

AbstractDue to the continued high incidence and mortality rate worldwide, there is a need to develop new strategies for the quick, precise, and valuable recognition of presenting injury pattern in traumatized and poly-traumatized patients. Extracellular vesicles (EVs) have been shown to facilitate intercellular communication processes between cells in close proximity as well as distant cells in healthy and disease organisms. miRNAs and proteins transferred by EVs play biological roles in maintaining normal organ structure and function under physiological conditions. In pathological conditions, EVs change the miRNAs and protein cargo composition, mediating or suppressing the injury consequences. Therefore, incorporating EVs with their unique protein and miRNAs signature into the list of promising new biomarkers is a logical next step. In this review, we discuss the general characteristics and technical aspects of EVs isolation and characterization. We discuss results of recent in vitro, in vivo, and patients study describing the role of EVs in different inflammatory diseases and traumatic organ injuries. miRNAs and protein signature of EVs found in patients with acute organ injury are also debated.

scholarly journals Modelling the Human Placental Interface In Vitro—A Review

Micromachines ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 884
Author(s):  
Marta Cherubini ◽  
Scott Erickson ◽  
Kristina Haase
Keyword(s):  
Drug Testing ◽  
Cell Types ◽  
In Vitro Models ◽  
Placental Development ◽  
Scientific Challenge ◽  
Induced Pluripotent ◽  
And Function ◽  
And Control

Acting as the primary link between mother and fetus, the placenta is involved in regulating nutrient, oxygen, and waste exchange; thus, healthy placental development is crucial for a successful pregnancy. In line with the increasing demands of the fetus, the placenta evolves throughout pregnancy, making it a particularly difficult organ to study. Research into placental development and dysfunction poses a unique scientific challenge due to ethical constraints and the differences in morphology and function that exist between species. Recently, there have been increased efforts towards generating in vitro models of the human placenta. Advancements in the differentiation of human induced pluripotent stem cells (hiPSCs), microfluidics, and bioprinting have each contributed to the development of new models, which can be designed to closely match physiological in vivo conditions. By including relevant placental cell types and control over the microenvironment, these new in vitro models promise to reveal clues to the pathogenesis of placental dysfunction and facilitate drug testing across the maternal–fetal interface. In this minireview, we aim to highlight current in vitro placental models and their applications in the study of disease and discuss future avenues for these in vitro models.

scholarly journals Therapeutic Application of Brain-Specific Angiogenesis Inhibitor 1 for Cancer Therapy

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3562
Author(s):  
Mitra Nair ◽  
Chelsea Bolyard ◽  
Tae Jin Lee ◽  
Balveen Kaur ◽  
Ji Young Yoo
Keyword(s):  
Angiogenesis Inhibitor ◽  
Suppressor Gene ◽  
Chemotherapeutic Agents ◽  
Tumor Models ◽  
In Vivo Models ◽  
Herpes Simplex Viruses ◽  
Multiple Tumor ◽  
And Function

Brain-specific angiogenesis inhibitor 1 (BAI1/ADGRB1) is an adhesion G protein-coupled receptor that has been found to play key roles in phagocytosis, inflammation, synaptogenesis, the inhibition of angiogenesis, and myoblast fusion. As the name suggests, it is primarily expressed in the brain, with a high expression in the normal adult and developing brain. Additionally, its expression is reduced in brain cancers, such as glioblastoma (GBM) and peripheral cancers, suggesting that BAI1 is a tumor suppressor gene. Several investigators have demonstrated that the restoration of BAI1 expression in cancer cells results in reduced tumor growth and angiogenesis. Its expression has also been shown to be inversely correlated with tumor progression, neovascularization, and peri-tumoral brain edema. One method of restoring BAI1 expression is by using oncolytic virus (OV) therapy, a strategy which has been tested in various tumor models. Oncolytic herpes simplex viruses engineered to express the secreted fragment of BAI1, called Vasculostatin (Vstat120), have shown potent anti-tumor and anti-angiogenic effects in multiple tumor models. Combining Vstat120-expressing oHSVs with other chemotherapeutic agents has also shown to increase the overall anti-tumor efficacy in both in vitro and in vivo models. In the current review, we describe the structure and function of BAI1 and summarize its application in the context of cancer treatment.

scholarly journals Alcohol-and-HIV-Induced Lysosomal Dysfunction Regulates Extracellular Vesicles Secretion in Vitro and in Liver-Humanized Mice

Biology ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 29
Author(s):  
Raghubendra Singh Dagur ◽  
Moses New-Aaron ◽  
Murali Ganesan ◽  
Weimin Wang ◽  
Svetlana Romanova ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Extracellular Vesicles ◽  
Treated Group ◽  
Human Hepatocytes ◽  
In Vivo Studies ◽  
People Living With Hiv ◽  
Humanized Mouse Model ◽  
And Function

Background: Alcohol abuse is common in people living with HIV-1 and dramaticallyenhances the severity of HIV-induced liver damage by inducing oxidative stress and lysosomaldysfunction in the liver cells. We hypothesize that the increased release of extracellular vesicles(EVs) in hepatocytes and liver humanized mouse model is linked to lysosome dysfunction. Methods:The study was performed on primary human hepatocytes and human hepatoma RLWXP-GFP (Huh7.5 cells stably transfected with CYP2E1 and XPack-GFP) cells and validated on ethanol-fed liverhumanizedfumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chainknockout (FRG-KO) mice. Cells and mice were infected with HIV-1ADA virus. Results: We observedan increase in the secretion of EVs associated with a decrease in lysosomal activity and expressionof lysosomal-associated membrane protein 1. Next-generation RNA sequencing of primary humanhepatocytes revealed 63 differentially expressed genes, with 13 downregulated and 50 upregulatedgenes in the alcohol–HIV-treated group. Upstream regulator analysis of differentially expressedgenes through Ingenuity Pathway Analysis identified transcriptional regulators affecting downstreamgenes associated with increased oxidative stress, lysosomal associated disease, and function andEVs biogenesis. Our in vitro findings were corroborated by in vivo studies on human hepatocytetransplantedhumanized mice, indicating that intensive EVs’ generation by human hepatocytes andtheir secretion to serum was associated with increased oxidative stress and reduction in lysosomalactivities triggered by HIV infection and ethanol diet. Conclusion: HIV-and-ethanol-metabolisminducedEVs release is tightly controlled by lysosome status in hepatocytes and participates in thedevelopment of double-insult-induced liver injury.

scholarly journals Phosphorylation-dependent interaction of the synaptic vesicle proteins cysteine string protein and synaptotagmin I

2002 ◽  
Vol 364 (2) ◽  
pp. 343-347 ◽  
Author(s):  
Gareth J.O. EVANS ◽  
Alan MORGAN
Keyword(s):  
Rat Brain ◽  
Direct Interaction ◽  
Secretory Vesicle ◽  
Brain Membrane ◽  
Synaptotagmin I ◽  
Phosphorylated Form ◽  
Order Of Magnitude ◽  
And Function

The secretory vesicle cysteine string proteins (CSPs) are members of the DnaJ family of chaperones, and function at late stages of Ca2+-regulated exocytosis by an unknown mechanism. To determine novel binding partners of CSPs, we employed a pull-down strategy from purified rat brain membrane or cytosolic proteins using recombinant hexahistidine-tagged (His6-)CSP. Western blotting of the CSP-binding proteins identified synaptotagmin I to be a putative binding partner. Furthermore, pull-down assays using cAMP-dependent protein kinase (PKA)-phosphorylated CSP recovered significantly less synaptotagmin. Complexes containing CSP and synaptotagmin were immunoprecipitated from rat brain membranes, further suggesting that these proteins interact in vivo. Binding assays in vitro using recombinant proteins confirmed a direct interaction between the two proteins and demonstrated that the PKA-phosphorylated form of CSP binds synaptotagmin with approximately an order of magnitude lower affinity than the non-phosphorylated form. Genetic studies have implicated each of these proteins in the Ca2+-dependency of exocytosis and, since CSP does not bind Ca2+, this novel interaction might explain the Ca2+-dependent actions of CSP.

scholarly journals DJ-1 regulates the integrity and function of ER-mitochondria association through interaction with IP3R3-Grp75-VDAC1

2019 ◽  
Vol 116 (50) ◽  
pp. 25322-25328 ◽  
Author(s):  
Yi Liu ◽  
Xiaopin Ma ◽  
Hisashi Fujioka ◽  
Jun Liu ◽  
Shengdi Chen ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Cellular Mechanism ◽  
Loss Of Function ◽  
Wild Type ◽  
Calcium Efflux ◽  
And Function ◽  
The Brain ◽  
Early Onset Parkinson’S Disease

Loss-of-function mutations in DJ-1 are associated with autosomal recessive early onset Parkinson’s disease (PD), yet the underlying pathogenic mechanism remains elusive. Here we demonstrate that DJ-1 localized to the mitochondria-associated membrane (MAM) both in vitro and in vivo. In fact, DJ-1 physically interacts with and is an essential component of the IP3R3-Grp75-VDAC1 complexes at MAM. Loss of DJ-1 disrupted the IP3R3-Grp75-VDAC1 complex and led to reduced endoplasmic reticulum (ER)-mitochondria association and disturbed function of MAM and mitochondria in vitro. These deficits could be rescued by wild-type DJ-1 but not by the familial PD-associated L166P mutant which had demonstrated reduced interaction with IP3R3-Grp75. Furthermore, DJ-1 ablation disturbed calcium efflux-induced IP3R3 degradation after carbachol treatment and caused IP3R3 accumulation at the MAM in vitro. Importantly, similar deficits in IP3R3-Grp75-VDAC1 complexes and MAM were found in the brain of DJ-1 knockout mice in vivo. The DJ-1 level was reduced in the substantia nigra of sporadic PD patients, which was associated with reduced IP3R3-DJ-1 interaction and ER-mitochondria association. Together, these findings offer insights into the cellular mechanism in the involvement of DJ-1 in the regulation of the integrity and calcium cross-talk between ER and mitochondria and suggests that impaired ER-mitochondria association could contribute to the pathogenesis of PD.

scholarly journals Mirtron-mediated RNA knockdown/replacement therapy for the treatment of dominant retinitis pigmentosa

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Harry O. Orlans ◽  
Michelle E. McClements ◽  
Alun R. Barnard ◽  
Cristina Martinez-Fernandez de la Camara ◽  
Robert E. MacLaren
Keyword(s):  
Retinitis Pigmentosa ◽  
Autosomal Dominant ◽  
Gene Mutations ◽  
Adeno Associated Virus ◽  
Treatment Development ◽  
Common Cause ◽  
Toxic Protein ◽  
Rna Knockdown

AbstractRhodopsin (RHO) gene mutations are a common cause of autosomal dominant retinitis pigmentosa (ADRP). The need to suppress toxic protein expression together with mutational heterogeneity pose challenges for treatment development. Mirtrons are atypical RNA interference effectors that are spliced from transcripts as short introns. Here, we develop a novel mirtron-based knockdown/replacement gene therapy for the mutation-independent treatment of RHO-related ADRP, and demonstrate efficacy in a relevant mammalian model. Splicing and potency of rhodopsin-targeting candidate mirtrons are initially determined, and a mirtron-resistant codon-modified version of the rhodopsin coding sequence is validated in vitro. These elements are then combined within a single adeno-associated virus (AAV) and delivered subretinally in a RhoP23H knock-in mouse model of ADRP. This results in significant mouse-to-human rhodopsin RNA replacement and is associated with a slowing of retinal degeneration. This provides proof of principle that synthetic mirtrons delivered by AAV are capable of reducing disease severity in vivo.

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