scholarly journals An Autophagy Modifier Screen Identifies Small Molecules Capable of Reducing Autophagosome Accumulation in a Model of CLN3-Mediated Neurodegeneration

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1531 ◽  
Author(s):  
Anton Petcherski ◽  
Uma Chandrachud ◽  
Elisabeth Butz ◽  
Madeleine Klein ◽  
Wen-Ning Zhao ◽  
...  
Keyword(s):  
Protein Function ◽  
Mevalonate Pathway ◽  
Cell Model ◽  
Major Gene ◽  
Neuronal Cell ◽  
Mitochondrial Atpase ◽  
Lysosomal Storage ◽  
Storage Material ◽  
Cln3 Disease ◽  
Cellular Components

Alterations in the autophagosomal–lysosomal pathway are a major pathophysiological feature of CLN3 disease, which is the most common form of childhood-onset neurodegeneration. Accumulating autofluorescent lysosomal storage material in CLN3 disease, consisting of dolichols, lipids, biometals, and a protein that normally resides in the mitochondria, subunit c of the mitochondrial ATPase, provides evidence that autophagosomal–lysosomal turnover of cellular components is disrupted upon loss of CLN3 protein function. Using a murine neuronal cell model of the disease, which accurately mimics the major gene defect and the hallmark features of CLN3 disease, we conducted an unbiased search for modifiers of autophagy, extending previous work by further optimizing a GFP-LC3 based assay and performing a high-content screen on a library of ~2000 bioactive compounds. Here we corroborate our earlier screening results and identify expanded, independent sets of autophagy modifiers that increase or decrease the accumulation of autophagosomes in the CLN3 disease cells, highlighting several pathways of interest, including the regulation of calcium signaling, microtubule dynamics, and the mevalonate pathway. Follow-up analysis on fluspirilene, nicardipine, and verapamil, in particular, confirmed activity in reducing GFP-LC3 vesicle burden, while also demonstrating activity in normalizing lysosomal positioning and, for verapamil, in promoting storage material clearance in CLN3 disease neuronal cells. This study demonstrates the potential for cell-based screening studies to identify candidate molecules and pathways for further work to understand CLN3 disease pathogenesis and in drug development efforts.

scholarly journals Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+Homeostasis, Autophagy, and CLN3 Protein Function

2015 ◽  
Vol 290 (23) ◽  
pp. 14361-14380 ◽  
Author(s):  
Uma Chandrachud ◽  
Mathew W. Walker ◽  
Alexandra M. Simas ◽  
Sasja Heetveld ◽  
Anton Petcherski ◽  
...  
Keyword(s):  
Protein Function ◽  
Cell Model ◽  
Neuronal Cell ◽  
Batten Disease

scholarly journals Lack of Prenylated Proteins, Autophagy Impairment and Apoptosis in SH-SY5Y Neuronal Cell Model of Mevalonate Kinase Deficiency

2017 ◽  
Vol 41 (4) ◽  
pp. 1649-1660 ◽  
Author(s):  
Paola Maura Tricarico ◽  
Alessandra Romeo ◽  
Rossella Gratton ◽  
Sergio Crovella ◽  
Fulvio Celsi
Keyword(s):  
Cell Death ◽  
Mevalonate Pathway ◽  
Cell Model ◽  
Neuroblastoma Cell ◽  
Neuronal Cell ◽  
Autophagic Flux ◽  
Mevalonate Kinase Deficiency ◽  
Mevalonate Kinase ◽  
Protein Prenylation ◽  
Prenylated Proteins

Background/Aims: Mevalonate Kinase Deficiency (MKD), is a hereditary disease due to mutations in mevalonate kinase gene (MVK). MKD has heterogeneous clinical phenotypes: the correlation between MVK mutations and MKD clinical phenotype is still to be fully elucidated. Deficiency of prenylated proteins has been hypothesized as possible MKD pathogenic mechanism. Based on this hypothesis and considering that neurologic impairment characterizes Mevalonic Aciduria (MA), the most severe form of MKD, we studied the effects of I268T and N301T MVK mutations on protein prenylation, autophagy and programmed cell death in SH-SY5Y neuroblastoma cell lines. Methods: SH-SY5Y cells were transiently transfected, with the pCMV-6 plasmid containing MVK wild type and the two mutated sequences. Protein prenylation levels were evaluated using GFP-RhoA-F to assess farnesylation, and GFP-RhoA to evaluate geranylgeranylation; autophagy was measured by evaluating LC3 and p62 protein levels, while Annexin V-FITC and Propidium Iodide staining allowed apoptosis detection. Results: MVK mutants’ over-expression causes decreased levels of farnesylation and geranylgeranylation, and also increased LC3 lipidation in SH-SY5Y, with concomitant p62 accumulation. Treatment with bafilomycin A1 (an inhibitor of vacuolar H+-ATPase, a late autophagy inhibitor) further increase LC3-II and p62 levels, suggesting that degradation of autophagolysosome could be impaired. SH-SY5Y, with both MVK mutants, showed apoptosis increase; the presence of N301T associated with augmented cell death. Conclusions: We hypothesize that mevalonate pathway impairment causes alteration of farnesylation and geranylgeranylation proteins and alteration of the autophagic flux; these changes can induce apoptosis, possibly more relevant in the presence of N301T mutation.

Electron Microscopy in the diagnosis of lysosomal storage diseases

1989 ◽  
Vol 47 ◽  
pp. 866-867
Author(s):  
Carole Vogler ◽  
Harvey S. Rosenberg
Keyword(s):  
Electron Microscopy ◽  
Lysosomal Enzyme ◽  
Rectal Mucosa ◽  
Lysosomal Storage Diseases ◽  
Cell Types ◽  
Lysosomal Storage ◽  
Storage Material ◽  
Ultrastructural Examination ◽  
Blood Leukocytes ◽  
Storage Diseases

Diagnostic procedures for evaluation of patients with lysosomal storage diseases (LSD) seek to identify a deficiency of a responsible lysosomal enzyme or accumulation of a substance that requires the missing enzyme for degradation. Most patients with LSD have progressive neurological degeneration and may have a variety of musculoskeletal and visceral abnormalities. In the LSD, the abnormally diminished lysosomal enzyme results in accumulation of unmetabolized catabolites in distended lysosomes. Because of the subcellular morphology and size of lysosomes, electron microscopy is an ideal tool to study tissue from patients with suspected LSD. In patients with LSD all cells lack the specific lysosomal enzyme but the distribution of storage material is dependent on the extent of catabolism of the substrate in each cell type under normal circumstances. Lysosmal storages diseases affect many cell types and tissues. Storage material though does not accumulate in all tissues and cell types and may be different biochemically and morphologically in different tissues.Conjunctiva, skin, rectal mucosa and peripheral blood leukocytes may show ultrastructural evidence of lysosomal storage even in the absence of clinical findings and thus any of these tissues can be used for ultrastructural examination in the diagnostic evaluation of patients with suspected LSD. Biopsy of skin and conjunctiva are easily obtained and provide multiple cell types including endothelium, epithelium, fibroblasts and nerves for ultrastructural study. Fibroblasts from skin and conjunctiva can also be utilized for the initiation of tissue cultures for chemical assays. Brain biopsy has been largely replaced by biopsy of more readily obtained tissue and by biochemical assays. Such assays though may give equivical or nondiagnostic results and in some lysosomal storage diseases an enzyme defect has not yet been identified and diagnoses can be made only by ultrastructural examination.

scholarly journals MitoQ Is Able to Modulate Apoptosis and Inflammation

2021 ◽  
Vol 22 (9) ◽  
pp. 4753
Author(s):  
Elisa Piscianz ◽  
Alessandra Tesser ◽  
Erika Rimondi ◽  
Elisabetta Melloni ◽  
Claudio Celeghini ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Mevalonate Pathway ◽  
Neuronal Cell ◽  
Reactive Oxygen ◽  
Mitochondrial Activity ◽  
Oxygen Species ◽  
Short Period ◽  
Inflammatory Stimuli ◽  
Apoptosis And Inflammation ◽  
The Impact

Mitoquinone (MitoQ) is a mitochondrial reactive oxygen species scavenger that is characterized by high bioavailability. Prior studies have demonstrated its neuroprotective potential. Indeed, the release of reactive oxygen species due to damage to mitochondrial components plays a pivotal role in the pathogenesis of several neurodegenerative diseases. The present study aimed to examine the impact of the inflammation platform activation on the neuronal cell line (DAOY) treated with specific inflammatory stimuli and whether MitoQ addition can modulate these deregulations. DAOY cells were pre-treated with MitoQ and then stimulated by a blockade of the cholesterol pathway, also called mevalonate pathway, using a statin, mimicking cholesterol deregulation, a common parameter present in some neurodegenerative and autoinflammatory diseases. To verify the role played by MitoQ, we examined the expression of genes involved in the inflammation mechanism and the mitochondrial activity at different time points. In this experimental design, MitoQ showed a protective effect against the blockade of the mevalonate pathway in a short period (12 h) but did not persist for a long time (24 and 48 h). The results obtained highlight the anti-inflammatory properties of MitoQ and open the question about its application as an effective adjuvant for the treatment of the autoinflammatory disease characterized by a cholesterol deregulation pathway that involves mitochondrial homeostasis.

scholarly journals A human model of Batten disease shows role of CLN3 in phagocytosis at the photoreceptor–RPE interface

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Cynthia Tang ◽  
Jimin Han ◽  
Sonal Dalvi ◽  
Kannan Manian ◽  
Lauren Winschel ◽  
...  
Keyword(s):  
Photoreceptor Cell ◽  
Vision Loss ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Cell Loss ◽  
Human Model ◽  
Lysosomal Storage ◽  
Rpe Cells ◽  
Cln3 Disease

AbstractMutations in CLN3 lead to photoreceptor cell loss in CLN3 disease, a lysosomal storage disorder characterized by childhood-onset vision loss, neurological impairment, and premature death. However, how CLN3 mutations cause photoreceptor cell death is not known. Here, we show that CLN3 is required for phagocytosis of photoreceptor outer segment (POS) by retinal pigment epithelium (RPE) cells, a cellular process essential for photoreceptor survival. Specifically, a proportion of CLN3 in human, mouse, and iPSC-RPE cells localized to RPE microvilli, the site of POS phagocytosis. Furthermore, patient-derived CLN3 disease iPSC-RPE cells showed decreased RPE microvilli density and reduced POS binding and ingestion. Notably, POS phagocytosis defect in CLN3 disease iPSC-RPE cells could be rescued by wild-type CLN3 gene supplementation. Altogether, these results illustrate a novel role of CLN3 in regulating POS phagocytosis and suggest a contribution of primary RPE dysfunction for photoreceptor cell loss in CLN3 disease that can be targeted by gene therapy.

scholarly journals The Novel Alpha-2 Adrenoceptor Inhibitor Beditin Reduces Cytotoxicity and Huntingtin Aggregates in Cell Models of Huntington’s Disease

2021 ◽  
Vol 14 (3) ◽  
pp. 257
Author(s):  
Elisabeth Singer ◽  
Lilit Hunanyan ◽  
Magda M. Melkonyan ◽  
Jonasz J. Weber ◽  
Lusine Danielyan ◽  
...  
Keyword(s):  
Huntington's Disease ◽  
Huntington’S Disease ◽  
Neurodegenerative Disorder ◽  
Cell Model ◽  
Resonance Energy Transfer ◽  
Neuronal Cell ◽  
Resonance Energy ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tunel Staining ◽  
Cell Models

Huntington’s disease (HD) is a monogenetic neurodegenerative disorder characterized by the accumulation of polyglutamine-expanded huntingtin (mHTT). There is currently no cure, and therefore disease-slowing remedies are sought to alleviate symptoms of the multifaceted disorder. Encouraging findings in Alzheimer’s and Parkinson’s disease on alpha-2 adrenoceptor (α2-AR) inhibition have shown neuroprotective and aggregation-reducing effects in cell and animal models. Here, we analyzed the effect of beditin, a novel α2- adrenoceptor (AR) antagonist, on cell viability and mHTT protein levels in cell models of HD using Western blot, time-resolved Foerster resonance energy transfer (TR-FRET), lactate dehydrogenase (LDH) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) cytotoxicity assays. Beditin decreases cytotoxicity, as measured by TUNEL staining and LDH release, in a neuronal progenitor cell model (STHdh cells) of HD and decreases the aggregation propensity of HTT exon 1 fragments in an overexpression model using human embryonic kidney (HEK) 293T cells. α2-AR is a promising therapeutic target for further characterization in HD models. Our data allow us to suggest beditin as a valuable candidate for the pharmaceutical manipulation of α2-AR, as it is capable of modulating neuronal cell survival and the level of mHTT.

scholarly journals Glucose regulates AMP-activated protein kinase activity and gene expression in clonal, hypothalamic neurons expressing proopiomelanocortin: additive effects of leptin or insulin

2007 ◽  
Vol 192 (3) ◽  
pp. 605-614 ◽  
Author(s):  
Fang Cai ◽  
Armen V Gyulkhandanyan ◽  
Michael B Wheeler ◽  
Denise D Belsham
Keyword(s):  
Protein Kinase ◽  
Energy Homeostasis ◽  
Cell Model ◽  
Neuronal Cell ◽  
Cell Types ◽  
Glucose Sensing ◽  
Protein Kinase Activity ◽  
Energy Status ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

The mammalian hypothalamus comprises an array of phenotypically distinct cell types that interpret peripheral signals of energy status and, in turn, elicits an appropriate response to maintain energy homeostasis. We used a clonal representative hypothalamic cell model expressing proopiomelanocortin (POMC; N-43/5) to study changes in AMP-activated protein kinase (AMPK) activity and glucose responsiveness. We have demonstrated the presence of cellular machinery responsible for glucose sensing in the cell line, including glucokinase, glucose transporters, and appropriate ion channels. ATP-sensitive potassium channels were functional and responded to glucose. The N-43/5 POMC neurons may therefore be an appropriate cell model to study glucose-sensing mechanisms in the hypothalamus. In N-43/5 POMC neurons, increasing glucose concentrations decreased phospho-AMPK activity. As a relevant downstream effect, we found that POMC transcription increased with 2.8 and 16.7 mM glucose. Upon addition of leptin, with either no glucose or with 5 mM glucose, we found that leptin decreased AMPK activity in N-43/5 POMC neurons, but had no significant effect at 25 mM glucose, whereas insulin decreased AMPK activity at only 5 mM glucose. These results demonstrate that individual hypothalamic neuronal cell types, such as the POMC neuron, can have distinct responses to peripheral signals that relay energy status to the brain, and will therefore be activated uniquely to control neuroendocrine function.

scholarly journals ACTN4 Mediates SEPT14 Mutation-Induced Sperm Head Defects

Biomedicines ◽  
2020 ◽  
Vol 8 (11) ◽  
pp. 518
Author(s):  
Yu-Hua Lin ◽  
Chia-Yen Huang ◽  
Chih-Chun Ke ◽  
Ya-Yun Wang ◽  
Tsung-Hsuan Lai ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cell Model ◽  
Membrane Dynamics ◽  
Sperm Head ◽  
Cellular Processes ◽  
Germ Cell Line ◽  
Head Formation ◽  
Fourth Component ◽  
Related Proteins ◽  
Cellular Components

Septins (SEPTs) are highly conserved GTP-binding proteins and the fourth component of the cytoskeleton. Polymerized SEPTs participate in the modulation of various cellular processes, such as cytokinesis, cell polarity, and membrane dynamics, through their interactions with microtubules, actin, and other cellular components. The main objective of this study was to dissect the molecular pathological mechanism of SEPT14 mutation-induced sperm head defects. To identify SEPT14 interactors, co-immunoprecipitation (co-IP) and nano-liquid chromatography-mass spectrometry/mass spectrometry were applied. Immunostaining showed that SEPT14 was significantly localized to the manchette structure. The SEPT14 interactors were identified and classified as (1) SEPT-, (2) microtubule-, (3) actin-, and (4) sperm structure-related proteins. One interactor, ACTN4, an actin-holding protein, was selected for further study. Co-IP experiments showed that SEPT14 interacts with ACTN4 in a male germ cell line. SEPT14 also co-localized with ACTN4 in the perinuclear and manchette regions of the sperm head in early elongating spermatids. In the cell model, mutated SEPT14 disturbed the localization pattern of ACTN4. In a clinical aspect, sperm with mutant SEPT14, SEPT14A123T (p.Ala123Thr), and SEPT14I333T (p.Ile333Thr), have mislocalized and fragmented ACTN4 signals. Sperm head defects in donors with SEPT14 mutations are caused by disruption of the functions of ACTN4 and actin during sperm head formation.

scholarly journals Heparan sulphate compounds regulate cholesterol metabolism in neurodegenerative disease models

2021 ◽  
Author(s):  
◽  
Rosemary Heathcott
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Neurodegenerative Disease ◽  
Cell Line ◽  
Cholesterol Metabolism ◽  
Neurodegenerative Disorder ◽  
Heparan Sulphate ◽  
Amyloid Β ◽  
Neuronal Cell ◽  
Lysosomal Storage

<p>Heparan sulphate proteoglycans (HSPG) are central to numerous processes of the mammalian cell. The highly charged negative side chains of the heparan sulphate (HS) oligosaccharides are essential for the regulatory and structural functions of the proteoglycan. Synthetic HS compounds have potential therapeutic value due to their ability to mimic naturally occurring HS. Niemann-Pick disease type C (NPC) is a fatal childhood neurodegenerative disease with characteristic cholesterol and sphingolipid accumulation in the late endosome or lysosome. Alzheimer’s disease, another neurodegenerative disorder, shares alterations of cholesterol and amyloid β metabolism with NPC. In this study,a set of novel heparan sulphate compounds with a range of structures and oligosaccharide side groups with a variety of degrees of sulphation was investigated with regards to their effects on cholesterol and amyloid β metabolism in cell line models of these two diseases. Fluorescent staining of cholesterol and confocal microscopy showed highly sulphated compounds reduce the accumulation of cholesterol in the perinuclear lysosomal storage organelles in patient fibroblast cell lines. The compounds had no effect on secreted amyloid β levels or amyloid precursor protein levels in a neuronal cell line model of early onset Alzheimer’s disease. The mechanism of cholesterol reduction is unclear but may be related to a reduction in HSPG-associated endocytosis of LDL/cholesterol.</p>

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