International consensus guidelines for the definition, detection, and interpretation of autophagy-dependent ferroptosis.

Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results.Abbreviation: 3-MA:3-methyladenine; 4HNE: 4-hydroxynonenal; ACD: accidentalcell death; ADF: autophagy-dependentferroptosis; ARE: antioxidant response element; BH2:dihydrobiopterin; BH4: tetrahydrobiopterin; BMDMs: bonemarrow-derived macrophages; CMA: chaperone-mediated autophagy; CQ:chloroquine; DAMPs: danger/damage-associated molecular patterns; EMT,epithelial-mesenchymal transition; EPR: electronparamagnetic resonance; ER, endoplasmic reticulum; FRET: Försterresonance energy transfer; GFP: green fluorescent protein;GSH: glutathione;IF: immunofluorescence; IHC: immunohistochemistry; IOP, intraocularpressure; IRI: ischemia-reperfusion injury; LAA: linoleamide alkyne;MDA: malondialdehyde; PGSK: Phen Green™ SK;RCD: regulatedcell death; PUFAs: polyunsaturated fatty acids; RFP: red fluorescentprotein;ROS: reactive oxygen species; TBA: thiobarbituricacid; TBARS: thiobarbituric acid reactive substances; TEM:transmission electron microscopy.

Variable Phenotype of Congenital Corneal Opacities in Biallelic CYP1B1 Pathogenic Variants.

PURPOSE: The aim of this study is to describe the variable phenotype of congenital corneal opacities occurring in patients with biallelic CYP1B1 pathogenic variants. METHODS: A retrospective chart review was conducted to identify patients with congenital corneal opacities and CYP1B1 pathogenic variants seen at UPMC Children's Hospital of Pittsburgh. Ophthalmic examination, high-frequency ultrasound, anterior segment optical coherence tomography, histopathologic images, and details of genetic testing were reviewed. RESULTS: Three children were identified. All presented with raised intraocular pressure. Two patients showed bilateral limbus-to-limbus avascular corneal opacification that did not resolve with intraocular pressure control; 1 showed unilateral avascular corneal opacity with a crescent of clear cornea, iridocorneal adhesions, iridolenticular adhesions, and classical features of congenital glaucoma in the fellow eye (enlarged corneal diameter, Haab striae, and clearing of the corneal clouding with appropriate intraocular pressure control). The first 2 patients were visually rehabilitated with penetrating keratoplasty. Histopathology revealed distinct features: a variably keratinized epithelium; a thick but discontinuous Bowman-like layer with areas of disruption and abnormal cellularity; Descemet membrane, when observed, showed reduced endothelial cells; and no pathological changes of Haab striae were identified. Two patients had compound heterozygous pathogenic variants in CYP1B1 causing premature stop codons, whereas 1 was homozygous for a pathogenic missense variant. CONCLUSIONS: Congenital corneal opacities seen in biallelic CYP1B1 pathogenic variants have a variable phenotype. One is that commonly termed as Peters anomaly type 1 (with iridocorneal adhesions, with or without iridolenticular adhesions) and the other is a limbus-to-limbus opacity, termed CYP1B1 cytopathy. Clinicians should be aware of this phenotypic variability.

iPSC-Derived Neurons from Patients with POLG Mutations Exhibit Decreased Mitochondrial Content and Dendrite Simplification.

Mutations in POLG, the gene encoding the catalytic subunit of DNA polymerase gamma, result in clinical syndromes characterized by mitochondrial DNA (mtDNA) depletion in affected tissues with variable organ involvement. The brain is one of the most affected organs, and symptoms include intractable seizures, developmental delay, dementia, and ataxia. Patient-derived induced pluripotent stem cells (iPSCs) provide opportunities to explore mechanisms in affected cell types and potential therapeutic strategies. Fibroblasts from two patients were reprogrammed to create new iPSC models of POLG-related mitochondrial diseases. Compared with iPSC-derived control neurons, mtDNA depletion was observed upon differentiation of the POLG-mutated lines to cortical neurons. POLG-mutated neurons exhibited neurite simplification with decreased mitochondrial content, abnormal mitochondrial structure and function, and increased cell death. Expression of the mitochondrial kinase PTEN-induced kinase 1 (PINK1) mRNA was decreased in patient neurons. Overexpression of PINK1 increased mitochondrial content and ATP:ADP ratios in neurites, decreasing cell death and rescuing neuritic complexity. These data indicate an intersection of polymerase gamma and PINK1 pathways that may offer a novel therapeutic option for patients affected by this spectrum of disorders.

Congenital corneal staphyloma in 8q21.11 microdeletion syndrome.

BACKGROUND: Although 8q21.11 microdeletion syndrome (8q21.11 DS) has been reported in association with congenital corneal opacities, reports of the clinicopathological features and management are scarce. METHODS: We reviewed medical records including ophthalmic evaluations, imaging, operative reports, and pathology reports of two unrelated patients referred to the Ophthalmology Clinic of UPMC Children's Hospital of Pittsburgh with a cytogenetic diagnosis of 8q21.11 DS. RESULTS: Ophthalmological evaluation of both children revealed bilateral enlarged, staphylomatous, and cloudy corneas with neovascularization. These findings were consistent with the diagnosis of congenital corneal staphyloma (CCS). In one patient, anterior segment optical coherence tomography and high-frequency ultrasound revealed materials consistent with lens remnants embedded in the cornea; this was confirmed by histopathology. In the second patient, lens was found to be adherent to the cornea during surgery. One eye underwent enucleation for corneal perforation secondary to elevated intraocular pressure. In the other eyes, treatment consisted of penetrating keratoplasty combined with vitrectomy. Ahmed tube was subsequently placed to control intraocular pressure. CONCLUSION: 8q21.11 microdeletion syndrome can be associated with bilateral CCS, likely related to a combination of anterior segment developmental anomalies and elevated intraocular pressure. Tectonic penetrating keratoplasty is necessary to prevent corneal perforation, together with a strict control of the intraocular pressure.

Congenital Corneal Opacity in 22q11.2 Deletion Syndrome: A Case Series.

PURPOSE: The purpose of this study was to describe the deep phenotype of congenital corneal opacities (CCO) in patients with 22q11.2 deletion syndrome (22q11.2 DS) and to identify putative regions or genes that could explain the CCO. METHODS: A retrospective chart review was conducted to identify patients with 22q11.2 DS seen in the ophthalmology clinic of a tertiary referral children's hospital. Thirty patients were identified, with molecular confirmation. Twenty-six did not show structural anterior segment anomalies aside from posterior embryotoxon (n = 4), whereas 4 had bilateral CCO, of which 3 had preoperative images. We reviewed medical, operative, and pathology reports; anterior segment optical coherence tomography; high-frequency ultrasound; histopathologic slides; and genetic testing. To identify putative genes responsible for CCO, chromosomal breakpoints in patients with and without CCO were compared. RESULTS: In the 3 patients with preoperative imaging and CCO, a pattern of paracentral corneal opacification with central clearing accompanied by iridocorneal or keratolenticular adhesions was observed. Anterior segment optical coherence tomography and histopathologic images showed central stromal thinning with a residual structure consistent with Descemet membrane. One patient presented at birth with unilateral corneal perforation, suggestive of likely stromal thinning. A comparison of the breakpoints across all cases failed to reveal unique regions or genes in patients with CCO. CONCLUSIONS: 22q11.2 DS can rarely be associated with CCO. We describe a consistent pattern of central clearing related to posterior stromal thinning, with or without ICA/KLA. Possible candidate genes for corneal opacification in 22q11.2 DS remain elusive.

Endogenous PTEN-Induced Kinase 1 Regulates Dendritic Architecture and Spinogenesis.

Mutations in PTEN-induced kinase 1 (PINK1) contribute to autosomal recessive Parkinson's disease with cognitive and neuropsychiatric comorbidities. Disturbances in dendritic and spine architecture are hallmarks of neurodegenerative and neuropsychiatric conditions, but little is known of the impact of PINK1 on these structures. We used Pink1 -/- mice to study the role of endogenous PINK1 in regulating dendritic architecture, spine density, and spine maturation. Pink1 -/- cortical neurons of unknown sex showed decreased dendritic arborization, affecting both apical and basal arbors. Dendritic simplification in Pink1 -/- neurons was primarily driven by diminished branching with smaller effects on branch lengths. Pink1 -/- neurons showed reduced spine density with a shift in morphology to favor filopodia at the expense of mushroom spines. Electrophysiology revealed significant reductions in miniature EPSC (mEPSC) frequency in Pink1 -/- neurons, consistent with the observation of decreased spine numbers. Transfecting with human PINK1 rescued changes in dendritic architecture, in thin, stubby, and mushroom spine densities, and in mEPSC frequency. Diminished spine density was also observed in Golgi-Cox stained adult male Pink1 -/- brains. Western blot study of Pink1 -/- brains of either sex revealed reduced phosphorylation of NSFL1 cofactor p47, an indirect target of PINK1. Transfection of Pink1 -/- neurons with a phosphomimetic p47 plasmid rescued dendritic branching and thin/stubby spine density with a partial rescue of mushroom spines, implicating a role for PINK1-regulated p47 phosphorylation in dendrite and spine development. These findings suggest that PINK1-dependent synaptodendritic alterations may contribute to the risk of cognitive and/or neuropsychiatric pathologies observed in PINK1-mutated families.SIGNIFICANCE STATEMENT Loss of PINK1 function has been implicated in both familial and sporadic neurodegenerative diseases. Yet surprisingly little is known of the impact of PINK1 loss on the fine structure of neurons. Neurons receive excitatory synaptic signals along a complex network of projections that form the dendritic tree, largely at tiny protrusions called dendritic spines. We studied cortical neurons and brain tissues from mice lacking PINK1. We discovered that PINK1 deficiency causes striking simplification of dendritic architecture associated with reduced synaptic input and decreased spine density and maturation. These changes are reversed by reintroducing human PINK1 or one of its downstream mediators into PINK1-deficient mouse neurons, indicating a conserved function, whose loss may contribute to neurodegenerative processes.

Mitochondria in neurodegeneration.

The brain is one of the most energetically demanding tissues in the human body, and mitochondrial pathology is strongly implicated in chronic neurodegenerative diseases. In contrast to acute brain injuries in which bioenergetics and cell death play dominant roles, studies modeling familial neurodegeneration implicate a more complex and nuanced relationship involving the entire mitochondrial life cycle. Recent literature on mitochondrial mechanisms in Parkinson's disease, Alzheimer's disease, frontotemporal dementia, Huntington's disease, and amyotrophic lateral sclerosis is reviewed with an emphasis on mitochondrial quality control, transport and synaptodendritic calcium homeostasis. Potential neuroprotective interventions include targeting the mitochondrial kinase PTEN-induced kinase 1 (PINK1), which plays a role in regulating not only multiple facets of mitochondrial biology, but also neuronal morphogenesis and dendritic arborization.

Molecular profiling of renal cell carcinoma presenting as iris metastasis.

Purpose: To describe a case of iris metastasis as the initial presentation of clear cell renal cell carcinoma, and to discuss molecular profiling of both the metastasis and primary kidney tumor. Observations: We report a patient with blurred vision who underwent ophthalmic examination and was found to have an iris mass, which was excised and diagnosed as a metastatic clear cell renal cell carcinoma by morphology and immunohistochemical analysis. As a result of the pathology findings, computed tomography imaging was performed, revealing a right kidney mass, which was also resected and shown to represent a high-grade carcinoma confined within the renal fascia without lymphovascular invasion. Molecular testing of the primary and metastatic tumors using a custom next-generation sequencing panel revealed similar mutational profiles but disclosed a TERT promoter mutation in the primary neoplasm, not present in the metastasis, suggesting seeding of an early lower grade neoplastic cell clone within the iris. Conclusions and importance: This report illustrates how pathological examination of a small iris lesion led to the discovery of a previously unknown systemic malignancy at a resectable stage. Molecular genetic profiling revealed that even lower grade clones within a high-grade neoplasm have metastatic potential.

Excitotoxicity, calcium and mitochondria: a triad in synaptic neurodegeneration.

Glutamate is the most commonly engaged neurotransmitter in the mammalian central nervous system, acting to mediate excitatory neurotransmission. However, high levels of glutamatergic input elicit excitotoxicity, contributing to neuronal cell death following acute brain injuries such as stroke and trauma. While excitotoxic cell death has also been implicated in some neurodegenerative disease models, the role of acute apoptotic cell death remains controversial in the setting of chronic neurodegeneration. Nevertheless, it is clear that excitatory synaptic dysregulation contributes to neurodegeneration, as evidenced by protective effects of partial N-methyl-D-aspartate receptor antagonists. Here, we review evidence for sublethal excitatory injuries in relation to neurodegeneration associated with Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and Huntington's disease. In contrast to classic excitotoxicity, emerging evidence implicates dysregulation of mitochondrial calcium handling in excitatory post-synaptic neurodegeneration. We discuss mechanisms that regulate mitochondrial calcium uptake and release, the impact of LRRK2, PINK1, Parkin, beta-amyloid and glucocerebrosidase on mitochondrial calcium transporters, and the role of autophagic mitochondrial loss in axodendritic shrinkage. Finally, we discuss strategies for normalizing the flux of calcium into and out of the mitochondrial matrix, thereby preventing mitochondrial calcium toxicity and excitotoxic dendritic loss. While the mechanisms that underlie increased uptake or decreased release of mitochondrial calcium vary in different model systems, a common set of strategies to normalize mitochondrial calcium flux can prevent excitatory mitochondrial toxicity and may be neuroprotective in multiple disease contexts.

Ocular Phenotype of Peters-Plus Syndrome.

PURPOSE: Peters-plus syndrome is a rare, autosomal recessive congenital disorder of glycosylation caused by mutations in the gene B3GLCT. A detailed description of the ocular findings is currently lacking in the scientific literature. We report a case series of Peters-plus syndrome with deep ocular phenotyping using anterior segment optical coherence tomography and ultrasound biomicroscopy. Where available, we describe the histology of host corneal buttons. METHODS: A retrospective chart review of patients with Peters-plus syndrome was conducted under the care of the senior author between January 2000 and June 2019. Demographic and clinical data including ocular and systemic features, ophthalmic imaging, and molecular diagnostic reports were collected. RESULTS: Four cases of Peters-plus syndrome were identified. Three patients were male and 1 was female. Five of the 8 eyes had an avascular paracentral ring opacity with relative central clearing. The paracentral opacity is due to iridocorneal adhesion and the relative central clearing associated with posterior stromal thinning. One eye had persistent fetal vasculature and microphthalmia, which has not previously been reported. One eye from each of 2 patients had a significantly different phenotype with a large vascularized central corneal opacity. CONCLUSIONS: The most common ocular phenotype seen in Peters-plus syndrome is an avascular paracentral ring opacity with relative central clearing. A different phenotype with a large vascularized corneal opacity may also be observed.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Neuronal autophagy and mitophagy in Parkinson's disease.

Autophagy is the process by which cells can selectively or non-selectively remove damaged proteins and organelles. As the cell's main means of sequestering damaged mitochondria for removal, mitophagy is central to cellular function and survival. Research on autophagy and mitochondrial quality control has increased exponentially in relation to the pathogenesis of numerous disease conditions, from cancer and immune diseases to chronic neurodegenerative diseases like Parkinson's disease (PD). Understanding how components of the autophagic/mitophagic machinery are affected during disease, as well as the contextual relationship of autophagy with determining neuronal health and function, is essential to the goal of designing therapies for human disease. In this review, we will summarize key signaling molecules that consign damaged mitochondria for autophagic degradation, describe the relationship of genes linked to PD to autophagy/mitophagy dysfunction, and discuss additional roles of both mitochondrial and cytosolic pools of PTEN-induced kinase 1 (PINK1) in mitochondrial homeostasis, dendritic morphogenesis and inflammation.

Transcriptome from opaque cornea of Fanconi anemia patient uncovers fibrosis and two connected players.

Congenital corneal opacities (CCO) are a group of blinding corneal disorders, where the underlying molecular mechanisms are poorly understood. Phenotyping through specialized imaging and histopathology analysis, together with assessment of key transcriptomic changes (including glycosaminoglycan metabolic enzymes) in cornea(s) with CCO from a case of Fanconi anemia is the approach taken in this study to identify causal mechanisms. Based on our findings, we propose a novel mechanism and two key players contributing to CCO.

Phospholipase iPLA2ß averts ferroptosis by eliminating a redox lipid death signal.

Ferroptosis, triggered by discoordination of iron, thiols and lipids, leads to the accumulation of 15-hydroperoxy (Hp)-arachidonoyl-phosphatidylethanolamine (15-HpETE-PE), generated by complexes of 15-lipoxygenase (15-LOX) and a scaffold protein, phosphatidylethanolamine (PE)-binding protein (PEBP)1. As the Ca2+-independent phospholipase A2ß (iPLA2ß, PLA2G6 or PNPLA9 gene) can preferentially hydrolyze peroxidized phospholipids, it may eliminate the ferroptotic 15-HpETE-PE death signal. Here, we demonstrate that by hydrolyzing 15-HpETE-PE, iPLA2ß averts ferroptosis, whereas its genetic or pharmacological inactivation sensitizes cells to ferroptosis. Given that PLA2G6 mutations relate to neurodegeneration, we examined fibroblasts from a patient with a Parkinson's disease (PD)-associated mutation (fPDR747W) and found selectively decreased 15-HpETE-PE-hydrolyzing activity, 15-HpETE-PE accumulation and elevated sensitivity to ferroptosis. CRISPR-Cas9-engineered Pnpla9R748W/R748W mice exhibited progressive parkinsonian motor deficits and 15-HpETE-PE accumulation. Elevated 15-HpETE-PE levels were also detected in midbrains of rotenone-infused parkinsonian rats and α-synuclein-mutant SncaA53T mice, with decreased iPLA2ß expression and a PD-relevant phenotype. Thus, iPLA2ß is a new ferroptosis regulator, and its mutations may be implicated in PD pathogenesis.

Sympathetic ophthalmia presenting 5 days after penetrating injury.

PURPOSE: To describe a rare case of early sympathetic ophthalmia that presented 5 days after penetrating injury. OBSERVATIONS: A 13-year-old boy presented with a penetrating left globe injury from a BB metallic projectile that was emergently repaired. Five days later, routine dilated exam of the right eye revealed interval development of vitritis over the posterior pole. Optical coherence tomography revealed fine, vitreous hyper-reflective material. Intravenous and topical steroid therapy was started, and the patient underwent prompt enucleation of the traumatized eye. Histopathologic examination of the globe demonstrated lymphocytic choroiditis and macrophage infiltration, consistent with prior reports of early sympathetic ophthalmia. The sympathizing eye maintained 20/20 acuity and never caused visual complaints. CONCLUSIONS AND IMPORTANCE: This is the earliest reported case of sympathetic ophthalmia, to our knowledge, and it presented without visual symptoms only five days after penetrating trauma. This case suggests that routine examination should start before the typical 14 days associated with development of sympathetic ophthalmia.

Chemical inhibition of FBXO7 reduces inflammation and confers neuroprotection by stabilizing the mitochondrial kinase PINK1.

Mitochondrial quality control is mediated by the PTEN-induced kinase 1 (PINK1), a cytoprotective protein that is dysregulated in inflammatory lung injury and neurodegenerative diseases. Here, we show that a ubiquitin E3 ligase receptor component, FBXO7, targets PINK1 for its cellular disposal. FBXO7, by mediating PINK1 ubiquitylation and degradation, was sufficient to induce mitochondrial injury and inflammation in experimental pneumonia. A computational simulation-based screen led to the identification of a small molecule, BC1464, which abrogated FBXO7 and PINK1 association, leading to increased cellular PINK1 concentrations and activities, and limiting mitochondrial damage. BC1464 exerted antiinflammatory activity in human tissue explants and murine lung inflammation models. Furthermore, BC1464 conferred neuroprotection in primary cortical neurons, human neuroblastoma cells, and patient-derived cells in several culture models of Parkinson's disease. The data highlight a unique opportunity to use small molecule antagonists that disrupt PINK1 interaction with the ubiquitin apparatus to enhance mitochondrial quality, limit inflammatory injury, and maintain neuronal viability.

Chronic treatment with the complex I inhibitor MPP+ depletes endogenous PTEN-induced kinase 1 (PINK1) via up-regulation of Bcl-2-associated athanogene 6 (BAG6).

Mitochondrial dysfunction is implicated in sporadic and familial Parkinson's disease (PD). However, the mechanisms that impair homeostatic responses to mitochondrial dysfunction remain unclear. Previously, we found that chronic, low-dose administration of the mitochondrial complex I inhibitor 1-methyl-4-phenylpyridinium (MPP+) dysregulates mitochondrial fission-fusion, mitophagy, and mitochondrial biogenesis. Given that PTEN-induced kinase 1 (PINK1) regulates mitochondrial function, dynamics, and turnover, we hypothesized that alterations in endogenous PINK1 levels contribute to depletion of mitochondria during chronic complex I injury. Here we found that chronic MPP+ treatment of differentiated SH-SY5Y neuronal cells significantly decreases PINK1 expression prior to reductions in other mitochondrial components. Furthermore, Bcl2-associated athanogene 6 (BAG6, BAT3, or Scythe), a protein involved in protein quality control and degradation, was highly up-regulated during the chronic MPP+ treatment. BAG6 interacted with PINK1, and BAG6 overexpression decreased the half-life of PINK1. Conversely, siRNA-mediated BAG6 knockdown prevented chronic MPP+ stress-induced loss of PINK1, reversed MPP+-provoked mitochondrial changes, increased cell viability, and prevented MPP+-induced dendrite shrinkage in primary neurons. These results indicate that BAG6 up-regulation during chronic complex I inhibition contributes to mitochondrial pathology by decreasing the levels of endogenous PINK1. Given that recessive mutations in PINK1 cause familial PD, the finding of accelerated PINK1 degradation in the chronic MPP+ model suggests that PINK1 loss of function represents a point of convergence between the neurotoxic and genetic causes of PD.

Propofol affects mouse embryonic fibroblast survival and proliferation in vitro via ATG5- and calcium-dependent regulation of autophagy.

Propofol is a commonly used intravenous anesthetic agent, which has been found to affect cell survival and proliferation especially in early life. Our previous studies show that propofol-induced neurodegeneration and neurogenesis are closely associated with cell autophagy. In the present study we explored the roles of autophagy-related gene 5 (ATG5) in propofol-induced autophagy in mouse embryonic fibroblasts (MEF) in vitro. We showed that ATG5 was functionally related to propofol-induced cell survival and damage: propofol significantly enhanced cell survival and proliferation at a clinically relevant dose (10 µM), but caused cell death at an extremely high concentration (200 µM) in ATG5-/- MEF, but not in WT cells. The dual effects found in ATG5-/- MEF could be blocked by intracellular Ca2+ channel antagonists. We also found that propofol evoked a moderate (promote cell growth) and extremely high (cause apoptosis) cytosolic Ca2+ elevation at the concentrations of 10 µM and 200 µM, respectively, only in ATG5-/- MEF. In addition, ATG5-/- MEF themselves released more Ca2+ in cytosolic space and endoplasmic reticulum compared with WT cells, suggesting that autophagy deficiency made intracellular calcium signaling more vulnerable to external stimuli (propofol). Altogether, our results reveal that ATG5 plays a crucial role in propofol regulation of cell survival and proliferation by affecting intracellular Ca2+ homeostasis.

Alzheimer's Disease Presenilin-1 Mutation Sensitizes Neurons to Impaired Autophagy Flux and Propofol Neurotoxicity: Role of Calcium Dysregulation.

BACKGROUND: Disruption of intracellular Ca2+ homeostasis and associated autophagy dysfunction contribute to neuropathology in Alzheimer's disease (AD). OBJECTIVE: To study the effects of propofol on cell viability via its effects on intracellular Ca2+ homeostasis, and the impact of autophagy, in a neuronal model of presenilin-mutated familial AD (FAD). METHODS: We treated PC12 cells, stably transfected with either mutated presenilin-1 (L286V) or wild type (WT) controls, with propofol at different doses and durations, in the presence or absence of extracellular Ca2+, antagonists of inositol trisphosphate receptors (InsP3R, xestospongin C) and/or ryanodine receptors (RYR, dantrolene), or an inhibitor of autophagy flux (Bafilomycin). We determined cell viability, cytosolic Ca2+ concentrations ([Ca2+]c), vATPase protein expression, and lysosomal acidification. RESULTS: The propofol dose- and time-dependently decreased cell viability significantly more in L286V than WT cells, especially at the pharmacological dose (>50µM), and together with bafilomycin (40 nM). Clinically used concentrations of propofol (<20µM) tended to increase cell viability. Propofol significantly increased [Ca2+]c more in L286V than in WT cells, which was associated with decrease of vATPase expression and localization to the lysosome. Both toxicity and increased Ca2+ levels were ameliorated by inhibiting InsP3R/RYR. However, the combined inhibition of both receptors paradoxically increased [Ca2+]c, by inducing Ca2+ influx from the extracellular space, causing greater cytotoxicity. CONCLUSION: Impairment in autophagy function acts to deteriorate cell death induced by propofol in FAD neuronal cells. Cell death is ameliorated by either RYR or InsP3R antagonists on their own, but not when both are co-administered.