DDX58 variant triggers IFN-ß-induced autophagy in trabecular meshwork and influences intraocular pressure.

Singleton-Merten syndrome (SMS) is a rare immunogenetic disorder affecting multiple systems, characterized by dental dysplasia, aortic calcification, glaucoma, skeletal abnormalities, and psoriasis. Glaucoma, a key feature of both classical and atypical SMS, remains poorly understood in terms of its molecular mechanism caused by DDX58 mutation. This study presented a novel DDX58 variant (c.1649A>C [p.Asp550Ala]) in a family with childhood glaucoma. Functional analysis showed that DDX58 variant caused an increase in IFN-stimulated gene expression and high IFN-ß-based type-I IFN. As the trabecular meshwork (TM) is responsible for controlling intraocular pressure (IOP), we examine the effect of IFN-ß on TM cells. Our study is the first to demonstrate that IFN-ß significantly reduced TM cell viability and function by activating autophagy. In addition, anterior chamber injection of IFN-ß remarkably increased IOP level in mice, which can be attenuated by treatments with autophagy inhibitor chloroquine. To uncover the specific mechanism underlying IFN-ß-induced autophagy in TM cells, we performed microarray analysis in IFN-ß-treated and DDX58 p.Asp550Ala TM cells. It showed that RSAD2 is necessary for IFN-ß-induced autophagy. Knockdown of RSAD2 by siRNA significantly decreased autophagy flux induced by IFN-ß. Our findings suggest that DDX58 mutation leads to the overproduction of IFN-ß, which elevates IOP by modulating autophagy through RSAD2 in TM cells.

NR2F1 shapes mitochondria in the mouse brain, providing new insights into Bosch-Boonstra-Schaaf optic atrophy syndrome.

The nuclear receptor NR2F1 acts as a strong transcriptional regulator in embryonic and postnatal neural cells. In humans, mutations in the NR2F1 gene cause Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS), a rare neurodevelopmental disorder characterized by multiple clinical features including vision impairment, intellectual disability and autistic traits. In this study, we identified, by genome-wide and in silico analyses, a set of nuclear-encoded mitochondrial genes as potential genomic targets under direct NR2F1 transcriptional control in neurons. By combining mouse genetic, neuroanatomical and imaging approaches, we demonstrated that conditional NR2F1 loss of function within the adult mouse hippocampal neurogenic niche results in a reduced mitochondrial mass associated with mitochondrial fragmentation and downregulation of key mitochondrial proteins in newborn neurons, the genesis, survival and functional integration of which are impaired. Importantly, we also found dysregulation of several nuclear-encoded mitochondrial genes and downregulation of key mitochondrial proteins in the brain of Nr2f1-heterozygous mice, a validated BBSOAS model. Our data point to an active role for NR2F1 in the mitochondrial gene expression regulatory network in neurons and support the involvement of mitochondrial dysfunction in BBSOAS pathogenesis.

Hydrogen sulfide alleviates mitochondrial damage and ferroptosis by regulating OPA3-NFS1 axis in doxorubicin-induced cardiotoxicity.

Ferroptosis is a major cause of cardiotoxicity induced by doxorubicin (DOX). Previous studies have shown that hydrogen sulfide (H2S) inhibits ferroptosis in cardiomyocytes and myoblasts, but the underlying mechanism has not been fully elucidated. In this study, we investigated the role of H2S in protecting against DOX-induced cardiotoxicity both in vivo and in vitro, and elucidated the potential mechanisms involved. We found that DOX downregulated the expression of glutathione peroxidase 4 (GPX4) and NFS1, and upregulated the expression of acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) expression level, resulting in increased lipid peroxidation and ferroptosis. Additionally, DOX inhibited MFN2 expression and increased DRP1 and FIS1 expression, leading to abnormal mitochondrial structure and function. In contrast, exogenous H2S inhibited DOX-induced ferroptosis by restoring GPX4 and NFS1 expression, and reducing lipid peroxidation in H9C2 cells. This effect was similar to that of the ferroptosis antagonist ferrostatin-1 (Fer-1) in protecting against DOX-induced cardiotoxicity. We further demonstrated that the protective effect of H2S was mediated by the key mitochondrial membrane protein optic atrophy 3 (OPA3), which was downregulated by DOX and restored by exogenous H2S. Overexpression of OPA3 alleviated DOX-induced mitochondrial dysfunction and ferroptosis both in vivo and in vitro. Mechanistically, NFS1 has an inhibitory effect on ferroptosis, and NFS1 deficiency increases the susceptibility of cardiomyocytes to ferroptosis. OPA3 is involved in the regulation of ferroptosis by interacting with NFS1. Post-translationally, DOX promoted OPA3 ubiquitination, while exogenous H2S antagonized OPA3 ubiquitination by promoting OPA3 s-sulfhydration. In summary, our findings suggested that H2S protects against DOX-induced cardiotoxicity by inhibiting ferroptosis via targeting the OPA3-NFS1 axis. This provides a potential therapeutic strategy for the treatment of DOX-induced cardiotoxicity.

Does Disruption of Optic Atrophy-1 (OPA1) Contribute to Cell Death in HL-1 Cardiomyocytes Subjected to Lethal Ischemia-Reperfusion Injury?

Disruption of mitochondrial structure/function is well-recognized to be a determinant of cell death in cardiomyocytes subjected to lethal episodes of ischemia-reperfusion (IR). However, the precise mitochondrial event(s) that precipitate lethal IR injury remain incompletely resolved. Using the in vitro HL-1 cardiomyocyte model, our aims were to establish whether: (1) proteolytic processing of optic atrophy protein-1 (OPA1), the inner mitochondrial membrane protein responsible for maintaining cristae junction integrity, plays a causal, mechanistic role in determining cardiomyocyte fate in cells subjected to lethal IR injury; and (2) preservation of OPA1 may contribute to the well-documented cardioprotection achieved with ischemic preconditioning (IPC) and remote ischemic conditioning. We report that HL-1 cells subjected to 2.5 h of simulated ischemia displayed increased activity of OMA1 (the metalloprotease responsible for proteolytic processing of OPA1) during the initial 45 min following reoxygenation. This was accompanied by processing of mitochondrial OPA1 (i.e., cleavage to yield short-OPA1 peptides) and release of short-OPA1 into the cytosol. However, siRNA-mediated knockdown of OPA1 content did not exacerbate lethal IR injury, and did not attenuate the cardioprotection seen with IPC and a remote preconditioning stimulus, achieved by transfer of 'reperfusate' medium (TRM-IPC) in this cell culture model. Taken together, our results do not support the concept that maintenance of OPA1 integrity plays a mechanistic role in determining cell fate in the HL-1 cardiomyocyte model of lethal IR injury, or that preservation of OPA1 underlies the cardioprotection seen with ischemic conditioning.

A Glucagon-like Peptide 1 Analog Protects Mitochondria and Attenuates Hypoxia-Reoxygenation Injury in Cultured Cardiomyocytes.

ABSTRACT: Glucagon-like peptide 1 (GLP-1) analogs improve glycemic control in diabetes and protect the heart against ischemia-reperfusion injury. However, the mechanisms underlying this protection remain unclear. Mitochondria are essential for myocyte homeostasis. Therefore, we herein examined the effects of a GLP-1 analog on mitochondria after the hypoxia-reoxygenation of rat neonatal cultured cardiomyocytes. Cardiomyocytes were subjected to hypoxia for 5 hours followed by reoxygenation for 30 minutes in the presence or absence of exendin-4 (50 nmol/L), a GLP-1 analog. Hypoxia-reoxygenation increased lactate dehydrogenase and caspase-3 activities, indicators of lethal myocyte injury and apoptosis, respectively, and exendin-4 attenuated these increases. The content of ATP in myocytes decreased after hypoxia-reoxygenation but was preserved by exendin-4. The membrane potential and shape of mitochondria were assessed using a fluorescent probe. Exendin-4 attenuated the hypoxia-reoxygenation-induced disruption of the mitochondrial membrane potential and shortening. Mitochondrial quality control-related factors, such as optic atrophy protein 1, mitofusin 2, dynamin-related protein 1, and parkin, were examined by Western blotting. Exendin-4 significantly increased the expression of the fusion proteins, optic atrophy protein 1 and mitofusin 2, and decreased that of the mitophagy-related protein, parkin, without altering dynamin-related protein 1 expression levels. Exendin-4 also preserved Akt phosphorylation levels after hypoxia-reoxygenation, whereas wortmannin, an inhibitor of the phosphoinositide 3-kinase-Akt pathway, blunted exendin-4-induced myocyte protection and its effects on mitochondrial quality control factors. In conclusion, exendin-4 protected mitochondria by preserving the phosphorylation of Akt and fusion proteins, leading to the attenuation of hypoxia-reoxygenation-induced injury in cultured myocytes.

Neuroglobin effectively halts vision loss in Harlequin mice at an advanced stage of optic nerve degeneration.

Mitochondrial diseases are among the most prevalent groups of inherited neurological disorders, affecting up to 1 in 5000 adults. Despite the progress achieved on the identification of gene mutations causing mitochondrial pathologies, they cannot be cured so far. Harlequin mice, a relevant model of mitochondrial pathology due to apoptosis inducing factor depletion, suffer from progressive disappearance of retinal ganglion cells leading to optic neuropathy. In our previous work, we showed that administering adeno-associated virus encompassing the coding sequences for neuroglobin, (a neuroprotective molecule belonging to the globin family) or apoptosis-inducing factor, before neurodegeneration onset, prevented retinal ganglion cell loss and preserved visual function. One of the challenges to develop an effective treatment for optic neuropathies is to consider that by the time patients become aware of their handicap, a large amount of nerve fibers has already disappeared. Gene therapy was performed in Harlequin mice aged between 4 and 5 months with either a neuroglobin or an apoptosis-inducing factor vector to determine whether the increased abundance of either one of these proteins in retinas could preserve visual function at this advanced stage of the disease. We demonstrated that gene therapy, by preserving the connectivity of transduced retinal ganglion cells and optic nerve bioenergetics, results in the enhancement of visual cortex activity, ultimately rescuing visual impairment. This study demonstrates that: (a) An increased abundance of neuroglobin functionally overcomes apoptosis-inducing factor absence in Harlequin mouse retinas at a late stage of neuronal degeneration; (b) The beneficial effect for visual function could be mediated by neuroglobin localization to the mitochondria, thus contributing to the maintenance of the organelle homeostasis.

BAP31: Physiological functions and roles in disease.

B-cell receptor-associated protein 31 (BAP31 or BCAP31) is a ubiquitously expressed transmembrane protein found mainly in the endoplasmic reticulum (ER), including in mitochondria-associated membranes (MAMs). It acts as a broad-specificity membrane protein chaperone and quality control factor, which can promote different fates for its clients, including ER retention, ER export, ER-associated degradation (ERAD), or evasion of degradation, and it also acts as a MAM tetherer and regulatory protein. It is involved in several cellular processes - it supports ER and mitochondrial homeostasis, promotes proliferation and migration, plays several roles in metabolism and the immune system, and regulates autophagy and apoptosis. Full-length BAP31 can be anti-apoptotic, but can also mediate activation of caspase-8, and itself be cleaved by caspase-8 into p20-BAP31, which promotes apoptosis by mobilizing ER calcium stores at MAMs. BAP31 loss-of-function mutations is the cause of 'deafness, dystonia, and central hypomyelination' (DDCH) syndrome, characterized by severe neurological symptoms and early death. BAP31 is furthermore implicated in a growing number of cancers and other diseases, and several viruses have been found to target it to promote their survival or life cycle progression. The purpose of this review is to provide an overview and examination of the basic properties, functions, mechanisms, and roles in disease of BAP31.

NDUFS3 depletion permits complex I maturation and reveals TMEM126A/OPA7 as an assembly factor binding the ND4-module intermediate.

Complex I (CI) is the largest enzyme of the mitochondrial respiratory chain, and its defects are the main cause of mitochondrial disease. To understand the mechanisms regulating the extremely intricate biogenesis of this fundamental bioenergetic machine, we analyze the structural and functional consequences of the ablation of NDUFS3, a non-catalytic core subunit. We show that, in diverse mammalian cell types, a small amount of functional CI can still be detected in the complete absence of NDUFS3. In addition, we determine the dynamics of CI disassembly when the amount of NDUFS3 is gradually decreased. The process of degradation of the complex occurs in a hierarchical and modular fashion in which the ND4 module remains stable and bound to TMEM126A. We, thus, uncover the function of TMEM126A, the product of a disease gene causing recessive optic atrophy as a factor necessary for the correct assembly and function of CI.

Optic atrophy-associated TMEM126A is an assembly factor for the ND4-module of mitochondrial complex I.

Mitochondrial disease is a debilitating condition with a diverse genetic etiology. Here, we report that TMEM126A, a protein that is mutated in patients with autosomal-recessive optic atrophy, participates directly in the assembly of mitochondrial complex I. Using a combination of genome editing, interaction studies, and quantitative proteomics, we find that loss of TMEM126A results in an isolated complex I deficiency and that TMEM126A interacts with a number of complex I subunits and assembly factors. Pulse-labeling interaction studies reveal that TMEM126A associates with the newly synthesized mitochondrial DNA (mtDNA)-encoded ND4 subunit of complex I. Our findings indicate that TMEM126A is involved in the assembly of the ND4 distal membrane module of complex I. In addition, we find that the function of TMEM126A is distinct from its paralogue TMEM126B, which acts in assembly of the ND2-module of complex I.

The Warburg Micro Syndrome-associated Rab3GAP-Rab18 module promotes autolysosome maturation through the Vps34 Complex I.

Warburg micro syndrome (WMS) is a hereditary autosomal neuromuscular disorder in humans caused by mutations in Rab18, Rab3GAP1, or Rab3GAP2 genes. Rab3GAP1/2 forms a heterodimeric complex, which acts as a guanosine nucleotide exchange factor and activates Rab18. Although the genetic causes of WMS are known, it is still unclear whether loss of the Rab3GAP-Rab18 module affects neuronal or muscle cell physiology or both, and how. In this work, we characterize a Rab3GAP2 mutant Drosophila line to establish a novel animal model for WMS. Similarly to symptoms of WMS, loss of Rab3GAP2 leads to highly decreased motility in Drosophila that becomes more serious with age. We demonstrate that these mutant flies are defective for autophagic degradation in multiple tissues including fat cells and muscles. Loss of Rab3GAP-Rab18 module members leads to perturbed autolysosome morphology due to destabilization of Rab7-positive autophagosomal and late endosomal compartments and perturbation of lysosomal biosynthetic transport. Importantly, overexpression of UVRAG or loss of Atg14, two alternative subunits of the Vps34/PI3K (vacuole protein sorting 34/phosphatidylinositol 3-kinase) complexes in fat cells, mimics the autophagic phenotype of Rab3GAP-Rab18 module loss. We find that GTP-bound Rab18 binds to Atg6/Beclin1, a permanent subunit of Vps34 complexes. Finally, we show that Rab3GAP2 and Rab18 are present on autophagosomal and autolysosomal membranes and colocalize with Vps34 Complex I subunits. Our data suggest that the Rab3GAP-Rab18 module regulates autolysosomal maturation through its interaction with the Vps34 Complex I, and perturbed autophagy due to loss of the Rab3GAP-Rab18 module may contribute to the development of WMS.

Haploinsufficiency due to a novel ACO2 deletion causes mitochondrial dysfunction in fibroblasts from a patient with dominant optic nerve atrophy.

ACO2 is a mitochondrial protein, which is critically involved in the function of the tricarboxylic acid cycle (TCA), the maintenance of iron homeostasis, oxidative stress defense and the integrity of mitochondrial DNA (mtDNA). Mutations in the ACO2 gene were identified in patients suffering from a broad range of symptoms, including optic nerve atrophy, cortical atrophy, cerebellar atrophy, hypotonia, seizures and intellectual disabilities. In the present study, we identified a heterozygous 51 bp deletion (c.1699_1749del51) in ACO2 in a family with autosomal dominant inherited isolated optic atrophy. A complementation assay using aco1-deficient yeast revealed a growth defect for the mutant ACO2 variant substantiating a pathogenic effect of the deletion. We used patient-derived fibroblasts to characterize cellular phenotypes and found a decrease of ACO2 protein levels, while ACO2 enzyme activity was not affected compared to two age- and gender-matched control lines. Several parameters of mitochondrial function, including mitochondrial morphology, mitochondrial membrane potential or mitochondrial superoxide production, were not changed under baseline conditions. However, basal respiration, maximal respiration, and spare respiratory capacity were reduced in mutant cells. Furthermore, we observed a reduction of mtDNA copy number and reduced mtDNA transcription levels in ACO2-mutant fibroblasts. Inducing oxidative stress led to an increased susceptibility for cell death in ACO2-mutant fibroblasts compared to controls. Our study reveals that a monoallelic mutation in ACO2 is sufficient to promote mitochondrial dysfunction and increased vulnerability to oxidative stress as main drivers of cell death related to optic nerve atrophy.

Cardiac phenotype in ATP1A3-related syndromes: A multicenter cohort study.

OBJECTIVE: To define the risks and consequences of cardiac abnormalities in ATP1A3-related syndromes. METHODS: Patients meeting clinical diagnostic criteria for rapid-onset dystonia-parkinsonism (RDP), alternating hemiplegia of childhood (AHC), and cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) with ATP1A3 genetic analysis and at least 1 cardiac assessment were included. We evaluated the cardiac phenotype in an Atp1a3 knock-in mouse (Mashl+/-) to determine the sequence of events in seizure-related cardiac death. RESULTS: Ninety-eight patients with AHC, 9 with RDP, and 3 with CAPOS (63 female, mean age 17 years) were included. Resting ECG abnormalities were found in 52 of 87 (60%) with AHC, 2 of 3 (67%) with CAPOS, and 6 of 9 (67%) with RDP. Serial ECGs showed dynamic changes in 10 of 18 patients with AHC. The first Holter ECG was abnormal in 24 of 65 (37%) cases with AHC and RDP with either repolarization or conduction abnormalities. Echocardiography was normal. Cardiac intervention was required in 3 of 98 (≈3%) patients with AHC. In the mouse model, resting ECGs showed intracardiac conduction delay; during induced seizures, heart block or complete sinus arrest led to death. CONCLUSIONS: We found increased prevalence of ECG dynamic abnormalities in all ATP1A3-related syndromes, with a risk of life-threatening cardiac rhythm abnormalities equivalent to that in established cardiac channelopathies (≈3%). Sudden cardiac death due to conduction abnormality emerged as a seizure-related outcome in murine Atp1a3-related disease. ATP1A3-related syndromes are cardiac diseases and neurologic diseases. We provide guidance to identify patients potentially at higher risk of sudden cardiac death who may benefit from insertion of a pacemaker or implantable cardioverter-defibrillator.

Acteoside inhibits autophagic apoptosis of retinal ganglion cells to rescue glaucoma-induced optic atrophy.

BACKGROUND: Glaucoma is the world's second biggest cause of blindness, and patients progressively lose their eyesight. The current clinical treatment for glaucoma involves controlling intraocular pressure with drugs or surgery; however, some patients still progressively lose their eyesight. This treatment is also similar to the treatment of traumatic optic neuropathy. Thus, saving retinal ganglion cells (RGCs) from apoptosis is essential. METHODS: The role of Acteoside on autophagy modulation in the 661 W cell line. RESULTS: In this study, we first find that Acteoside inhibits autophagy, Rapamycin alleviates this inhibition and the PI3K inhibitor, 3-MA or LY294002, synergistically promotes it. In a mechanistic study, we find that Optineurin (OPTN) mediates Acteoside regulation of autophagy. OPTN overexpression or knockdown activates or inhibits autophagy, respectively. OPTN is inhibited by autophagy inhibitors, such as Acteoside and 3-MA and is promoted by the autophagy activator, Rapamycin. Meanwhile, PI3K and AKT are elevated by Acteoside and 3-MA and inhibited by Rapamycin. Finally, we find that Acteoside inhibits apoptosis in parallel to autophagy and that this inhibition is also mediated by OPTN. CONCLUSION: In summary, we conclude that Acteoside inhibits autophagy-induced apoptosis in RGCs through the OPTN and PI3K/AKT/mTOR pathway, and glaucoma patients may benefit from Acteoside treatment alone or in combination with other autophagy inhibitors.

Rab18: new insights into the function of an essential protein.

Rab18 is one of the small number of conserved Rab proteins which have been traced to the last eukaryotic common ancestor. It is found in organisms ranging from humans to trypanosomes, and localizes to multiple organelles, including most notably endoplasmic reticulum and lipid droplets. In humans, absence of Rab18 leads to a severe illness known as Warburg-Micro syndrome. Despite this evidence that Rab18 is essential, its role in cells remains mysterious. However, recent studies identifying effectors and interactors of Rab18, are now shedding light on its mechanism of action, suggesting functions related to organelle tethering and to autophagy. In this review, we examine the variety of roles proposed for Rab18 with a focus on new evidence giving insights into the molecular mechanisms it utilizes. Based on this summary of our current understanding, we identify priority areas for further research.

Rab18 Collaborates with Rab7 to Modulate Lysosomal and Autophagy Activities in the Nervous System: an Overlapping Mechanism for Warburg Micro Syndrome and Charcot-Marie-Tooth Neuropathy Type 2B.

Mutations in RAB18, a member of small G protein, cause Warburg micro syndrome (WARBM), whose clinical features include vision impairment, postnatal microcephaly, and lower limb spasticity. Previously, our Rab18-/- mice exhibited hind limb weakness and spasticity as well as signs of axonal degeneration in the spinal cord and lumbar spinal nerves. However, the cellular and molecular function of RAB18 and its roles in the pathogenesis of WARBM are still not fully understood. Using immunofluorescence staining and expression of Rab18 and organelle markers, we find that Rab18 associates with lysosomes and actively traffics along neurites in cultured neurons. Interestingly, Rab18-/- neurons exhibit impaired lysosomal transport. Using autophagosome marker LC3-II, we show that Rab18 dysfunction leads to aberrant autophagy activities in neurons. Electron microscopy further reveals accumulation of lipofuscin-like granules in the dorsal root ganglion of Rab18-/- mice. Surprisingly, Rab18 colocalizes, cofractionates, and coprecipitates with the lysosomal regulator Rab7, mutations of which cause Charcot-Marie-Tooth (CMT) neuropathy type 2B. Moreover, Rab7 is upregulated in Rab18-deficient neurons, suggesting a compensatory effect. Together, our results suggest that the functions of RAB18 and RAB7 in lysosomal and autophagic activities may constitute an overlapping mechanism underlying WARBM and CMT pathogenesis in the nervous system.

Revealing Clusters of Connected Pathways Through Multisource Data Integration in Huntington's Disease and Spastic Ataxia.

The advancement of scientific and medical research over the past years has generated a wealth of experimental data from multiple technologies, including genomics, transcriptomics, proteomics, and other forms of -omics data, which are available for a number of diseases. The integration of such multisource data is a key component toward the success of precision medicine. In this paper, we are investigating a multisource data integration method developed by our group, regarding its ability to drive to clusters of connected pathways under two different approaches: first, a disease-centric approach, where we integrate data around a disease, and second, a gene-centric approach, where we integrate data around a gene. We have used as a paradigm for the first approach Huntington's disease (HD), a disease with a plethora of available data, whereas for the second approach the GBA2, a gene that is related to spastic ataxia (SA), a phenotype with sparse availability of data. Our paper shows that valuable information at the level of disease-related pathway clusters can be obtained for both HD and SA. New pathways that classical pathway analysis methods were unable to reveal, emerged as necessary "connectors" to build connected pathway stories formed as pathway clusters. The capability to integrate multisource molecular data, concluding to something more than the sum of the existing information, empowers precision and personalized medicine approaches.

Functional consequences of the CAPOS mutation E818K of Na+,K+-ATPase.

The cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) syndrome is caused by the single mutation E818K of the α3-isoform of Na+,K+-ATPase. Here, using biochemical and electrophysiological approaches, we examined the functional characteristics of E818K, as well as of E818Q and E818A mutants. We found that these amino acid substitutions reduce the apparent Na+ affinity at the cytoplasmic-facing sites of the pump protein and that this effect is more pronounced for the lysine and glutamine substitutions (3-4-fold) than for the alanine substitution. The electrophysiological measurements indicated a more conspicuous, ∼30-fold reduction of apparent Na+ affinity for the extracellular-facing sites in the CAPOS mutant, which was related to an accelerated transition between the phosphoenzyme intermediates E1P and E2P. The apparent affinity for K+ activation of the ATPase activity was unaffected by these substitutions, suggesting that primarily the Na+-specific site III is affected. Furthermore, the apparent affinities for ATP and vanadate were WT-like in E818K, indicating a normal E1-E2 equilibrium of the dephosphoenzyme. Proton-leak currents were not increased in E818K. However, the CAPOS mutation caused a weaker voltage dependence of the pumping rate and a stronger inhibition by cytoplasmic K+ than the WT enzyme, which together with the reduced Na+ affinity of the cytoplasmic-facing sites precluded proper pump activation under physiological conditions. The functional deficiencies could be traced to the participation of Glu-818 in an intricate hydrogen-bonding/salt-bridge network, connecting it to key residues involved in Na+ interaction at site III.

Spatiotemporal Expression Changes of PACAP and Its Receptors in Retinal Ganglion Cells After Optic Nerve Crush.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been demonstrated to play a crucial part in protecting retinal ganglion cells (RGCs) from apoptosis in various retinal injury animal models. PACAP has two basic groups of receptors: PACAP receptor type 1 (PAC1R) and vasoactive intestinal polypeptide/PACAP receptors (VPAC1R and VPAC2R). However, few studies illustrated the spatial and temporal expression changes of endogenous PACAP and its receptors in a rodent optic nerve crush (ONC) model. In this study, a significant upregulation of PACAP and PAC1R in the retina after ONC was observed in both protein and RNA levels. The peak level of PACAP and PAC1R expression could be found on the fifth day following ONC. In addition, immunofluorescent labeling indicated that PACAP and PAC1R were localized mainly in RGCs. On the contrary, VPAC1R and VPAC2R were hardly detected in the retina. Collectively, the spatiotemporal expression of PACAP and its high-affinity receptor PAC1R were remarkably changed after ONC, and mainly expressed in the ganglion cell layer of the retina. This suggested that the upregulation of PACAP and PAC1R may play a vital role in RGC death after ONC.

Biochemical Characterization of the GBA2 c.1780G>C Missense Mutation in Lymphoblastoid Cells from Patients with Spastic Ataxia.

The GBA2 gene encodes the non-lysosomal glucosylceramidase (NLGase), an enzyme that catalyzes the conversion of glucosylceramide (GlcCer) to ceramide and glucose. Mutations in GBA2 have been associated with the development of neurological disorders such as autosomal recessive cerebellar ataxia, hereditary spastic paraplegia, and Marinesco-Sjogren-Like Syndrome. Our group has previously identified the GBA2 c.1780G>C [p.Asp594His] missense mutation, in a Cypriot consanguineous family with spastic ataxia. In this study, we carried out a biochemical characterization of lymphoblastoid cell lines (LCLs) derived from three patients of this family. We found that the mutation strongly reduce NLGase activity both intracellularly and at the plasma membrane level. Additionally, we observed a two-fold increase of GlcCer content in LCLs derived from patients compared to controls, with the C16 lipid being the most abundant GlcCer species. Moreover, we showed that there is an apparent compensatory effect between NLGase and the lysosomal glucosylceramidase (GCase), since we found that the activity of GCase was three-fold higher in LCLs derived from patients compared to controls. We conclude that the c.1780G>C mutation results in NLGase loss of function with abolishment of the enzymatic activity and accumulation of GlcCer accompanied by a compensatory increase in GCase.