Reverting the mode of action of the mitochondrial FOF1-ATPase by Legionella pneumophila preserves its replication niche.

Legionella pneumophila, the causative agent of Legionnaires' disease, a severe pneumonia, injects via a type 4 secretion system (T4SS) more than 300 proteins into macrophages, its main host cell in humans. Certain of these proteins are implicated in reprogramming the metabolism of infected cells by reducing mitochondrial oxidative phosphorylation (OXPHOS) early after infection. Here. we show that despite reduced OXPHOS, the mitochondrial membrane potential (Δψm) is maintained during infection of primary human monocyte-derived macrophages (hMDMs). We reveal that L. pneumophila reverses the ATP-synthase activity of the mitochondrial FOF1-ATPase to ATP-hydrolase activity in a T4SS-dependent manner, which leads to a conservation of the Δψm, preserves mitochondrial polarization, and prevents macrophage cell death. Analyses of T4SS effectors known to target mitochondrial functions revealed that LpSpl is partially involved in conserving the Δψm, but not LncP and MitF. The inhibition of the L. pneumophila-induced 'reverse mode' of the FOF1-ATPase collapsed the Δψm and caused cell death in infected cells. Single-cell analyses suggested that bacterial replication occurs preferentially in hMDMs that conserved the Δψm and showed delayed cell death. This direct manipulation of the mode of activity of the FOF1-ATPase is a newly identified feature of L. pneumophila allowing to delay host cell death and thereby to preserve the bacterial replication niche during infection.

Loss of Soluble (Pro)renin Receptor Attenuates Angiotensin-II Induced Hypertension and Renal Injury.

[Figure: see text].

Impaired F1Fo-ATP-Synthase Dimerization Leads to the Induction of Cyclophilin D-Mediated Autophagy-Dependent Cell Death and Accelerated Aging.

Mitochondrial F1Fo-ATP-synthase dimers play a critical role in shaping and maintenance of mitochondrial ultrastructure. Previous studies have revealed that ablation of the F1Fo-ATP-synthase assembly factor PaATPE of the ascomycete Podospora anserina strongly affects cristae formation, increases hydrogen peroxide levels, impairs mitochondrial function and leads to premature cell death. In the present study, we investigated the underlying mechanistic basis. Compared to the wild type, we observed a slight increase in non-selective and a pronounced increase in mitophagy, the selective vacuolar degradation of mitochondria. This effect depends on the availability of functional cyclophilin D (PaCYPD), the regulator of the mitochondrial permeability transition pore (mPTP). Simultaneous deletion of PaAtpe and PaAtg1, encoding a key component of the autophagy machinery or of PaCypD, led to a reduction of mitophagy and a partial restoration of the wild-type specific lifespan. The same effect was observed in the PaAtpe deletion strain after inhibition of PaCYPD by its specific inhibitor, cyclosporin A. Overall, our data identify autophagy-dependent cell death (ADCD) as part of the cellular response to impaired F1Fo-ATP-synthase dimerization, and emphasize the crucial role of functional mitochondria in aging.

Bioenergetic consequences of FoF1-ATP synthase/ATPase deficiency in two life cycle stages of Trypanosoma brucei.

Mitochondrial ATP synthase is a reversible nanomotor synthesizing or hydrolyzing ATP depending on the potential across the membrane in which it is embedded. In the unicellular parasite Trypanosoma brucei, the direction of the complex depends on the life cycle stage of this digenetic parasite: in the midgut of the tsetse fly vector (procyclic form), the FoF1-ATP synthase generates ATP by oxidative phosphorylation, whereas in the mammalian bloodstream form, this complex hydrolyzes ATP and maintains mitochondrial membrane potential (ΔΨm). The trypanosome FoF1-ATP synthase contains numerous lineage-specific subunits whose roles remain unknown. Here, we seek to elucidate the function of the lineage-specific protein Tb1, the largest membrane-bound subunit. In procyclic form cells, Tb1 silencing resulted in a decrease of FoF1-ATP synthase monomers and dimers, rerouting of mitochondrial electron transfer to the alternative oxidase, reduced growth rate and cellular ATP levels, and elevated ΔΨm and total cellular reactive oxygen species levels. In bloodstream form parasites, RNAi silencing of Tb1 by ∼90% resulted in decreased FoF1-ATPase monomers and dimers, but it had no apparent effect on growth. The same findings were obtained by silencing of the oligomycin sensitivity-conferring protein, a conserved subunit in T. brucei FoF1-ATP synthase. However, as expected, nearly complete Tb1 or oligomycin sensitivity-conferring protein suppression was lethal because of the inability to sustain ΔΨm. The diminishment of FoF1-ATPase complexes was further accompanied by a decreased ADP/ATP ratio and reduced oxygen consumption via the alternative oxidase. Our data illuminate the often diametrically opposed bioenergetic consequences of FoF1-ATP synthase loss in insect versus mammalian forms of the parasite.

ATP synthase deficiency due to m.8528T>C mutation - a novel cause of severe neonatal hyperammonemia requiring hemodialysis.

OBJECTIVES: Hyperammonemia in a newborn is a serious condition, which requires prompt intervention as it can lead to severe neurological impairment and death if left untreated. The most common causes of hyperammonemia in a newborn are acute liver failure and inherited metabolic disorders. Several mitochondrial disorders have been described as a cause of severe neonatal hyperammonemia. CASE PRESENTATION: Here we describe a new case of adenosine-triphosphate (ATP) synthase deficiency due to m.8528T>C mutation as a novel cause of severe neonatal hyperammonemia. So far six patients with this mutation have been described but none of them was reported to need hemodialysis in the first days of life. CONCLUSION: This broadens the so far known differential diagnosis of severe neonatal hyperammonemia requiring hemodialysis.

Review of clinical and molecular variability in autosomal recessive cutis laxa 2A.

ATP6V0A2-related cutis laxa, also known as autosomal recessive cutis laxa type 2A (ARCL2A), is a subtype of hereditary cutis laxa originally characterized by skin, skeletal, and neurological involvement, and a combined defect of N-glycosylation and O-glycosylation. The associated clinical spectrum subsequently expanded to a less severe phenotype dominated by cutaneous involvement. At the moment, ARCL2A was described in a few case reports and series only. An Italian adult woman ARCL2A with a phenotype restricted to skin and the two novel c.3G>C and c.1101dup ATP6V0A2 variants has been reported. A systematic literature review allowed us to identify 69 additional individuals from 64 families. Available data were scrutinized in order to describe the clinical and molecular variability of ARCL2A. About 78.3% of known variants were predicted null alleles, while 11 were missense and 2 affected noncanonical splice sites. Age at ascertainment appeared as the unique phenotypic discriminator with earlier age more commonly associated with facial dysmorphism (p .02), high/cleft palate (p .005), intellectual disability/global developmental delay (p .013), and seizures (p .024). No specific genotype-phenotype correlations were identified. This work confirmed the existence of an attenuated phenotype associated with ATP6V0A2 biallelic variants and offers an updated critique to the clinical and molecular variability of ARCL2A.

Astrocytes Protect Human Dopaminergic Neurons from α-Synuclein Accumulation and Propagation.

The pathologic hallmark of Parkinson's disease is the accumulation of α-synuclein-containing Lewy bodies/neurites almost exclusively in neurons, and rarely in glial cells. However, emerging evidence suggests that glia such as astrocytes play an important role in the development of α-synuclein pathology. Using induced pluripotent stem-derived dopaminergic neurons and astrocytes from healthy subjects and patients carrying mutations in lysosomal ATP13A2, a monogenic form of synucleinopathy, we found that astrocytes rapidly internalized α-synuclein, and exhibited higher lysosomal degradation rates compared with neurons. Moreover, coculturing astrocytes and neurons led to decreased accumulation of α-synuclein in neurons and consequently diminished interneuronal transfer of α-synuclein. These protective functions of astrocytes were attenuated by ATP13A2 deficiency, suggesting that the loss of ATP13A2 function in astrocytes at least partially contributes to neuronal α-synuclein pathology. Together, our results highlight the importance of lysosomal function in astrocytes in the pathogenesis of synucleinopathies.SIGNIFICANCE STATEMENT While most neurodegenerative disorders are characterized by the accumulation of aggregated mutant proteins exclusively in neurons, the contribution of glial cells in this process remains poorly explored. Here, we demonstrate that astrocytes contribute to the removal of extracellular α-synuclein and that disruption of this pathway caused by mutations in the Parkinson's disease-linked gene ATP13A2 result in α-synuclein accumulation in human dopaminergic neurons. We found that astrocytes also protect neurons from α-synuclein propagation, whereas ATP13A2 deficiency in astrocytes compromises this protective function. These results highlight astrocyte-mediated α-synuclein clearance as a potential therapeutic target in disorders characterized by the accumulation of α-synuclein, including Parkinson's disease.

ATP13A2 deficiency disrupts lysosomal polyamine export.

ATP13A2 (PARK9) is a late endolysosomal transporter that is genetically implicated in a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome-a parkinsonism with dementia1-and early-onset Parkinson's disease2. ATP13A2 offers protection against genetic and environmental risk factors of Parkinson's disease, whereas loss of ATP13A2 compromises lysosomes3. However, the transport function of ATP13A2 in lysosomes remains unclear. Here we establish ATP13A2 as a lysosomal polyamine exporter that shows the highest affinity for spermine among the polyamines examined. Polyamines stimulate the activity of purified ATP13A2, whereas ATP13A2 mutants that are implicated in disease are functionally impaired to a degree that correlates with the disease phenotype. ATP13A2 promotes the cellular uptake of polyamines by endocytosis and transports them into the cytosol, highlighting a role for endolysosomes in the uptake of polyamines into cells. At high concentrations polyamines induce cell toxicity, which is exacerbated by ATP13A2 loss due to lysosomal dysfunction, lysosomal rupture and cathepsin B activation. This phenotype is recapitulated in neurons and nematodes with impaired expression of ATP13A2 or its orthologues. We present defective lysosomal polyamine export as a mechanism for lysosome-dependent cell death that may be implicated in neurodegeneration, and shed light on the molecular identity of the mammalian polyamine transport system.

(Pro)renin receptor knockdown in the paraventricular nucleus of the hypothalamus attenuates hypertension development and AT1 receptor-mediated calcium events.

Activation of the brain renin-angiotensin system (RAS) is a pivotal step in the pathogenesis of hypertension. The paraventricular nucleus (PVN) of the hypothalamus is a critical part of the angiotensinergic sympatho-excitatory neuronal network involved in neural control of blood pressure and hypertension. However, the importance of the PVN (pro)renin receptor (PVN-PRR)-a key component of the brain RAS-in hypertension development has not been examined. In this study, we investigated the involvement and mechanisms of the PVN-PRR in DOCA-salt-induced hypertension, a mouse model of hypertension. Using nanoinjection of adeno-associated virus-mediated Cre recombinase expression to knock down the PRR specifically in the PVN, we report here that PVN-PRR knockdown attenuated the enhanced blood pressure and sympathetic tone associated with hypertension. Mechanistically, we found that PVN-PRR knockdown was associated with reduced activation of ERK (extracellular signal-regulated kinase)-1/2 in the PVN and rostral ventrolateral medulla during hypertension. In addition, using the genetically encoded Ca2+ biosensor GCaMP6 to monitor Ca2+-signaling events in the neurons of PVN brain slices, we identified a reduction in angiotensin II type 1 receptor-mediated Ca2+ activity as part of the mechanism by which PVN-PRR knockdown attenuates hypertension. Our study demonstrates an essential role of the PRR in PVN neurons in hypertension through regulation of ERK1/2 activation and angiotensin II type 1 receptor-mediated Ca2+ activity. NEW & NOTEWORTHY PRR knockdown in PVN neurons attenuates the development of DOCA-salt hypertension and autonomic dysfunction through a decrease in ERK1/2 activation in the PVN and RVLM during hypertension. In addition, PRR knockdown reduced AT1aR expression and AT1R-mediated calcium activity during hypertension. Furthermore, we characterized the neuronal targeting specificity of AAV serotype 2 in the mouse PVN and validated the advantages of the genetically encoded calcium biosensor GCaMP6 in visualizing neuronal calcium activity in the PVN.

ATP13A2 facilitates HDAC6 recruitment to lysosome to promote autophagosome-lysosome fusion.

Mutations in ATP13A2 cause Kufor-Rakeb syndrome, an autosomal recessive form of juvenile-onset atypical Parkinson's disease (PD). Recent work tied ATP13A2 to autophagy and other cellular features of neurodegeneration, but how ATP13A2 governs numerous cellular functions in PD pathogenesis is not understood. In this study, the ATP13A2-deficient mouse developed into aging-dependent phenotypes resembling those of autophagy impairment. ATP13A2 deficiency impaired autophagosome-lysosome fusion in cultured cells and in in vitro reconstitution assays. In ATP13A2-deficient cells or Drosophila melanogaster or mouse tissues, lysosomal localization and activity of HDAC6 were reduced, with increased acetylation of tubulin and cortactin. Wild-type HDAC6, but not a deacetylase-inactive mutant, restored autophagosome-lysosome fusion, antagonized cortactin hyperacetylation, and promoted lysosomal localization of cortactin in ATP13A2-deficient cells. Mechanistically, ATP13A2 facilitated recruitment of HDAC6 and cortactin to lysosomes. Cortactin overexpression in cultured cells reversed ATP13A2 deficiency-associated impairment of autophagosome-lysosome fusion. PD-causing ATP13A2 mutants failed to rescue autophagosome-lysosome fusion or to promote degradation of protein aggregates and damaged mitochondria. These results suggest that ATP13A2 recruits HDAC6 to lysosomes to deacetylate cortactin and promotes autophagosome-lysosome fusion and autophagy. This study identifies ATP13A2 as an essential molecular component for normal autophagy flux in vivo and implies potential treatments targeting HDAC6-mediated autophagy for PD.

Collecting duct principal, but not intercalated, cell prorenin receptor regulates renal sodium and water excretion.

The collecting duct is the predominant nephron site of prorenin and prorenin receptor (PRR) expression. We previously demonstrated that the collecting duct PRR regulates epithelial Na+ channel (ENaC) activity and water transport; however, which cell type is involved remains unclear. Herein, we examined the effects of principal cell (PC) or intercalated cell (IC) PRR deletion on renal Na+ and water handling. PC or IC PRR knockout (KO) mice were obtained by crossing floxed PRR mice with mice harboring Cre recombinase under the control of the AQP2 or B1 subunit of the H+ ATPase promoters, respectively. PC KO mice had reduced renal medullary ENaC-α abundance and increased urinary Na+ losses on a low-Na+ diet compared with controls. Conversely, IC KO mice had no apparent differences in Na+ balance or ENaC abundance compared with controls. Acute treatment with prorenin increased ENaC channel number and open probability in acutely isolated cortical collecting ducts from control and IC PRR KO, but not PC PRR KO, mice. Furthermore, compared with controls, PC KO, but not IC KO mice, had increased urine volume, reduced urine osmolality, and reduced abundance of renal medullary AQP2. Taken together, these findings indicate that PC, but not IC, PRR modulates ENaC activity, urinary Na+ excretion, and water transport.

The macula densa prorenin receptor is essential in renin release and blood pressure control.

The prorenin receptor (PRR) was originally proposed to be a member of the renin-angiotensin system (RAS); however, recent work questioned their association. The present paper describes a functional link between the PRR and RAS in the renal juxtaglomerular apparatus (JGA), a classic anatomical site of the RAS. PRR expression was found in the sensory cells of the JGA, the macula densa (MD), and immunohistochemistry-localized PRR to the MD basolateral cell membrane in mouse, rat, and human kidneys. MD cell PRR activation led to MAP kinase ERK1/2 signaling and stimulation of PGE2 release, the classic pathway of MD-mediated renin release. Exogenous renin or prorenin added to the in vitro microperfused JGA-induced acute renin release, which was inhibited by removing the MD or by the administration of a PRR decoy peptide. To test the function of MD PRR in vivo, we established a new mouse model with inducible conditional knockout (cKO) of the PRR in MD cells based on neural nitric oxide synthase-driven Cre-lox recombination. Deletion of the MD PRR significantly reduced blood pressure and plasma renin. Challenging the RAS by low-salt diet + captopril treatment caused further significant reductions in blood pressure, renal renin, cyclooxygenase-2, and microsomal PGE synthase expression in cKO vs. wild-type mice. These results suggest that the MD PRR is essential in a novel JGA short-loop feedback mechanism, which is integrated within the classic MD mechanism to control renin synthesis and release and to maintain blood pressure.

Effects of Disruption of PMC1 in the tfp1∆/∆ Mutant on Calcium Homeostasis, Oxidative and Osmotic Stress Resistance in Candida albicans.

The vacuolar-type H+-ATPase (V-ATPase) is essential for many cell processes. Our previous study has demonstrated that Tfp1 is a putative subunit of V-ATPase, loss of which causes disorders in calcium homeostasis and decreased resistance to oxidative stress. In this study, we found that further deletion of PMC1, a vacuolar calcium pump, in tfp1∆/∆ mutant led to more severe dysregulation of calcium homeostasis. Besides, the tfp1∆/∆pmc1∆/∆ mutant was more sensitive to H2O2 and had a higher ROS level. As is known, V-ATPase mutants are sensitive to NaCl, and PMC1 mutant is resistant against NaCl. However, the tfp1∆/∆pmc1∆/∆ mutant exhibited sensitivity to NaCl. Mechanism study demonstrated that their sensitivity was associated with reduced osmotic resistance caused by relatively low expression of GPD1. In addition, we first found that NaCl addition significantly declined ROS levels in tfp1∆/∆ and tfp1∆/∆pmc1∆/∆ mutants. In tfp1∆/∆ mutant, decreased ROS levels were relevant to enhanced antioxidant activities. However, in tfp1∆/∆pmc1∆/∆ mutant, reduced ROS resulted from decreased total calcium content, revealing that NaCl affected ROS levels in the two mutants through different mechanisms. Taken together, our data indicated that loss of both TFP1 and PMC1 further affected calcium homeostasis and other cellular processes in Candida albicans and provides a potential antifungal target.

Mutations in the X-linked ATP6AP2 cause a glycosylation disorder with autophagic defects.

The biogenesis of the multi-subunit vacuolar-type H+-ATPase (V-ATPase) is initiated in the endoplasmic reticulum with the assembly of the proton pore V0, which is controlled by a group of assembly factors. Here, we identify two hemizygous missense mutations in the extracellular domain of the accessory V-ATPase subunit ATP6AP2 (also known as the [pro]renin receptor) responsible for a glycosylation disorder with liver disease, immunodeficiency, cutis laxa, and psychomotor impairment. We show that ATP6AP2 deficiency in the mouse liver caused hypoglycosylation of serum proteins and autophagy defects. The introduction of one of the missense mutations into Drosophila led to reduced survival and altered lipid metabolism. We further demonstrate that in the liver-like fat body, the autophagic dysregulation was associated with defects in lysosomal acidification and mammalian target of rapamycin (mTOR) signaling. Finally, both ATP6AP2 mutations impaired protein stability and the interaction with ATP6AP1, a member of the V0 assembly complex. Collectively, our data suggest that the missense mutations in ATP6AP2 lead to impaired V-ATPase assembly and subsequent defects in glycosylation and autophagy.

Atp6ap2 ablation in adult mice impairs viability through multiple organ deficiencies.

ATP6AP2 codes for the (pro)renin receptor and is an essential component of vacuolar H+ ATPase. Activating (pro)renin for conversion of Angiotensinogen to Angiotensin makes ATP6AP2 attractive for drug intervention. Tissue-specific ATP6AP2 inactivation in mouse suggested a strong impact on various organs. Consistent with this, we found that embryonic ablation of Atp6ap2 resulted in both male hemizygous lethality and female haploinsufficiency. Next, we examined the phenotype of an induced inactivation in the adult animal, most akin to detect potential effect of functional interference of ATP6AP2 through drug therapy. Induced ablation of Atp6ap2, even without equal efficiency in all tissues (aorta, brain and kidney), resulted in rapid lethality marked by weight loss, changes in nutritional as well as blood parameters, leukocyte depletion, and bone marrow hypoplasia. Upon Atp6ap2 ablation, the colon demonstrated a rapid disruption of crypt morphology, aberrant proliferation, cell-death activation, as well as generation of microadenomas. Consequently, disruption of ATP6AP2 is extremely poorly tolerated in the adult, and severely affects various organ systems demonstrating that ATP6AP2 is an essential gene implicated in basic cellular mechanisms and necessary for multiple organ function. Accordingly, any potential drug targeting of this gene product must be strictly assessed for safety.

Collecting duct prorenin receptor knockout reduces renal function, increases sodium excretion, and mitigates renal responses in ANG II-induced hypertensive mice.

Augmented intratubular angiotensin (ANG) II is a key determinant of enhanced distal Na+ reabsorption via activation of epithelial Na+ channels (ENaC) and other transporters, which leads to the development of high blood pressure (BP). In ANG II-induced hypertension, there is increased expression of the prorenin receptor (PRR) in the collecting duct (CD), which has been implicated in the stimulation of the sodium transporters and resultant hypertension. The impact of PRR deletion along the nephron on BP regulation and Na+ handling remains controversial. In the present study, we investigate the role of PRR in the regulation of renal function and BP by using a mouse model with specific deletion of PRR in the CD (CDPRR-KO). At basal conditions, CDPRR-KO mice had decreased renal function and lower systolic BP associated with higher fractional Na+ excretion and lower ANG II levels in urine. After 14 days of ANG II infusion (400 ng·kg-1·min-1), the increases in systolic BP and diastolic BP were mitigated in CDPRR-KO mice. CDPRR-KO mice had lower abundance of cleaved αENaC and γENaC, as well as lower ANG II and renin content in urine compared with wild-type mice. In isolated CD from CDPRR-KO mice, patch-clamp studies demonstrated that ANG II-dependent stimulation of ENaC activity was reduced because of fewer active channels and lower open probability. These data indicate that CD PRR contributes to renal function and BP responses during chronic ANG II infusion by enhancing renin activity, increasing ANG II, and activating ENaC in the distal nephron segments.

Disruption of the vacuolar-type H+-ATPase complex in liver causes MTORC1-independent accumulation of autophagic vacuoles and lysosomes.

The vacuolar-type H+-translocating ATPase (v-H+-ATPase) has been implicated in the amino acid-dependent activation of the mechanistic target of rapamycin complex 1 (MTORC1), an important regulator of macroautophagy. To reveal the mechanistic links between the v-H+-ATPase and MTORC1, we destablilized v-H+-ATPase complexes in mouse liver cells by induced deletion of the essential chaperone ATP6AP2. ATP6AP2-mutants are characterized by massive accumulation of endocytic and autophagic vacuoles in hepatocytes. This cellular phenotype was not caused by a block in endocytic maturation or an impaired acidification. However, the degradation of LC3-II in the knockout hepatocytes appeared to be reduced. When v-H+-ATPase levels were decreased, we observed lysosome association of MTOR and normal signaling of MTORC1 despite an increase in autophagic marker proteins. To better understand why MTORC1 can be active when v-H+-ATPase is depleted, the activation of MTORC1 was analyzed in ATP6AP2-deficient fibroblasts. In these cells, very little amino acid-elicited activation of MTORC1 was observed. In contrast, insulin did induce MTORC1 activation, which still required intracellular amino acid stores. These results suggest that in vivo the regulation of macroautophagy depends not only on v-H+-ATPase-mediated regulation of MTORC1.

Loss of ATP13A2 impairs glycolytic function in Kufor-Rakeb syndrome patient-derived cell models.

BACKGROUND: Kufor-Rakeb syndrome (KRS) is an autosomal recessive, juvenile-onset Parkinson's disease (PD) caused by loss-of-function mutations in ATP13A2 (PARK9). Impaired energy metabolism is considered a pathogenic mechanism in PD and mitochondrial dysfunction resulting from Zn(2+) dyshomeostasis has been found in KRS patient-derived cells. In addition to mitochondrial energy production, glycolysis plays an important role in cellular energy metabolism and glucose hypometabolism has been reported in PD. However, glycolytic status in KRS remains undetermined despite its potential importance. METHODS: We assessed glycolytic function in ATP13A2-deficient KRS patient-derived human olfactory neurosphere cells and fibroblasts and determined the effect of pyruvate supplementation on improving cellular energy production. RESULTS: We found impaired extracellular acidification, reduction in pyruvate production and a decrease in the NAD(+)/NADH ratio, indicative of glycolytic dysfunction. In addition, gene expression analysis revealed an altered expression profile for several glycolytic enzymes. Glycolytic dysfunction was aggravated when the intracellular Zn(2+) concentration was increased, while ATP13A2 overexpression and pyruvate supplementation blocked the observed Zn(2+)-mediated toxicity. Moreover, supplementation with pyruvate significantly increased basal mitochondrial ATP production and abolished Zn(2+)-induced cell death. CONCLUSIONS: These findings indicate that glycolytic dysfunction contributes to pathogenesis and pyruvate supplementation improves overall cellular bioenergetics in our KRS patient-derived cell model, highlighting a therapeutic potential.

A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells.

GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid assays to identify novel GLP1R interactors in both mouse and human islets. Among these, ATP6ap2 (ATPase H(+)-transporting lysosomal accessory protein 2) was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion; however, siRNA knockdown decreased both glucose-stimulated and GLP1-induced insulin secretion. Decreases in GLP1-induced insulin secretion were accompanied by attenuated GLP1 stimulated cAMP accumulation. Because ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knockdown but paradoxically increased proinsulin secretion. Importantly, as a GLP1R interactor, ATP6ap2 was required for GLP1-induced Ca(2+) influx, in part explaining decreased insulin secretion in ATP6ap2 knockdown cells. Taken together, our findings identify a group of proteins that interact with the GLP1R. We further show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signaling and insulin processing to affect insulin secretion.

Parkinson's disease-associated human ATP13A2 (PARK9) deficiency causes zinc dyshomeostasis and mitochondrial dysfunction.

Human ATP13A2 (PARK9), a lysosomal type 5 P-type ATPase, has been associated with autosomal recessive early-onset Parkinson's disease (PD). ATP13A2 encodes a protein that is highly expressed in neurons and is predicted to function as a cation pump, although the substrate specificity remains unclear. Accumulation of zinc and mitochondrial dysfunction are established aetiological factors that contribute to PD; however, their underlying molecular mechanisms are largely unknown. Using patient-derived human olfactory neurosphere cultures, which harbour loss-of-function mutations in both alleles of ATP13A2, we identified a low intracellular free zinc ion concentration ([Zn(2+)]i), altered expression of zinc transporters and impaired sequestration of Zn(2+) into autophagy-lysosomal pathway-associated vesicles, indicating that zinc dyshomeostasis occurs in the setting of ATP13A2 deficiency. Pharmacological treatments that increased [Zn(2+)]i also induced the production of reactive oxygen species and aggravation of mitochondrial abnormalities that gave rise to mitochondrial depolarization, fragmentation and cell death due to ATP depletion. The toxic effect of Zn(2+) was blocked by ATP13A2 overexpression, Zn(2+) chelation, antioxidant treatment and promotion of mitochondrial fusion. Taken together, these results indicate that human ATP13A2 deficiency results in zinc dyshomeostasis and mitochondrial dysfunction. Our data provide insights into the molecular mechanisms of zinc dyshomeostasis in PD and its contribution to mitochondrial dysfunction with ATP13A2 as a molecular link between the two distinctive aetiological factors of PD.