Parental histone deposition on the replicated strands promotes error-free DNA damage tolerance and regulates drug resistance.

Ctf4 is a conserved replisome component with multiple roles in DNA metabolism. To investigate connections between Ctf4-mediated processes involved in drug resistance, we conducted a suppressor screen of ctf4Δ sensitivity to the methylating agent MMS. We uncovered that mutations in Dpb3 and Dpb4 components of polymerase ε result in the development of drug resistance in ctf4Δ via their histone-binding function. Alleviated sensitivity to MMS of the double mutants was not associated with rescue of ctf4Δ defects in sister chromatid cohesion, replication fork architecture, or template switching, which ensures error-free replication in the presence of genotoxic stress. Strikingly, the improved viability depended on translesion synthesis (TLS) polymerase-mediated mutagenesis, which was drastically increased in ctf4 dpb3 double mutants. Importantly, mutations in Mcm2-Ctf4-Polα and Dpb3-Dpb4 axes of parental (H3-H4)2 deposition on lagging and leading strands invariably resulted in reduced error-free DNA damage tolerance through gap filling by template switch recombination. Overall, we uncovered a chromatin-based drug resistance mechanism in which defects in parental histone transfer after replication fork passage impair error-free recombination bypass and lead to up-regulation of TLS-mediated mutagenesis and drug resistance.

Platinum-induced mitochondrial DNA mutations confer lower sensitivity to paclitaxel by impairing tubulin cytoskeletal organization.

Development of chemoresistance is a cogent clinical issue in oncology, whereby combination of anticancer drugs is usually preferred also to enhance efficacy. Paclitaxel (PTX), combined with carboplatin, represents the standard first-line chemotherapy for different types of cancers. We here depict a double-edge role of mitochondrial DNA (mtDNA) mutations induced in cancer cells after treatment with platinum. MtDNA mutations were positively selected by PTX, and they determined a decrease in the mitochondrial respiratory function, as well as in proliferative and tumorigenic potential, in terms of migratory and invasive capacity. Moreover, cells bearing mtDNA mutations lacked filamentous tubulin, the main target of PTX, and failed to reorient the Golgi body upon appropriate stimuli. We also show that the bioenergetic and cytoskeletal phenotype were transferred along with mtDNA mutations in transmitochondrial hybrids, and that this also conferred PTX resistance to recipient cells. Overall, our data show that platinum-induced deleterious mtDNA mutations confer resistance to PTX, and confirm what we previously reported in an ovarian cancer patient treated with carboplatin and PTX who developed a quiescent yet resistant tumor mass harboring mtDNA mutations.

COQ4 mutations cause a broad spectrum of mitochondrial disorders associated with CoQ10 deficiency.

Primary coenzyme Q10 (CoQ10) deficiencies are rare, clinically heterogeneous disorders caused by mutations in several genes encoding proteins involved in CoQ10 biosynthesis. CoQ10 is an essential component of the electron transport chain (ETC), where it shuttles electrons from complex I or II to complex III. By whole-exome sequencing, we identified five individuals carrying biallelic mutations in COQ4. The precise function of human COQ4 is not known, but it seems to play a structural role in stabilizing a multiheteromeric complex that contains most of the CoQ10 biosynthetic enzymes. The clinical phenotypes of the five subjects varied widely, but four had a prenatal or perinatal onset with early fatal outcome. Two unrelated individuals presented with severe hypotonia, bradycardia, respiratory insufficiency, and heart failure; two sisters showed antenatal cerebellar hypoplasia, neonatal respiratory-distress syndrome, and epileptic encephalopathy. The fifth subject had an early-onset but slowly progressive clinical course dominated by neurological deterioration with hardly any involvement of other organs. All available specimens from affected subjects showed reduced amounts of CoQ10 and often displayed a decrease in CoQ10-dependent ETC complex activities. The pathogenic role of all identified mutations was experimentally validated in a recombinant yeast model; oxidative growth, strongly impaired in strains lacking COQ4, was corrected by expression of human wild-type COQ4 cDNA but failed to be corrected by expression of COQ4 cDNAs with any of the mutations identified in affected subjects. COQ4 mutations are responsible for early-onset mitochondrial diseases with heterogeneous clinical presentations and associated with CoQ10 deficiency.

Exome sequence reveals mutations in CoA synthase as a cause of neurodegeneration with brain iron accumulation.

Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of disorders with progressive extrapyramidal signs and neurological deterioration, characterized by iron accumulation in the basal ganglia. Exome sequencing revealed the presence of recessive missense mutations in COASY, encoding coenzyme A (CoA) synthase in one NBIA-affected subject. A second unrelated individual carrying mutations in COASY was identified by Sanger sequence analysis. CoA synthase is a bifunctional enzyme catalyzing the final steps of CoA biosynthesis by coupling phosphopantetheine with ATP to form dephospho-CoA and its subsequent phosphorylation to generate CoA. We demonstrate alterations in RNA and protein expression levels of CoA synthase, as well as CoA amount, in fibroblasts derived from the two clinical cases and in yeast. This is the second inborn error of coenzyme A biosynthesis to be implicated in NBIA.

Mitochondrial iron and energetic dysfunction distinguish fibroblasts and induced neurons from pantothenate kinase-associated neurodegeneration patients.

Pantothenate kinase-associated neurodegeneration is an early onset autosomal recessive movement disorder caused by mutation of the pantothenate kinase-2 gene, which encodes a mitochondrial enzyme involved in coenzyme A synthesis. The disorder is characterised by high iron levels in the brain, although the pathological mechanism leading to this accumulation is unknown. To address this question, we tested primary skin fibroblasts from three patients and three healthy subjects, as well as neurons induced by direct fibroblast reprogramming, for oxidative status, mitochondrial functionality and iron parameters. The patients' fibroblasts showed altered oxidative status, reduced antioxidant defence, and impaired cytosolic and mitochondrial aconitase activities compared to control cells. Mitochondrial iron homeostasis and functionality analysis of patient fibroblasts indicated increased labile iron pool content and reactive oxygen species development, altered mitochondrial shape, decreased membrane potential and reduced ATP levels. Furthermore, analysis of induced neurons, performed at a single cell level, confirmed some of the results obtained in fibroblasts, indicating an altered oxidative status and signs of mitochondrial dysfunction, possibly due to iron mishandling. Thus, for the first time, altered biological processes have been identified in vitro in live diseased neurons. Moreover, the obtained induced neurons can be considered a suitable human neuronal model for the identification of candidate therapeutic compounds for this disease.

Pantethine treatment is effective in recovering the disease phenotype induced by ketogenic diet in a pantothenate kinase-associated neurodegeneration mouse model.

Pantothenate kinase-associated neurodegeneration, caused by mutations in the PANK2 gene, is an autosomal recessive disorder characterized by dystonia, dysarthria, rigidity, pigmentary retinal degeneration and brain iron accumulation. PANK2 encodes the mitochondrial enzyme pantothenate kinase type 2, responsible for the phosphorylation of pantothenate or vitamin B5 in the biosynthesis of co-enzyme A. A Pank2 knockout (Pank2(-/-)) mouse model did not recapitulate the human disease but showed azoospermia and mitochondrial dysfunctions. We challenged this mouse model with a low glucose and high lipid content diet (ketogenic diet) to stimulate lipid use by mitochondrial beta-oxidation. In the presence of a shortage of co-enzyme A, this diet could evoke a general impairment of bioenergetic metabolism. Only Pank2(-/-) mice fed with a ketogenic diet developed a pantothenate kinase-associated neurodegeneration-like syndrome characterized by severe motor dysfunction, neurodegeneration and severely altered mitochondria in the central and peripheral nervous systems. These mice also showed structural alteration of muscle morphology, which was comparable with that observed in a patient with pantothenate kinase-associated neurodegeneration. We here demonstrate that pantethine administration can prevent the onset of the neuromuscular phenotype in mice suggesting the possibility of experimental treatment in patients with pantothenate kinase-associated neurodegeneration.

Pantothenate kinase-associated neurodegeneration: altered mitochondria membrane potential and defective respiration in Pank2 knock-out mouse model.

Neurodegeneration with brain iron accumulation (NBIA) comprises a group of neurodegenerative disorders characterized by high brain content of iron and presence of axonal spheroids. Mutations in the PANK2 gene, which encodes pantothenate kinase 2, underlie an autosomal recessive inborn error of coenzyme A metabolism, called pantothenate kinase-associated neurodegeneration (PKAN). PKAN is characterized by dystonia, dysarthria, rigidity and pigmentary retinal degeneration. The pathogenesis of this disorder is poorly understood and, although PANK2 is a mitochondrial protein, perturbations in mitochondrial bioenergetics have not been reported. A knock-out (KO) mouse model of PKAN exhibits retinal degeneration and azoospermia, but lacks any neurological phenotype. The absence of a clinical phenotype has partially been explained by the different cellular localization of the human and murine PANK2 proteins. Here we demonstrate that the mouse Pank2 protein localizes to mitochondria, similar to its human orthologue. Moreover, we show that Pank2-defective neurons derived from KO mice have an altered mitochondrial membrane potential, a defect further corroborated by the observations of swollen mitochondria at the ultra-structural level and by the presence of defective respiration.

Absence of an orphan mitochondrial protein, c19orf12, causes a distinct clinical subtype of neurodegeneration with brain iron accumulation.

The disease classification neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of progressive neurodegenerative disorders characterized by brain iron deposits in the basal ganglia. For about half of the cases, the molecular basis is currently unknown. We used homozygosity mapping followed by candidate gene sequencing to identify a homozygous 11 bp deletion in the orphan gene C19orf12. Mutation screening of 23 ideopathic NBIA index cases revealed two mutated alleles in 18 of them, and one loss-of-function mutation is the most prevalent. We also identified compound heterozygous missense mutations in a case initially diagnosed with Parkinson disease at age 49. Psychiatric signs, optic atrophy, and motor axonal neuropathy were common findings. Compared to the most prevalent NBIA subtype, pantothenate kinase associated neurodegeneration (PKAN), individuals with two C19orf12 mutations were older at age of onset and the disease progressed more slowly. A polyclonal antibody against the predicted membrane spanning protein showed a mitochondrial localization. A histopathological examination in a single autopsy case detected Lewy bodies, tangles, spheroids, and tau pathology. The mitochondrial localization together with the immunohistopathological findings suggests a pathomechanistic overlap with common forms of neurodegenerative disorders.

Alteration of the coenzyme A biosynthetic pathway in neurodegeneration with brain iron accumulation syndromes.

NBIA (neurodegeneration with brain iron accumulation) comprises a heterogeneous group of neurodegenerative diseases having as a common denominator, iron overload in specific brain areas, mainly basal ganglia and globus pallidus. In the past decade a bunch of disease genes have been identified, but NBIA pathomechanisms are still not completely clear. PKAN (pantothenate kinase-associated neurodegeneration), an autosomal recessive disorder with progressive impairment of movement, vision and cognition, is the most common form of NBIA. It is caused by mutations in the PANK2 (pantothenate kinase 2) gene, coding for a mitochondrial enzyme that phosphorylates vitamin B5 in the first reaction of the CoA (coenzyme A) biosynthetic pathway. A distinct form of NBIA, denominated CoPAN (CoA synthase protein-associated neurodegeneration), is caused by mutations in the CoASY (CoA synthase) gene coding for a bifunctional mitochondrial enzyme, which catalyses the final steps of CoA biosynthesis. These two inborn errors of CoA metabolism further support the concept that dysfunctions in CoA synthesis may play a crucial role in the pathogenesis of NBIA.

Metabolic consequences of mitochondrial coenzyme A deficiency in patients with PANK2 mutations.

Pantothenate kinase-associated neurodegeneration (PKAN) is a rare, inborn error of metabolism characterized by iron accumulation in the basal ganglia and by the presence of dystonia, dysarthria, and retinal degeneration. Mutations in pantothenate kinase 2 (PANK2), the rate-limiting enzyme in mitochondrial coenzyme A biosynthesis, represent the most common genetic cause of this disorder. How mutations in this core metabolic enzyme give rise to such a broad clinical spectrum of pathology remains a mystery. To systematically explore its pathogenesis, we performed global metabolic profiling on plasma from a cohort of 14 genetically defined patients and 18 controls. Notably, lactate is elevated in PKAN patients, suggesting dysfunctional mitochondrial metabolism. As predicted, but never previously reported, pantothenate levels are higher in patients with premature stop mutations in PANK2. Global metabolic profiling and follow-up studies in patient-derived fibroblasts also reveal defects in bile acid conjugation and lipid metabolism, pathways that require coenzyme A. These findings raise a novel therapeutic hypothesis, namely, that dietary fats and bile acid supplements may hold potential as disease-modifying interventions. Our study illustrates the value of metabolic profiling as a tool for systematically exploring the biochemical basis of inherited metabolic diseases.

Rad51-mediated replication of damaged templates relies on monoSUMOylated DDK kinase.

DNA damage tolerance (DDT), activated by replication stress during genome replication, is mediated by translesion synthesis and homologous recombination (HR). Here we uncover that DDK kinase, essential for replication initiation, is critical for replication-associated recombination-mediated DDT. DDK relies on its multi-monoSUMOylation to facilitate HR-mediated DDT and optimal retention of Rad51 recombinase at replication damage sites. Impairment of DDK kinase activity, reduced monoSUMOylation and mutations in the putative SUMO Interacting Motifs (SIMs) of Rad51 impair replication-associated recombination and cause fork uncoupling with accumulation of large single-stranded DNA regions at fork branching points. Notably, genetic activation of salvage recombination rescues the uncoupled fork phenotype but not the recombination-dependent gap-filling defect of DDK mutants, revealing that the salvage recombination pathway operates preferentially proximal to fork junctions at stalled replication forks. Overall, we uncover that monoSUMOylated DDK acts with Rad51 in an axis that prevents replication fork uncoupling and mediates recombination-dependent gap-filling.

APOE epsilon-4 allele and cytokine production in Alzheimer's disease.

OBJECTIVE: The APOE epsilon-4 allele has consistently emerged as a susceptibility factor for Alzheimer's disease (AD). Pro-inflammatory cytokines are detectable at abnormal levels in AD, and are thought to play a pathophysiological role. Animal studies have shown dose-dependent correlations between the number of APOE epsilon-4 alleles and the levels of pro-inflammatory cytokines. The aims of this study were to investigate the influence of APOE genotypes on TNF-alpha, IL-6, and IL-1beta secreted by peripheral blood mononuclear cells (PBMC) from human patients with AD and to analyze the correlation between cytokine production and AD clinical features. METHODS: Outpatients with AD (n = 40) were clinically evaluated for cognitive decline (MMSE) and psychiatric symptoms (Cornell Scale for Depression in Dementia; Neuropsychiatric Inventory) and genotyped for APOE variants. PBMCs were isolated from the donors and used to assess spontaneous and PMA-stimulated secretion of TNF-alpha, IL-6, and IL-1beta. Cytokine production was determined by immuno-enzymatic assays (ELISA). RESULTS: In comparison with their counterparts without APOE4, patients with at least one copy of the APOE epsilon-4 allele showed higher spontaneous (p = 0.037) and PMA-induced (p = 0.039) production of IL-1beta after controlling for clinical variables. Significant correlations were reported between NPI scores (psychotic symptoms) and IL-6 production. CONCLUSION: These preliminary findings suggest the involvement of inflammatory response in the pathogenic effect of the APOE epsilon-4 allele in AD, although their replication in larger samples is mandatory. The modest correlations between pro-inflammatory cytokines released at peripheral level and AD features emphasizes the need for further research to elucidate the role of neuroinflammation in pathophysiology of AD.

Lack of association between interleukin-1 alpha rs1800587 polymorphism and Alzheimer's disease in two Independent European samples.

Interleukin-1 (IL1) can contribute to pathophysiology of Alzheimer's disease (AD) by promoting deposition of amyloid-beta in the brain. The gene encoding IL1 alpha (IL1A) has a common polymorphism in its 5' regulatory region (rs1800587) with possible functional effects. IL1A T/T genotype has been associated with AD but the overall effect is modest and negative studies have been published. The aim of this study was to investigate the association of the IL1A rs1800587 polymorphism with AD in two independent case-control groups from Greece (Athens) and Italy (Faenza and Granarolo). Preliminary results from the ongoing sample (110 patients with sporadic AD and 130 nonpsychiatric controls) showed no association between IL1A variants and AD, however C/T heterozygotes had more severe depression in AD (Cornell Scale for Depression in Dementia) compared to other genotypes (F = 4.56, d.f = 1, p = 0.037) after controlling for age, illness duration and cognitive impairment (MMSE). Despite the small sample size and the possibility of a false negative finding, our preliminary data support the hypothesis the IL1A rs1800587 variants are not associated with AD. The effect of the IL1A on depressive symptomatology warrants further investigations, however the lack of a gene-dose relationship would suggest a false positive.

Alteration of mitochondrial membrane inner potential in three Italian patients with megaconial congenital muscular dystrophy carrying new mutations in CHKB gene.

Congenital Muscular Dystrophies (CMDs) are a heterogeneous group of autosomal recessive disorders presenting at birth with psychomotor delay, cognitive impairment, muscle weakness and hypotonia. Here we described an alteration of mitochondrial inner membrane potential and mitochondrial network in cells derived from Italian patients carrying three novel mutations in CHKB gene, recently associated with "megaconial CMD". On the bases of our findings, we hypothesize that the mitochondrial membrane potential alteration, presumably as a consequence of the altered biosynthesis of phosphatidylcholine, could be responsible for the peculiar morphological aspect of mitochondria in this disease and might be involved in the disease pathogenesis.

AND-1 fork protection function prevents fork resection and is essential for proliferation.

AND-1/Ctf4 bridges the CMG helicase and DNA polymerase alpha, facilitating replication. Using an inducible degron system in avian cells, we find that AND-1 depletion is incompatible with proliferation, owing to cells accumulating in G2 with activated DNA damage checkpoint. Replication without AND-1 causes fork speed slow-down and accumulation of long single-stranded DNA (ssDNA) gaps at the replication fork junction, with these regions being converted to DNA double strand breaks (DSBs) in G2. Strikingly, resected forks and DNA damage accumulation in G2, but not fork slow-down, are reverted by treatment with mirin, an MRE11 nuclease inhibitor. Domain analysis of AND-1 further revealed that the HMG box is important for fast replication but not for proliferation, whereas conversely, the WD40 domain prevents fork resection and subsequent DSB-associated lethality. Thus, our findings uncover a fork protection function of AND-1/Ctf4 manifested via the WD40 domain that is essential for proliferation and averts genome instability.

Gene-specific mitochondria dysfunctions in human TARDBP and C9ORF72 fibroblasts.

Dysregulation of RNA metabolism represents an important pathogenetic mechanism in both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) due to the involvement of the DNA/RNA-binding proteins TDP-43 and FUS and, more recently, of C9ORF72. A potential link between dysregulation of RNA metabolism and mitochondrial dysfunction is recently emerged in TDP-43 disease models. To further investigate the possible relationship between these two pathogenetic mechanisms in ALS/FTD, we studied mitochondria functionality in human mutant TARDBP(p.A382T) and C9ORF72 fibroblasts grown in galactose medium to induce a switch from a glycolytic to an oxidative metabolism. In this condition we observed significant changes in mitochondria morphology and ultrastructure in both mutant cells with a fragmented mitochondria network particularly evident in TARDBP(p.A382T) fibroblasts. From analysis of the mitochondrial functionality, a decrease of mitochondria membrane potential with no alterations in oxygen consumption rate emerged in TARDBP fibroblasts. Conversely, an increased oxygen consumption and mitochondria hyperpolarization were observed in C9ORF72 fibroblasts in association to increased ROS and ATP content. We found evidence of autophagy/mitophagy in dynamic equilibrium with the biogenesis of novel mitochondria, particularly in mutant C9ORF72 fibroblasts where an increase of mitochondrial DNA content and mass, and of PGC1-α protein was observed. Our imaging and biochemical data show that wild-type and mutant TDP-43 proteins do not localize at mitochondria so that the molecular mechanisms responsible for such mitochondria impairment remain to be further elucidated. For the first time our findings assess a link between C9ORF72 and mitochondria dysfunction and indicate that mitochondria functionality is affected in TARDBP and C9ORF72 fibroblasts with gene-specific features in oxidative conditions. As in neuronal metabolism mitochondria are actively used for ATP production, we speculate that TARDBP and C9ORF72 mutations might trigger cell death by impairing not only RNA metabolism, but also mitochondria activity in ALS/FTD neurons.

Coenzyme A corrects pathological defects in human neurons of PANK2-associated neurodegeneration.

Pantothenate kinase-associated neurodegeneration (PKAN) is an early onset and severely disabling neurodegenerative disease for which no therapy is available. PKAN is caused by mutations in PANK2, which encodes for the mitochondrial enzyme pantothenate kinase 2. Its function is to catalyze the first limiting step of Coenzyme A (CoA) biosynthesis. We generated induced pluripotent stem cells from PKAN patients and showed that their derived neurons exhibited premature death, increased ROS production, mitochondrial dysfunctions-including impairment of mitochondrial iron-dependent biosynthesis-and major membrane excitability defects. CoA supplementation prevented neuronal death and ROS formation by restoring mitochondrial and neuronal functionality. Our findings provide direct evidence that PANK2 malfunctioning is responsible for abnormal phenotypes in human neuronal cells and indicate CoA treatment as a possible therapeutic intervention.

Modeling human Coenzyme A synthase mutation in yeast reveals altered mitochondrial function, lipid content and iron metabolism.

Mutations in nuclear genes associated with defective coenzyme A biosynthesis have been identified as responsible for some forms of neurodegeneration with brain iron accumulation (NBIA), namely PKAN and CoPAN. PKAN are defined by mutations in PANK2, encoding the pantothenate kinase 2 enzyme, that account for about 50% of cases of NBIA, whereas mutations in CoA synthase COASY have been recently reported as the second inborn error of CoA synthesis leading to CoPAN. As reported previously, yeast cells expressing the pathogenic mutation exhibited a temperature-sensitive growth defect in the absence of pantothenate and a reduced CoA content. Additional characterization revealed decreased oxygen consumption, reduced activities of mitochondrial respiratory complexes, higher iron content, increased sensitivity to oxidative stress and reduced amount of lipid droplets, thus partially recapitulating the phenotypes found in patients and establishing yeast as a potential model to clarify the pathogenesis underlying PKAN and CoPAN diseases.

Interleukin-1α, interleukin-1ß and tumor necrosis factor-α genetic variants and risk of dementia in the very old: evidence from the "Monzino 80-plus" prospective study.

The association among single nucleotide polymorphisms in inflammatory genes as interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1ß) or tumor necrosis factor alpha (TNF-α) and dementia has been explored mostly in Alzheimer's disease, while few data addressing their association with dementia in very old people are available. We performed a prospective, door-to-door population-based study of 80 years or older residents in eight municipalities of Varese province, Italy (the Monzino 80-plus study). No difference was found by a cross-sectional approach comparing IL-1α rs1800587, IL-1ß rs3087258 and TNF-α rs1799724 genotypic and allelic frequencies between those affected and not affected by dementia. After a 5-year follow-up, the elderly carriers of T-allele of TNF-α rs1799724 were at an increased risk of dementia (p = 0.03). This association was no more significant adjusting for the apolipoprotein E epsilon-4 allele (APOE-ε4, p = 0.26), which was an independent predictor of dementia onset (p = 0.0002). In short, in this Italian population of oldest olds, dementia was associated to the APOE-ε4 allele only.

C19orf12 and FA2H mutations are rare in Italian patients with neurodegeneration with brain iron accumulation.

Neurodegeneration with brain iron accumulation (NBIA) defines a wide spectrum of clinical entities characterized by iron accumulation in specific regions of the brain, predominantly in the basal ganglia. We evaluated the presence of FA2H and C19orf12 mutations in a cohort of 46 Italian patients with early onset NBIA, which were negative for mutations in the PANK2 and PLA2G6 genes. Follow-up molecular genetic and in vitro analyses were then performed. We did not find any mutations in the FA2H gene, although we identified 3 patients carrying novel mutations in the C19orf12 gene. The recent discovery of new genes responsible for NBIA extends the spectrum of the genetic investigation now available for these disorders and makes it possible to delineate a clearer clinical-genetic classification of different forms of this syndrome. A large fraction of patients still remain without a molecular genetics diagnosis, suggesting that additional NBIA genes are still to be discovered.