Anna Maria Porcelli

Syndromic parkinsonism and dementia associated with OPA1 missense mutations.

Mounting evidence links neurodegenerative disorders such as Parkinson disease and Alzheimer disease with mitochondrial dysfunction, and recent emphasis has focused on mitochondrial dynamics and quality control. Mitochondrial dynamics and mtDNA maintenance is another link recently emerged, implicating mutations in the mitochondrial fusion genes OPA1 and MFN2 in the pathogenesis of multisystem syndromes characterized by neurodegeneration and accumulation of mtDNA multiple deletions in postmitotic tissues. Here, we report 2 Italian families affected by dominant chronic progressive external ophthalmoplegia (CPEO) complicated by parkinsonism and dementia.

Different mtDNA mutations modify tumor progression in dependence of the degree of respiratory complex I impairment

Mitochondrial DNA mutations are currently investigated as modifying factors impinging on tumor growth and aggressiveness, having been found in virtually all cancer types and most commonly affecting genes encoding mitochondrial complex I (CI) subunits. However, it is still unclear whether they exert a pro- or anti-tumorigenic effect. We here analyzed the impact of three homoplasmic mtDNA mutations (m.3460G>A/MT-ND1, m.3571insC/MT-ND1 and m.3243A>G/MT-TL1) on osteosarcoma progression, chosen since they induce different degrees of oxidative phosphorylation impairment. In fact, the m.3460G>A/MT-ND1 mutation caused only a reduction in CI activity, whereas the m.3571insC/MT-ND1 and the m.3243A>G/MT-TL1 mutations induced a severe structural and functional CI alteration. As a consequence, this severe CI dysfunction determined an energetic defect associated with a compensatory increase in glycolytic metabolism and AMP-activated protein kinase activation. Osteosarcoma cells carrying such marked CI impairment displayed a reduced tumorigenic potential both in vitro and in vivo, when compared with cells with mild CI dysfunction, suggesting that mtDNA mutations may display diverse impact on tumorigenic potential depending on the type and severity of the resulting oxidative phosphorylation dysfunction. The modulation of tumor growth was independent from reactive oxygen species production but correlated with hypoxia-inducible factor 1α stabilization, indicating that structural and functional integrity of CI and oxidative phosphorylation are required for hypoxic adaptation and tumor progression.

Complex I impairment in mitochondrial diseases and cancer: parallel roads leading to different outcomes.

Respiratory chain complex I (CI) dysfunctions have been recognized as one of the most frequent causes of mitochondrial neuro-muscular disorders. Moreover, latest reports reveal that CI impairment is a major contributing factor in many other pathological processes, including cancer. In fact, energy depletion, oxidative stress and metabolites unbalance are frequently associated with CI functional and structural alterations. The occurrence of mitochondrial DNA (mtDNA) mutations is a shared feature in neuro-muscular diseases and cancer; however, the two diverging phenotypes arise depending on the mutation type (disassembling versus non-disassembling mutations), the mutant load and the cytotype. In this review, we unify our knowledge on CI impairment caused by mutations in structural CI genes and assembly chaperones, both in mitochondrial disorders and cancer, stratifying such mutations based on their functional versus structural effects. We summarize shared and specific metabolic consequences of CI dysfunction in these pathologies, which allow us to draw two parallel roads that lead to different clinical outcomes. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

Gamma rays induce a p53-independent mitochondrial biogenesis that is counter-regulated by HIF1α

Mitochondrial biogenesis is an orchestrated process that presides to the regulation of the organelles homeostasis within a cell. We show that γ-rays, at doses commonly used in the radiation therapy for cancer treatment, induce an increase in mitochondrial mass and function, in response to a genotoxic stress that pushes cells into senescence, in the presence of a functional p53. Although the main effector of the response to γ-rays is the p53-p21 axis, we demonstrated that mitochondrial biogenesis is only indirectly regulated by p53, whose activation triggers a murine double minute 2 (MDM2)-mediated hypoxia-inducible factor 1α (HIF1α) degradation, leading to the release of peroxisome-proliferator activated receptor gamma co-activator 1β inhibition by HIF1α, thus promoting mitochondrial biogenesis. Mimicking hypoxia by HIF1α stabilization, in fact, blunts the mitochondrial response to γ-rays as well as the induction of p21-mediated cell senescence, indicating prevalence of the hypoxic over the genotoxic response. Finally, we also show in vivo that post-radiotherapy mitochondrial DNA copy number increase well correlates with lack of HIF1α increase in the tissue, concluding this may be a useful molecular tool to infer the trigger of a hypoxic response during radiotherapy, which may lead to failure of activation of cell senescence.

Targeting respiratory complex I to prevent the Warburg effect.

In the last 10 years, studies of energetic metabolism in different tumors clearly indicate that the definition of Warburg effect, i.e. the glycolytic shift cells undergo upon transformation, ought to be revisited considering the metabolic plasticity of cancer cells. In fact, recent findings show that the shift from glycolysis to re-established oxidative metabolism is required for certain steps of tumor progression, suggesting that mitochondrial function and, in particular, respiratory complex I are crucial for metabolic and hypoxic adaptation. Based on these evidences, complex I can be considered a lethality target for potential anticancer strategies. In conclusion, in this mini review we summarize and discuss why it is not paradoxical to develop pharmacological and genome editing approaches to target complex I as novel adjuvant therapies for cancer treatment. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.

Dysregulation of Parkin-mediated mitophagy in thyroid Hürthle cell tumors.

Abnormal accumulation of defective mitochondria is the hallmark of oncocytes, which are frequently observed in thyroid Hürthle cell lesions. Autophagy is an essential cellular catabolic mechanism for the degradation of dysfunctional organelles and has been implicated in several human diseases. It is yet unknown how autophagic turnover of defective mitochondria in Hürthle cell tumors is regulated. We characterized the expression patterns of molecular markers including Beclin1, LC3, PINK1 and Parkin, which are required for autophagy or mitophagy, in human oncocytic lesions of the thyroid. To undertake mechanistic studies, we investigated autophagy and mitophagy using XTC.UC1 cells, the only in vitro model of Hürthle cell tumors. Beclin1 and LC3 were highly expressed in oncocytes of Hürthle cell tumors. XTC.UC1 showed autophagic responses to starvation and rapamycin treatment, whereas they displayed ineffective activation of mitophagy, which is triggered by the coordinated action of PINK1 and Parkin in response to CCCP. This resulted in a decreased turnover of abnormal mitochondria. The mechanisms underlying defective mitophagy and mitochondrial turnover were investigated by genetic analysis of the PARK2 gene in XTC.UC1 and Hürthle cell tumor tissues. XTC.UC1 and several tumors harbored the V380L mutation, resulting in dysfunctional autoubiquitination and decreased E3 ligase activity. Consistently, oncocytes in Hürthle cell tumors displayed comparable expression of PINK1 but decreased Parkin expression in comparison to normal thyrocytes. The introduction of wild-type Parkin sensitized XTC.UC1 to death induced by CCCP. This study provides a possible etiological basis for oncocytic formation in heterogeneous Hürthle cell tumors through insufficient mitophagy leading to ineffective turnover of aberrant mitochondria caused by dysfunctional Parkin-mediated pathways of mitochondria quality control.

Evidence of association of human papillomavirus with prognosis worsening in glioblastoma multiforme

BACKGROUNDnGlioblastoma multiforme (GBM) is the most malignant brain tumor in adults, but its etiology still remains unknown. Recently, a role of viruses such as cytomegalovirus and JC virus in gliomagenesis has been suggested. Since human papillomavirus (HPV) is considered the most common oncogenic virus in humans, we evaluated its occurrence in GBM samples.nnnMATERIAL AND METHODSnFifty-two formalin-fixed paraffin-embedded primary glioblastoma specimens were retrospectively analyzed. The presence of HPV genome on tumor DNA was assessed by MY/GP nested PCR. Confirmation of HPV detection was obtained by chromogenic in situ hybridization (CISH) and immunohistochemistry (IHC) with an antibody directed against the L1 capsidic protein. Finally, univariate and multivariate proportional-hazards models were used to compare the risk of death among HPV-positive and HPV-negative patients.nnnRESULTSnStrikingly, viral DNA was detected after PCR in 12 cases (23%). HPV16 genome was present in 25% infected samples, whereas the remaining samples tested positive for HPV6. CISH confirmed positivity in all infected samples for which enough material was available. Moreover, IHC positivity suggested that production of viral proteins from HPV genome is an ongoing process in GBM cancer cells. Finally an association between HPV infection and a worse prognosis was found in patients upon age stratification with a univariate analysis (HR, 2.10; 95% CI, 1.00-4.44; log-rank P = .045).nnnCONCLUSIONSnHPV infection status may be considered an independent prognostic factor in GBM patients and suggests that prevention may be considered, should HPV be recognized as a causative agent in gliomagenesis.

Cybrid studies establish the causal link between the mtDNA m.3890G>A/MT-ND1 mutation and optic atrophy with bilateral brainstem lesions

Complex I (CI) deficiency is a frequent cause of mitochondrial disorders and, in most cases, is due to mutations in CI subunit genes encoded by mitochondrial DNA (mtDNA). In this study, we establish the pathogenic role of the heteroplasmic mtDNA m.3890G>A/MT-ND1 (p.R195Q) mutation, which affects an extremely conserved amino acid position in ND1 subunit of CI. This mutation was found in a young-adult male with optic atrophy resembling Lebers hereditary optic neuropathy (LHON) and bilateral brainstem lesions. The only previously reported case with this mutation was a girl with fatal infantile Leigh syndrome with bilateral brainstem lesions. Transfer of the mutant mtDNA in the cybrid cell system resulted in a marked reduction of CI activity and CI-dependent ATP synthesis in the presence of a normally assembled enzyme. These findings establish the pathogenicity of the m.3890G>A/MT-ND1 mutation and remark the link between CI mutations affecting the mtDNA-encoded ND subunits and LHON-like optic atrophy, which may be complicated by bilateral and symmetric lesions affecting the central nervous system. Peculiar to this mutation is the distribution of the brainstem lesions, with sparing of the striatum in both patients.

Genome-wide expression profiling and functional characterization of SCA28 lymphoblastoid cell lines reveal impairment in cell growth and activation of apoptotic pathways

BackgroundSCA28 is an autosomal dominant ataxia associated with AFG3L2 gene mutations. We performed a whole genome expression profiling using lymphoblastoid cell lines (LCLs) from four SCA28 patients and six unrelated healthy controls matched for sex and age.MethodsGene expression was evaluated with the Affymetrix GeneChip Human Genome U133A 2.0 Arrays and data were validated by real-time PCR.ResultsWe found 66 genes whose expression was statistically different in SCA28 LCLs, 35 of which were up-regulated and 31 down-regulated. The differentially expressed genes were clustered in five functional categories: (1) regulation of cell proliferation; (2) regulation of programmed cell death; (3) response to oxidative stress; (4) cell adhesion, and (5) chemical homeostasis. To validate these data, we performed functional experiments that proved an impaired SCA28 LCLs growth compared to controls (pu2009<u20090.005), an increased number of cells in the G0/G1 phase (pu2009<u20090.001), and an increased mortality because of apoptosis (pu2009<u20090.05). We also showed that respiratory chain activity and reactive oxygen species levels was not altered, although lipid peroxidation in SCA28 LCLs was increased in basal conditions (pu2009<u20090.05). We did not detect mitochondrial DNA large deletions. An increase of TFAM, a crucial protein for mtDNA maintenance, and of DRP1, a key regulator of mitochondrial dynamic mechanism, suggested an alteration of fission/fusion pathways.ConclusionsWhole genome expression profiling, performed on SCA28 LCLs, allowed us to identify five altered functional categories that characterize the SCA28 LCLs phenotype, the first reported in human cells to our knowledge.

A practical approach for the detection of DNA nanostructures in single live human cells by fluorescence microscopy.

In the last decade, in vivo studies have revealed that even subtle differences in size, concentration of components, cell cycle stage, make the cells in a population respond differently to the same stimulus. In order to characterize such complexity of behavior and shed more light on the functioning and communication amongst cells, researchers are developing strategies to study single live cells in a population. In this paper, we describe the methods to design and prepare DNA-based fluorescent tetrahedral nanostructures, to deliver them to live cells and characterize such cells with epifluorescence microscopy. We report that HeLa cells internalize these nanostructures spontaneously with a higher efficiency with respect to single-stranded or double-stranded oligonucleotides. Our findings suggest that DNA tetrahedra could serve as a platform for the realization of a series of multifunctional intracellular biosensors for the analysis of single live cells.