PINK1 Protects against Staurosporine-Induced Apoptosis by Interacting with Beclin1 and Impairing Its Pro-Apoptotic Cleavage.

PINK1 is a causative gene for Parkinson's disease and the corresponding protein has been identified as a master regulator of mitophagy-the autophagic degradation of damaged mitochondria. It interacts with Beclin1 to regulate autophagy and initiate autophagosome formation, even outside the context of mitophagy. Several other pro-survival functions of this protein have been described and indicate that it might play a role in other disorders, such as cancer and proliferative diseases. In this study, we investigated a novel anti-apoptotic function of PINK1. To do so, we used SH-SY5Y neuroblastoma cells, a neuronal model used in Parkinson's disease and cancer studies, to characterize the pro-survival functions of PINK1 in response to the apoptosis inducer staurosporine. In this setting, we found that staurosporine induces apoptosis but not mitophagy, and we demonstrated that PINK1 protects against staurosporine-induced apoptosis by impairing the pro-apoptotic cleavage of Beclin1. Our data also show that staurosporine-induced apoptosis is preceded by a phase of enhanced autophagy, and that PINK1 in this context regulates the switch from autophagy to apoptosis. PINK1 protein levels progressively decrease after treatment, inducing this switch. The PINK1-Beclin1 interaction is crucial in exerting this function, as mutants that are unable to interact do not show the anti-apoptotic effect. We characterized a new anti-apoptotic function of PINK1 that could provide options for treatment in proliferative or neurodegenerative diseases.

PINK1 and BECN1 relocalize at mitochondria-associated membranes during mitophagy and promote ER-mitochondria tethering and autophagosome formation.

Mitophagy is a highly specialized process to remove dysfunctional or superfluous mitochondria through the macroautophagy/autophagy pathway, aimed at protecting cells from the damage of disordered mitochondrial metabolism and apoptosis induction. PINK1, a neuroprotective protein mutated in autosomal recessive Parkinson disease, has been implicated in the activation of mitophagy by selectively accumulating on depolarized mitochondria, and promoting PARK2/Parkin translocation to them. While these steps have been characterized in depth, less is known about the process and site of autophagosome formation upon mitophagic stimuli. A previous study reported that, in starvation-induced autophagy, the proautophagic protein BECN1/Beclin1 (which we previously showed to interact with PINK1) relocalizes at specific regions of contact between the endoplasmic reticulum (ER) and mitochondria called mitochondria-associated membranes (MAM), from which the autophagosome originates. Here we show that, following mitophagic stimuli, autophagosomes also form at MAM; moreover, endogenous PINK1 and BECN1 were both found to relocalize at MAM, where they promoted the enhancement of ER-mitochondria contact sites and the formation of omegasomes, that represent autophagosome precursors. PARK2 was also enhanced at MAM following mitophagy induction. However, PINK1 silencing impaired BECN1 enrichment at MAM independently of PARK2, suggesting a novel role for PINK1 in regulating mitophagy. MAM have been recently implicated in many key cellular events. In this light, the observed prevalent localization of PINK1 at MAM may well explain other neuroprotective activities of this protein, such as modulation of mitochondrial calcium levels, mitochondrial dynamics, and apoptosis.

Candidate genes for Parkinson disease: Lessons from pathogenesis.

Parkinson disease (PD) is a multifactorial neurodegenerative disease characterized by the progressive loss of specific neuronal populations and accumulation of Lewy bodies in the brain, leading to motor and non-motor symptoms. In a small subset of patients, PD is dominantly or recessively inherited, while a number of susceptibility genetic loci have been identified through genome wide association studies. The discovery of genes mutated in PD and functional studies on their protein products have provided new insights into the molecular events leading to neurodegeneration, suggesting that few interconnected molecular pathways may be deranged in all forms of PD, triggering neuronal loss. Here, we summarize the most relevant findings implicating the main PD-related proteins in biological processes such as mitochondrial dysfunction, misfolded protein damage, alteration of cellular clearance systems, abnormal calcium handling and altered inflammatory response, which represent key targets for neuroprotection.

A variant in the carboxyl-terminus of connexin 40 alters GAP junctions and increases risk for tetralogy of Fallot.

GJA5 gene (MIM no. 121013), localized at 1q21.1, encodes for the cardiac gap junction protein connexin 40. In humans, copy number variants of chromosome 1q21.1 have been associated with variable phenotypes comprising congenital heart disease (CHD), including isolated TOF. In mice, the deletion of Gja5 can cause a variety of complex CHDs, in particular of the cardiac outflow tract, corresponding to TOF in many cases. In the present study, we screened for mutations in the GJA5 gene 178 unrelated probands with isolated TOF. A heterozygous nucleotide change (c.793C>T) in exon 2 of the gene leading to the p.Pro265Ser variant at the carboxyl-terminus of the protein was found in two unrelated sporadic patients, one with classic anatomy and one with pulmonary atresia. This GJA5 missense substitution was not observed in 1568 ethnically-matched control chromosomes. Immunofluorescent staining and confocal microscopy revealed that cells expressing the mutant protein form sparse or no visible gap-junction plaques in the region of cell-cell contact. Moreover, analysis of the transfer of the gap junction permanent tracer lucifer yellow showed that cells expressing the mutant protein have a reduced rate of dye transfer compared with wild-type cells. Finally, use of a zebrafish model revealed that microinjection of the GJA5-p.Pro265Ser mutant disrupts overall morphology of the heart tube in the 37% (22/60) of embryos, compared with the 6% (4/66) of the GJA5 wild-type-injected embryos. These findings implicate GJA5 gene as a novel susceptibility gene for TOF.

Molecular pathways in sporadic PD.

Over the last decade, several autosomal dominant and recessive genes causative of Parkinson's disease (PD) have been identified. The functional studies on their protein products and the pathogenetic effect related to their mutations have greatly contributed to understand the many cellular pathways leading to neurodegeneration, that include oxidative stress damage, mitochondrial dysfunction, misfolded protein stress and impairment of cellular clearance systems, namely the ubiquitin-proteasome system (UPS) and the autophagy pathway. Although mendelian genes are responsible only for a small subset of PD patients, it is expected that the same pathogenetic mechanisms could play a relevant role also in the more frequent sporadic PD, that is currently recognized as a multifactorial disorder. In this model, different genetic and environmental factors, either playing a protective or a susceptibility role, variably interact to reach a threshold of disease over which PD will become clinically manifest. As an example, mutations or multiplication of the alpha-synuclein gene cause autosomal dominant PD, while common genetic variants at the same locus have been consistently associated to the risk of developing PD by genome-wide association studies. These findings are opening novel interesting perspectives to identify critical molecular pathways leading to neurodegeneration.

Late onset sporadic Parkinson's disease caused by PINK1 mutations: clinical and functional study.

Homozygous or compound heterozygous mutations in the PINK1 gene represent the second most frequent cause of autosomal recessive parkinsonism after Parkin. The phenotype differs from idiopathic Parkinson's disease for earlier onset, slower disease progression, and better response to therapy. Indeed, the rare patients with onset above 50 years are usually relatives of early-onset probands. Here, we report the first occurrence of compound heterozygous PINK1 mutations in a sporadic patient with a phenotype indistinguishable from idiopathic Parkinson's disease (PD), with onset in the late seventh decade, rapid progression and good response to levodopa that waned with time. Both mutations (p.A244G and p.V317I) were found to abolish the protective effect of wild-type PINK1 against staurosporine-induced apoptosis. These findings further expand the clinical spectrum of PINK1-related parkinsonism to include late onset, typical PD, and underline the existing difficulties in discriminating between mendelian parkinsonism and idiopathic PD.

Heterochromatic gene repression of the retinoic acid pathway in acute myeloid leukemia.

Alteration of lineage-specific transcriptional programs for hematopoiesis causes differentiation block and promotes leukemia development. Here, we show that AML1/ETO, the most common translocation fusion product in acute myeloid leukemia (AML), counteracts the activity of retinoic acid (RA), a transcriptional regulator of myelopoiesis. AML1/ETO participates in a protein complex with the RA receptor alpha (RARalpha) at RA regulatory regions on RARbeta2, which is a key RA target gene mediating RA activity/resistance in cells. At these sites, AML1/ETO recruits histone deacetylase, DNA methyltransferase, and DNA-methyl-CpG binding activities that promote a repressed chromatin conformation. The link among AML1/ETO, heterochromatic RARbeta2 repression, RA resistance, and myeloid differentiation block is indicated by the ability of either siRNA-AML1/ETO or the DNA methylation inhibitor 5-azacytidine to revert these epigenetic alterations and to restore RA differentiation response in AML1/ETO blasts. Finally, RARbeta2 is commonly silenced by hypermethylation in primary AML blasts but not in normal hematopoietic precursors, thus suggesting a role for the epigenetic repression of the RA signaling pathway in myeloid leukemogenesis.

A minicircuitry comprised of microRNA-223 and transcription factors NFI-A and C/EBPalpha regulates human granulopoiesis.

MicroRNAs play important roles in cell differentiation by acting as translational inhibitors of specific target genes. Here we show that human granulocytic differentiation is controlled by a regulatory circuitry involving miR-223 and two transcriptional factors, NFI-A and C/EBPalpha. The two factors compete for binding to the miR-223 promoter: NFI-A maintains miR-223 at low levels, whereas its replacement by C/EBPalpha, following retinoic acid (RA)-induced differentiation, upregulates miR-223 expression. The competition by C/EBPalpha and the granulocytic differentiation are favored by a negative-feedback loop in which miR-223 represses NFI-A translation. In line with this, both RNAi against NFI-A and ectopic expression of miR-223 in acute promyelocytic leukemia (APL) cells enhance differentiation, whereas miR-223 knockdown inhibits the differentiation response to RA. Altogether, our data indicate that miR-223 plays a crucial role during granulopoiesis and point to the NFI-A repression as an important molecular pathway mediating gene reprogramming in this cell lineage.

Histone deacetylase inhibitor valproic acid enhances the cytokine-induced expansion of human hematopoietic stem cells.

Ex vivo amplification of human hematopoietic stem cells (HSC) without loss of their self-renewing potential represents an important target for transplantation, gene and cellular therapies. Valproic acid is a safe and widely used neurologic agent that acts as a potent inhibitor of histone deacetylase activities. Here, we show that valproic acid addition to liquid cultures of human CD34+ cells isolated from cord blood, mobilized peripheral blood, and bone marrow strongly enhances the ex vivo expansion potential of different cytokine cocktails as shown by morphologic, cytochemical, immunophenotypical, clonogenic, and gene expression analyses. Notably, valproic acid highly preserves the CD34 positivity after 1 week (range, 40-89%) or 3 weeks (range, 21-52%) amplification cultures with two (Flt3L + thrombopoietin) or four cytokines (Flt3L + thrombopoietin + stem cell factor + interleukin 3). Moreover, valproic acid treatment increases histone H4 acetylation levels at specific regulatory sites on HOXB4, a transcription factor gene with a key role in the regulation of HSC self-renewal and AC133, a recognized marker gene for stem cell populations. Overall, our results relate the changes induced by valproic acid on chromatin accessibility with the enhancement of the cytokine effect on the maintenance and expansion of a primitive hematopoietic stem cell population. These findings underscore the potentiality of novel epigenetic approaches to modify HSC fate in vitro.

Molecular signature of retinoic acid treatment in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia characterized by a block of differentiation at the promyelocytic stage. APL patients respond to pharmacological concentrations of all-trans retinoic acid (RA) and disease remission correlates with terminal differentiation of leukemic blasts. The PML/RAR oncogenic transcription factor is responsible for both the pathogenesis of APL and for its sensitivity to RA. In order to identify physiological targets of RA therapy, we analysed gene expression profiles of RA-treated APL blasts and found 1056 common target genes. Comparing these results to those obtained in RA-treated U937 cell lines revealed that transcriptional response to RA is largely dependent on the expression of PML/RAR. Several genes involved in the control of differentiation and stem cell renewal are early targets of RA regulation, and may be important effectors of RA response. Modulation of chromatin modifying genes was also observed, suggesting that specific structural changes in local chromatin domains may be required to promote RA-mediated differentiation. Computational analysis of upstream genomic regions in RA target genes revealed nonrandom distribution of transcription factor binding sites, indicating that specific transcriptional regulatory complexes may be involved in determining RA response.

Targeting fusion protein/corepressor contact restores differentiation response in leukemia cells.

The AML1/ETO and PML/RARalpha leukemia fusion proteins induce acute myeloid leukemia by acting as transcriptional repressors. They interact with corepressors, such as N-CoR and SMRT, that recruit a multiprotein complex containing histone deacetylases on crucial myeloid differentiation genes. This leads to gene repression contributing to generate a differentiation block. We expressed in leukemia cells containing PML/RARalpha and AML1/ETO N-CoR protein fragments derived from fusion protein/corepressor interaction surfaces. This blocks N-CoR/SMRT binding by these fusion proteins, and disrupts the repressor protein complex. In consequence, the expression of genes repressed by these fusion proteins increases and differentiation response to vitamin D3 and retinoic acid is restored in previously resistant cells. The alteration of PML/RARalpha-N-CoR/SMRT connections triggers proteasomal degradation of the fusion protein. The N-CoR fragments are biologically effective also when directly transduced by virtue of a protein transduction domain. Our data indicate that fusion protein activity is permanently required to maintain the leukemia phenotype and show the route to developing a novel therapeutic approach for leukemia, based on its molecular pathogenesis.

Inhibitors of histone deacetylases induce tumor-selective apoptosis through activation of the death receptor pathway.

Histone deacetylases (HDACs) regulate transcription and specific cellular functions, such as tumor suppression by p53, and are frequently altered in cancer. Inhibitors of HDACs (HDACIs) possess antitumor activity and are well tolerated, supporting the idea that their use might develop as a specific strategy for cancer treatment. The molecular basis for their selective antitumor activity is, however, unknown. We investigated the effects of HDACIs on leukemias expressing the PML-RAR or AML1-ETO oncoproteins, known to initiate leukemogenesis through deregulation of HDACs. Here we report that: (i) HDACIs induce apoptosis of leukemic blasts, although oncogene expression is not sufficient to confer HDACI sensitivity to normal cells; (ii) apoptosis is p53 independent and depends, both in vitro and in vivo, upon activation of the death receptor pathway (TRAIL and Fas signaling pathways); (iii) TRAIL, DR5, FasL and Fas are upregulated by HDACIs in the leukemic cells, but not in normal hematopoietic progenitors. These results show that sensitivity to HDACIs in leukemias is a property of the fully transformed phenotype and depends on activation of a specific death pathway.

Acute myeloid leukemia fusion proteins deregulate genes involved in stem cell maintenance and DNA repair.

Acute myelogenous leukemias (AMLs) are genetically heterogeneous and characterized by chromosomal rearrangements that produce fusion proteins with aberrant transcriptional regulatory activities. Expression of AML fusion proteins in transgenic mice increases the risk of myeloid leukemias, suggesting that they induce a preleukemic state. The underlying molecular and biological mechanisms are, however, unknown. To address this issue, we performed a systematic analysis of fusion protein transcriptional targets. We expressed AML1/ETO, PML/RAR, and PLZF/RAR in U937 hemopoietic precursor cells and measured global gene expression using oligonucleotide chips. We identified 1,555 genes regulated concordantly by at least two fusion proteins that were further validated in patient samples and finally classified according to available functional information. Strikingly, we found that AML fusion proteins induce genes involved in the maintenance of the stem cell phenotype and repress DNA repair genes, mainly of the base excision repair pathway. Functional studies confirmed that ectopic expression of fusion proteins constitutively activates pathways leading to increased stem cell renewal (e.g., the Jagged1/Notch pathway) and provokes accumulation of DNA damage. We propose that expansion of the stem cell compartment and induction of a mutator phenotype are relevant features underlying the leukemic potential of AML-associated fusion proteins.