Nicoleta Moisoi

The mitochondrial protease HtrA2 is regulated by Parkinson's disease-associated kinase PINK1

In mice, targeted deletion of the serine protease HtrA2 (also known as Omi) causes mitochondrial dysfunction leading to a neurodegenerative disorder with parkinsonian features. In humans, point mutations in HtrA2 are a susceptibility factor for Parkinsons disease (PARK13 locus). Mutations in PINK1, a putative mitochondrial protein kinase, are associated with the PARK6 autosomal recessive locus for susceptibility to early-onset Parkinsons disease. Here we determine that HtrA2 interacts with PINK1 and that both are components of the same stress-sensing pathway. HtrA2 is phosphorylated on activation of the p38 pathway, occurring in a PINK1-dependent manner at a residue adjacent to a position found mutated in patients with Parkinsons disease. HtrA2 phosphorylation is decreased in brains of patients with Parkinsons disease carrying mutations in PINK1. We suggest that PINK1-dependent phosphorylation of HtrA2 might modulate its proteolytic activity, thereby contributing to an increased resistance of cells to mitochondrial stress.

Neuroprotective Role of the Reaper-Related Serine Protease HtrA2/Omi Revealed by Targeted Deletion in Mice

ABSTRACT The serine protease HtrA2/Omi is released from the mitochondrial intermembrane space following apoptotic stimuli. Once in the cytosol, HtrA2/Omi has been implicated in promoting cell death by binding to inhibitor of apoptosis proteins (IAPs) via its amino-terminal Reaper-related motif, thus inducing caspase activity, and also in mediating caspase-independent death through its own protease activity. We report here the phenotype of mice entirely lacking expression of HtrA2/Omi due to targeted deletion of its gene, Prss25. These animals, or cells derived from them, show no evidence of reduced rates of cell death but on the contrary suffer loss of a population of neurons in the striatum, resulting in a neurodegenerative disorder with a parkinsonian phenotype that leads to death of the mice around 30 days after birth. The phenotype of these mice suggests that it is the protease function of this protein and not its IAP binding motif that is critical. This conclusion is reinforced by the finding that simultaneous deletion of the other major IAP binding protein, Smac/DIABLO, does not obviously alter the phenotype of HtrA2/Omi knockout mice or cells derived from them. Mammalian HtrA2/Omi is therefore likely to function in vivo in a manner similar to that of its bacterial homologues DegS and DegP, which are involved in protection against cell stress, and not like the proapoptotic Reaper family proteins in Drosophila melanogaster.

Mitochondrial dysfunction triggered by loss of HtrA2 results in the activation of a brain-specific transcriptional stress response.

Cellular stress responses can be activated following functional defects in organelles such as mitochondria and the endoplasmic reticulum. Mitochondrial dysfunction caused by loss of the serine protease HtrA2 leads to a progressive movement disorder in mice and has been linked to parkinsonian neurodegeneration in humans. Here, we demonstrate that loss of HtrA2 results in transcriptional upregulation of nuclear genes characteristic of the integrated stress response, including the transcription factor CHOP, selectively in the brain. We also show that loss of HtrA2 results in the accumulation of unfolded proteins in the mitochondria, defective mitochondrial respiration and enhanced production of reactive oxygen species that contribute to the induction of CHOP expression and to neuronal cell death. CHOP expression is also significantly increased in Parkinsons disease patients’ brain tissue. We therefore propose that this brain-specific transcriptional response to stress may be important in the advance of neurodegenerative diseases.

Comprehensive Expression Analyses of Neural Cell-Type-Specific miRNAs Identify New Determinants of the Specification and Maintenance of Neuronal Phenotypes

MicroRNAs (miRNAs) have been shown to play important roles in both brain development and the regulation of adult neural cell functions. However, a systematic analysis of brain miRNA functions has been hindered by a lack of comprehensive information regarding the distribution of miRNAs in neuronal versus glial cells. To address this issue, we performed microarray analyses of miRNA expression in the four principal cell types of the CNS (neurons, astrocytes, oligodendrocytes, and microglia) using primary cultures from postnatal d 1 rat cortex. These analyses revealed that neural miRNA expression is highly cell-type specific, with 116 of the 351 miRNAs examined being differentially expressed fivefold or more across the four cell types. We also demonstrate that individual neuron-enriched or neuron-diminished RNAs had a significant impact on the specification of neuronal phenotype: overexpression of the neuron-enriched miRNAs miR-376a and miR-434 increased the differentiation of neural stem cells into neurons, whereas the opposite effect was observed for the glia-enriched miRNAs miR-223, miR-146a, miR-19, and miR-32. In addition, glia-enriched miRNAs were shown to inhibit aberrant glial expression of neuronal proteins and phenotypes, as exemplified by miR-146a, which inhibited neuroligin 1-dependent synaptogenesis. This study identifies new nervous system functions of specific miRNAs, reveals the global extent to which the brain may use differential miRNA expression to regulate neural cell-type-specific phenotypes, and provides an important data resource that defines the compartmentalization of brain miRNAs across different cell types.

Genetic analysis of mitochondrial protein misfolding in Drosophila melanogaster

Protein misfolding has a key role in several neurological disorders including Parkinsons disease. Although a clear mechanism for such proteinopathic diseases is well established when aggregated proteins accumulate in the cytosol, cell nucleus, endoplasmic reticulum and extracellular space, little is known about the role of protein aggregation in the mitochondria. Here we show that mutations in both human and fly PINK1 result in higher levels of misfolded components of respiratory complexes and increase in markers of the mitochondrial unfolded protein response. Through the development of a genetic model of mitochondrial protein misfolding employing Drosophila melanogaster, we show that the in vivo accumulation of an unfolded protein in mitochondria results in the activation of AMP-activated protein kinase-dependent autophagy and phenocopies of pink1 and parkin mutants. Parkin expression acts to clear mitochondria with enhanced levels of misfolded proteins by promoting their autophagic degradation in vivo, and refractory to Sigma P (ref(2)P), the Drosophila orthologue of mammalian p62, is a critical downstream effector of this quality control pathway. We show that in flies, a pathway involving pink1, parkin and ref(2)P has a role in the maintenance of a viable pool of cellular mitochondria by promoting organellar quality control.

Drosophila HtrA2 is dispensable for apoptosis but acts downstream of PINK1 independently from Parkin.

High temperature requirement A2 (HtrA2/Omi) is a mitochondrial protease that exhibits proapoptotic and cell-protective properties and has been linked to Parkinsons disease (PD). Impaired mitochondrial function is a common trait in PD patients, and is likely to play a significant role in pathogenesis of parkinsonism, but the molecular mechanisms remain poorly understood. Genetic studies in Drosophila have provided valuable insight into the function of other PD-linked genes, in particular PINK1 and parkin, and their role in maintaining mitochondrial integrity. Recently, HtrA2 was shown to be phosphorylated in a PINK1-dependent manner, suggesting it might act in the PINK1 pathway. Here, we describe the characterization of mutations in Drosophila HtrA2, and genetic analysis of its function with PINK1 and parkin. Interestingly, we find HtrA2 appears to be dispensable for developmental or stress-induced apoptosis. In addition, we found HtrA2 mutants share some phenotypic similarities with parkin and PINK1 mutants, suggesting that it may function in maintaining mitochondrial integrity. Our genetic interaction studies, including analysis of double-mutant combinations and epistasis experiments, suggest HtrA2 acts downstream of PINK1 but in a pathway parallel to Parkin.

HtrA2 deficiency causes mitochondrial uncoupling through the F1F0-ATP synthase and consequent ATP depletion

Loss of the mitochondrial protease HtrA2 (Omi) in mice leads to mitochondrial dysfunction, neurodegeneration and premature death, but the mechanism underlying this pathology remains unclear. Using primary cultures from wild-type and HtrA2-knockout mice, we find that HtrA2 deficiency significantly reduces mitochondrial membrane potential in a range of cell types. This depolarisation was found to result from mitochondrial uncoupling, as mitochondrial respiration was increased in HtrA2-deficient cells and respiratory control ratio was dramatically reduced. HtrA2-knockout cells exhibit increased proton translocation through the ATP synthase, in combination with decreased ATP production and truncation of the F1 α-subunit, suggesting the ATP synthase as the source of the proton leak. Uncoupling in the HtrA2-deficient mice is accompanied by altered breathing pattern and, on a cellular level, ATP depletion and vulnerability to chemical ischaemia. We propose that this vulnerability may ultimately cause the neurodegeneration observed in these mice.

MAP4K3 modulates cell death via the post-transcriptional regulation of BH3-only proteins

Intracellular signal transduction networks involving protein kinases are important modulators of cell survival and cell death in multicellular organisms. Functional compromise of these networks has been linked to aberrant apoptosis in diseases such as cancer. To identify novel kinase regulators of cell death, we conducted an RNAi-based screen to identify modulators of the intrinsic apoptosis pathway. Using this approach, we identified MAP4K3 as a novel apoptosis inducer. Here, we present evidence that this pro-apoptotic kinase orchestrates activation of BAX via the concerted posttranscriptional modulation of PUMA, BAD, and BIM. Additionally, we found decreased levels of this kinase in pancreatic cancer samples, suggesting a tumor suppressor role for MAP4K3 in pancreatic tumorigenesis.

Loss of PINK1 enhances neurodegeneration in a mouse model of Parkinson's disease triggered by mitochondrial stress.

Parkinsons disease (PD) shows a complex etiology, where both genetic and environmental factors contribute to initiation and advance of pathology. Mitochondrial dysfunction and mutation of genes implicated in mitochondria quality control are recognized contributors to etiopathology and progression of PD. Here we report the development and characterization of a genetic mouse model of PD with a combined etiology comprising: 1) induction of mitochondrial stress achieved through the expression of a mitochondrial matrix protein that accumulates in an unfolded state and 2) deletion of PINK1 gene. Using this model we address the role of PINK1 in mitochondrial quality control and disease progression. To induce mitochondrial stress specifically in catecholaminergic neurons we generated transgenic animals where the conditional expression of mitochondrial unfolded ornithine transcarbamylase (dOTC) is achieved under the tyrosine hydroxylase (Th) promoter. The mice were characterized in terms of survival, growth and motor behaviour. The characterization was followed by analysis of cell death induced in dopaminergic neurons and responsiveness to l-dopa. We demonstrate that accumulation of dOTC in dopaminergic neurons causes neurodegeneration and motor behaviour impairment that illustrates a parkinsonian phenotype. This associates with l-dopa responsiveness validating the model as a model of PD. The combined transgenic model where dOTC is overexpressed in PINK1 KO background presents increased neurodegeneration as compared to dOTC transgenic in wild-type background. Moreover, this combined model does not show responsiveness to l-dopa. Our in vivo data show that loss of PINK1 accelerates neurodegenerative phenotypes induced by mitochondrial stress triggered by the expression of an unfolded protein in this organelle.

Compartmental stress responses correlate with cell survival in bystander effects induced by the DNA damage agent, bleomycin

Physical or chemical stress applied to a cell system trigger a signal cascade that is transmitted to the neighboring cell population in a process known as bystander effect. Despite its wide occurrence in biological systems this phenomenon is mainly documented in cancer treatments. Thus understanding whether the bystander effect acts as an adaptive priming element for the neighboring cells or a sensitization factor is critical in designing treatment strategies. Here we characterize the bystander effects induced by bleomycin, a DNA-damaging agent, and compartmental stress responses associated with this phenomenon. Mouse fibroblasts were treated with increasing concentrations of bleomycin and assessed for DNA damage, cell death and induction of compartmental stress response (endoplasmic reticulum, mitochondrial and cytoplasmic stress). Preconditioned media were used to analyze bystander damage using the same end-points. Bleomycin induced bystander response was reflected primarily in increased DNA damage. This was dependent on the concentration of bleomycin and time of media conditioning. Interestingly, we found that ROS but not NO are involved in the transmission of the bystander effect. Consistent transcriptional down-regulation of the stress response factors tested (i.e. BiP, mtHsp60, Hsp70) occurred in the direct effect indicating that bleomycin might induce an arrest of transcription correlated with decreased survival. We observed the opposite trend in the bystander effect, with specific stress markers appearing increased and correlated with increased survival. These data shed new light on the potential role of stress pathways activation in bystander effects and their putative impact on the pro-survival pro-death balance.