Presenilin-associated Rhomboid-like Research Articles

Loss of parla Function Results in Inactivity, Olfactory Impairment, and Dopamine Neuron Loss in Zebrafish.

The presenilin-associated rhomboid-like (PARL) gene was found to contribute to mitochondrial morphology and function and was linked to familial Parkinson’s disease (PD). The PARL gene product is a mitochondrial intramembrane cleaving protease that acts on a number of mitochondrial proteins involved in mitochondrial morphology, apoptosis, and mitophagy. To date, functional and genetic studies of PARL have been mainly performed in mammals. However, little is known about PARL function and its role in dopaminergic (DA) neuron development in vertebrates. The zebrafish genome comprises two PARL paralogs: parla and parlb. Here, we established a loss-of-function mutation in parla via CRISPR/Cas9-mediated mutagenesis. We examined DA neuron numbers in the adult brain and expression of genes associated with DA neuron function in larvae and adults. We show that loss of parla function results in loss of DA neurons, mainly in the olfactory bulb. Changes in the levels of tyrosine hydroxylase transcripts supported this neuronal loss. Expression of fis1, a gene involved in mitochondrial fission, was increased in parla mutants. Finally, we showed that loss of parla function translates into impaired olfaction and altered locomotion parameters. These results suggest a role for parla in the development and/or maintenance of DA neuron function in zebrafish.

Expression and Purification of Human Mitochondrial Intramembrane Protease PARL.

Rhomboid proteases are a ubiquitous superfamily of serine intramembrane peptidases that play a role in a wide variety of cellular processes. The mammalian mitochondrial rhomboid protease, Presenilin-Associated Rhomboid Like (PARL), is a critical regulator of mitochondrial homeostasis through the cleavage of its substrates, which have roles in mitochondrial quality control and apoptosis. However, neither structural nor functional information for this important protease is available, because the expression of eukaryotic membrane proteins to sufficient levels in an active form often represents a major bottleneck for in vitro studies. Here we present an optimized protocol for expression and purification of the human PARL protease using the eukaryotic expression host Pichia pastoris. The PARL gene construct was generated in tandem with green fluorescent protein (GFP), which allowed for the selection of high expressing clones and monitoring during the large-scale expression and purification steps. We discuss the production protocol with precise details for each step. The protocol yields 1mg of pure PARL per liter of yeast culture.

Influence of Familial Parkinson's Disease Mutations on Mitochondrial Localization and Secondary Structure of PINK1

Mutations in the PARK6 gene, encoding for the protein PINK1 (Phosphatase-Tensin homologue (PTEN)-induced protein kinase), are associated with early-onset familial Parkinson's disease (PD). PINK1 is found in multiple cellular locations, including the cytosol and both mitochondrial membranes (outer mitochondrial membrane, OMM; and inner mitochondrial membrane, IMM). PINK1 must enter into the IMM in order to be cleaved by PARL (Presenilin-associated rhomboid-like), an IMM protease. Cleavage of PINK1 by PARL activates the degradation of PINK1, indicating a healthy, polarized mitochondrion. Improper processing of PINK1 PD mutants leads to unprocessed PINK1 accumulating on the OMM which induces mitophagy, or the selective autophagy of mitochondria. We hypothesize that accumulation of unprocessed PINK1 on the OMM is either due to improper localization of PINK1 or due to the inability of PARL to cleave the mutated protein. Many traditional protein localization techniques including confocal microscopy cannot distinguish between the inner and outer mitochondrial membrane. Super-resolution microscopy will be used to determine the precise localization patterns to the appropriate mitochondrial membrane of both wildtype PINK1 and PINK1 with point mutations associated to PD. Preliminary confocal microscopy determined that PINK1 constructs are capable of being translated in vivo in HeLa cells. These preliminary results have proved to be promising and further experiments will be conducted using super-resolution microscopy and structured illumination microscopy to determine precise mitochondrial localization patterns in order to determine if PD mutations lead to improper localization of PINK1. Molecular dynamics simulations on PINK1 transmembrane segments harbouring familial Parkinson's disease mutations will be conducted to determine if localization defects are due to structural perturbations. This work sets the stage for an understanding for the role of PINK1 mutations in Parkinson's disease at a molecular level.

Mutation analyses and association studies to assess the role of the presenilin-associated rhomboid-like gene in Parkinson's disease

Presenilin-associated rhomboid-like (PARL), a serine protease located in the inner mitochondrial membrane, has been shown to genetically interact and process PTEN-induced putative kinase a protein known for its critical role in mitochondrial homeostasis and early-onset forms of Parkinson's disease (PD). The identification of a PD-associated variant in the PARL gene (p.Ser77Asn) led us to assess the relevance of PARL for PD pathogenesis using a mutation screening of the coding sequences and adjacent intronic sequences. We investigated 3 single nucleotide polymorphisms (rs3792589, rs13091, and rs3732581), a synonymous base substitution (Leu79Leu) and the previously described p.Ser77Asn mutation, which were subsequently screened in more than 2000 patients and controls. Not detecting the p.Ser77Asn mutation in our cohort, nor a robust association between variations in the PARL gene and PD, the role of disease causing genetic variants in the PARL gene could not be further substantiated in our samples. Our findings indicate that PARL mutations are a rare cause of PD and genetic variants are neither strong nor common risk factors in PD.

In utero exposure to prepregnancy maternal obesity and postweaning high-fat diet impair regulators of mitochondrial dynamics in rat placenta and offspring

The proportion of pregnant women who are obese at conception continues to rise. Compelling evidence suggests the intrauterine environment is an important determinant of offspring health. Maternal obesity and unhealthy diets are shown to promote metabolic programming in the offspring. Mitochondria are maternally inherited, and we have previously shown impaired mitochondrial function in rat offspring exposed to maternal obesity in utero. Mitochondrial health is maintained by mitochondrial dynamics, or the processes of fusion and fission, which serve to repair damaged mitochondria, remove irreparable mitochondria, and maintain mitochondrial morphology. An imbalance between fusion and fission has been associated with obesity, insulin resistance, and reproduction complications. In the present study, we examined the influence of maternal obesity and postweaning high-fat diet (HFD) on key regulators of mitochondrial fusion and fission in rat offspring at important developmental milestones which included postnatal day (PND)35 (2 wk HFD) and PND130 (∼16 wk HFD). Our results indicate HFD-fed offspring had reduced mRNA expression of presenilin-associated rhomboid-like (PARL), optic atrophy (OPA)1, mitofusin (Mfn)1, Mfn2, fission (Fis)1, and nuclear respiratory factor (Nrf)1 at PND35, while OPA1 and Mfn2 remained decreased at PND130. Putative transcriptional regulators of mitochondrial dynamics were reduced in rat placenta and offspring liver and skeletal muscle [peroxisome proliferator-activated receptor gamma coactivator (PGC1)α, PGC1β, and estrogen-related receptor (ERR)α], consistent with indirect calorimetry findings revealing reduced energy expenditure and impaired fat utilization. Overall, maternal obesity detrimentally alters mitochondrial targets that may contribute to impaired mitochondrial health and increased obesity susceptibility in later life.

PARL and HtrA2: Another Intricate Ischemic Neuronal Apoptotic Process Starting within Mitochondria

Mitochondria integrity is essential for maintaining neuronal viability. Under ischemic stress, neurons undergo apoptosis with death signaling processes originating within mitochondria. The intricate actions of dysfunctional mitochondria in causing ischemic neuronal death were delineated in a recent review in this Journal1 as targets for developing neuroprotective strategies. The featured article by Yoshioka et al2 in this issue reports a novel death signaling process in striatal neurons under ischemic stress, involving presenilin-associated rhomboid-like (PARL) protein and high temperature requirement factor A2 (HtrA2). In elegantly designed experimental paradigms, the authors have established that PARL, an inner mitochondrial membrane protein, has a pivotal role in limiting striatal neuronal apoptosis by binding to HtrA2. Under global ischemia, PARL is degraded to release the processed form of HtrA2 from mitochondria into cytosol to bind to an inhibitor of apoptosis, the X-chromosome-linked inhibitor-of-apoptosis protein (XIAP). This results in striatal neuronal apoptosis. Presenilin-associated rhomboid-like protein is an intramembrane serine protease in the rhomboid subfamily in mitochondria. The complex name containing Presenilin is based on the original findings in yeast two-hybrid studies using a C-terminal region of Presenilin-2 as the bait to identify PARL.3 Presenilin-associated rhomboid-like protein has been shown to interact with a number of other mitochondrial proteins to constrain proapoptotic activities, starting within mitochondria. Presenilin-associated rhomboid-like protein acts in concert with OPA1, another antiapoptotic protein in mitochondria, to serve as ‘a lid on apoptosis',4 or with HtrA2 to act as ‘gate keepers of mitochondrial dynamics and apoptosis'.3 This is the first report linking PARL–HtraA2 interaction inside mitochondria to neuronal apoptosis in a setting of global cerebral ischemia. It adds another death signaling cascade that starts within mitochondria to those described in a comprehensive review of neuroprotective strategies targeting mitochondria.1 The novel findings reported in this featured article raise a number of questions for future exploration. First, is Hax 1, a mitochondrial protein resembling antiapoptotic proteins in the Bcl-2 family carrying BH-1and BH-2 domains, involved in PARL–HtrA2 interaction in ischemic striatal neuronal apoptosis? Hax 1 has been shown to present HtrA2 to PARL to retain the former in the mitochondrial intermembrane space for maintaining cell viability.5 It would be interesting to know if Hax 1 is engaged in this death signaling process in striatal neurons. Second, is this novel apoptotic cascade restricted to striatal neurons only? Does PARL–HtrA2 interaction contribute to cortical neuronal apoptosis under global ischemic stress as well? Presenilin-associated rhomboid-like protein has been linked to Parkinson's disease involving Parkin and PINK1.6 It is not impossible that the antiapoptotic action of PARL is restricted only to striatal neurons. Finally, to what extent can ischemic striatal neuronal apoptosis be ascribed to PARL–HtrA2 interaction, beyond well-established death signaling processes originating within mitochondria? In summary, this featured article reports a novel death signaling cascade in ischemic striatal neurons apoptosis. It broadens our insight into the complex mechanisms of ischemic neuronal death involving mitochondria.

The Role of Parl and HtrA2 in Striatal Neuronal Injury After Transient Global Cerebral Ischemia

The presenilin-associated rhomboid-like (PARL) protein and high temperature requirement factor A2 (HtrA2) are key regulators of mitochondrial integrity and play pivotal roles in apoptosis. However, their roles after cerebral ischemia have not been thoroughly elucidated. To clarify these roles, mice were subjected to transient global cerebral ischemia, and striatal neuronal injury was assessed. Western blot and coimmunoprecipitation analyses revealed that PARL and processed HtrA2 localized to mitochondria, and that PARL was bound to HtrA2 in sham animals. Expression of PARL and processed HtrA2 in mitochondria significantly decreased 6 to 72 hours after ischemia, and the binding of PARL to HtrA2 disappeared after ischemia. In contrast, expression of processed HtrA2 increased 24 hours after ischemia in the cytosol, where HtrA2 was bound to X chromosome-linked inhibitor-of-apoptosis protein (XIAP). Administration of PARL small interfering RNA inhibited HtrA2 processing and worsened ischemic neuronal injury. Our results show that downregulation of PARL after ischemia is a key step in ischemic neuronal injury, and that it decreases HtrA2 processing and increases neuronal vulnerability. In addition, processed HtrA2 released into the cytosol after ischemia contributes to neuronal injury via inhibition of XIAP.

Rhomboid proteins: a role in keratinocyte proliferation and cancer

The Rhomboids represent a relatively recently discovered family of proteins, consisting in a variety of intramembrane serine proteases and their inactive homologues, the iRhoms. Rhomboids typically contain six or seven transmembrane domains (TMD) and have been classified into four subgroups: Secretase A and B, Presenilin-Associated-Rhomboid-Like (PARL) and iRhoms. Although the iRhoms, iRhom1 and iRhom2, have lost their protease activity during evolution, they retain key non-protease functions and have been implicated in the regulation of epidermal growth factor (EGF) signalling. EGF is moreover a substrate of RHBDL2, their active Rhomboid relative. Other substrates of RHBDL2 include members of the EphrinB family and thrombomodulin. RHBDL2 has also previously been demonstrated to be important in wound healing in cutaneous keratinocytes through the cleavage of thrombomodulin. Additional roles for these intriguing proteins seem likely to be revealed in the future. This review focuses on our current understanding of Rhomboids and, in particular, on RHBDL2 and iRhom2 and their roles in cellular processes and human disease.

Rhomboid Protease PARL Mediates the Mitochondrial Membrane Potential Loss-induced Cleavage of PGAM5

Regulated intramembrane proteolysis is a widely conserved mechanism for controlling diverse biological processes. Considering that proteolysis is irreversible, it must be precisely regulated in a context-dependent manner. Here, we show that phosphoglycerate mutase 5 (PGAM5), a mitochondrial Ser/Thr protein phosphatase, is cleaved in its N-terminal transmembrane domain in response to mitochondrial membrane potential (ΔΨ(m)) loss. This ΔΨ(m) loss-dependent cleavage of PGAM5 was mediated by presenilin-associated rhomboid-like (PARL). PARL is a mitochondrial resident rhomboid serine protease and has recently been reported to mediate the cleavage of PINK1, a mitochondrial Ser/Thr protein kinase, in healthy mitochondria with intact ΔΨ(m). Intriguingly, we found that PARL dissociated from PINK1 and reciprocally associated with PGAM5 in response to ΔΨ(m) loss. These results suggest that PARL mediates differential cleavage of PINK1 and PGAM5 depending on the health status of mitochondria. Our data provide a prototypical example of stress-dependent regulation of PARL-mediated regulated intramembrane proteolysis.

Zebrafish Parla‐ and Parlb‐deficiency affects dopaminergic neuron patterning and embryonic survival

Many genes associated with familial Parkinson's disease contribute to mitochondrial morphology and function. Some of these genes, for example, Pink1 and Parkin, are part of a common pathway. The presenilin-associated rhomboid-like (PARL) gene was recently linked to familial Parkinson's disease. The PARL gene product is found in the inner mitochondrial membrane and cleaves the optic atrophy 1 protein, involved in mitochondrial morphology and apoptosis. In Drosophila, the PARL-related rhomboid-7 gene acts upstream of pink1 and parkin. However, such a genetic relationship is still unknown in vertebrates. Here, we show that the zebrafish genome comprises two parl paralogs: parla and parlb. Morpholino-mediated loss of parla and/or parlb function resulted in mild neurodegeneration, as evidenced by a lower density of dopaminergic neurons. Patterning of dopaminergic neurons was also perturbed in the ventral diencephalon. Morphants exhibited extensive cell death throughout the entire body as well as increased larval mortality. The morphant phenotype could be rescued by injection of human PARL mRNA, but not catalytically inactive PARL, suggesting functional conservation between the human and zebrafish proteins. More importantly, the zebrafish pink1 mRNA as well as the human PINK1 mRNA, but not kinase-dead nor Parkinson's disease-linked mutant PINK1 mRNA, also rescued the morphant phenotype, providing evidence that Parl genes may function upstream of Pink1, as part of a conserved pathway in vertebrates.

The antiapoptotic OPA1/Parl couple participates in mitochondrial adaptation to heat shock

The mitochondria-shaping protein optic atrophy 1 (OPA1) has genetically distinguishable roles in mitochondrial morphology and apoptosis. The latter depends on the presenilin associated rhomboid like (PARL) protease, essential for the accumulation of a soluble intermembrane space form of OPA1 (IMS-OPA1). Here we show that OPA1 and PARL participate in the heat shock response, a stereotypical cellular process of adaptation to thermal stress. Upon heat shock, long forms of OPA1 are lost and mitochondria fragment. However, mitochondrial fusion is dispensable to maintain viability, whereas IMS-OPA1 is required. Upon conditioning—a process of mild heat shock and recovery—IMS-OPA1 accumulates, OPA1 oligomers increase and mitochondria release less cytochrome c, ultimately resulting in cellular resistance to subsequent apoptotic inducers. In Parl−/− cells accumulation of IMS-OPA1 is blunted and conditioning fails to protect from cytochrome c release and apoptosis. Thus, the OPA1/PARL dependent pathway of cristae remodeling is implicated in heat shock. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Evaluation of nine candidate genes in patients with normal tension glaucoma: a case control study

BackgroundNormal tension glaucoma is a major subtype of glaucoma, associated with intraocular pressures that are within the statistically normal range of the population. Monogenic forms following classical inheritance patterns are rare in this glaucoma subtype. Instead, multigenic inheritance is proposed for the majority of cases. The present study tested common sequence variants in candidate genes for association with normal tension glaucoma in the German population.MethodsNinety-eight SNPs were selected to tag the common genetic variation in nine genes, namely OPTN (optineurin), RDX (radixin), SNX16 (sorting nexin 16), OPA1 (optic atrophy 1), MFN1 (mitofusin 1), MFN2 (mitofusin 2), PARL (presenilin associated, rhomboid-like), SOD2 (superoxide dismutase 2, mitochondrial) and CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1). These SNPs were genotyped in 285 cases and 282 fully evaluated matched controls. Statistical analyses comprised single polymorphism association as well as haplogroup based association testing.ResultsResults suggested that genetic variation in five of the candidate genes (RDX, SNX16, OPA1, SOD2 and CYP1B1) is unlikely to confer major risk to develop normal tension glaucoma in the German population. In contrast, we observed a trend towards association of single SNPs in OPTN, MFN1, MFN2 and PARL. The SNPs of OPTN, MFN2 and PARL were further analysed by multimarker haplotype-based association testing. We identified a risk haplotype being more frequent in patients and a vice versa situation for the complementary protective haplotype in each of the three genes.ConclusionCommon variants of OPTN, PARL, MFN1 and MFN2 should be analysed in other cohorts to confirm their involvement in normal tension glaucoma.

Association of PARL rs3732581 genetic variant with insulin levels, metabolic syndrome and coronary artery disease

PARL (presenilin-associated rhomboid-like) is a mitochondrial protein involved in mitochondrial membrane remodelling, and maps to a quantitative trait locus (3q27) associated with metabolic traits. Recently the rs3732581 (Leu262Val) variant was found to be associated with increased levels of plasma insulin, a finding not replicated in a larger cohort. The aim of the current study was to investigate the associations between rs3732581 and levels of plasma insulin, metabolic syndrome (MetS) and its components, and cardiovascular disease. The CUPID population consisted of 556 subjects with angiographically proven CAD and the CUDAS cohort consisted of 1,109 randomly selected individuals from Perth, Western Australia. Samples were genotyped using mutation-specific PCR. No significant associations were observed between rs3732581 and levels of plasma insulin, glucose, BMI or MetS in either population. However, carriers of the minor allele had significantly lower mean intima-media thickness (IMT) [0.69 mm, 95% CI (0.69, 0.70 mm); P = 0.004], compared with major allele homozygotes [mean IMT = 0.71 mm, 95% CI (0.70, 0.72 mm)] in the CUDAS population. Further analysis using a recessive model showed homozygous carriers of the minor allele were predisposed to CAD [OR 1.55, 95% CI (1.11, 2.16); P = 0.01]. Despite the functional evidence for a role of PARL in regulating insulin levels, no association with rs3732581 was found in the current study. Additionally, there were no associations with glucose levels, BMI or MetS. There were significant effects of the variant on mean IMT and risk of CAD. A role for PARL in metabolic conditions cannot be excluded and more comprehensive genetic studies are warranted.

Phosphorylation and cleavage of presenilin-associated rhomboid-like protein (PARL) promotes changes in mitochondrial morphology

Remodeling of mitochondria is a dynamic process coordinated by fusion and fission of the inner and outer membranes of the organelle, mediated by a set of conserved proteins. In metazoans, the molecular mechanism behind mitochondrial morphology has been recruited to govern novel functions, such as development, calcium signaling, and apoptosis, which suggests that novel mechanisms should exist to regulate the conserved membrane fusion/fission machinery. Here we show that phosphorylation and cleavage of the vertebrate-specific Pbeta domain of the mammalian presenilin-associated rhomboid-like (PARL) protease can influence mitochondrial morphology. Phosphorylation of three residues embedded in this domain, Ser-65, Thr-69, and Ser-70, impair a cleavage at position Ser(77)-Ala(78) that is required to initiate PARL-induced mitochondrial fragmentation. Our findings reveal that PARL phosphorylation and cleavage impact mitochondrial dynamics, providing a blueprint to study the molecular evolution of mitochondrial morphology.

Mitochondrial Rhomboid PARL Regulates Cytochrome c Release during Apoptosis via OPA1-Dependent Cristae Remodeling

Rhomboids, evolutionarily conserved integral membrane proteases, participate in crucial signaling pathways. Presenilin-associated rhomboid-like (PARL) is an inner mitochondrial membrane rhomboid of unknown function, whose yeast ortholog is involved in mitochondrial fusion. Parl-/- mice display normal intrauterine development but from the fourth postnatal week undergo progressive multisystemic atrophy leading to cachectic death. Atrophy is sustained by increased apoptosis, both in and ex vivo. Parl-/- cells display normal mitochondrial morphology and function but are no longer protected against intrinsic apoptotic death stimuli by the dynamin-related mitochondrial protein OPA1. Parl-/- mitochondria display reduced levels of a soluble, intermembrane space (IMS) form of OPA1, and OPA1 specifically targeted to IMS complements Parl-/- cells, substantiating the importance of PARL in OPA1 processing. Parl-/- mitochondria undergo faster apoptotic cristae remodeling and cytochrome c release. These findings implicate regulated intramembrane proteolysis in controlling apoptosis.