Mitochondrial Proteomic Studies of Genetic Animal Models of PD
Several elegantly designed studies have applied a proteomics protocol to genetically modified model organisms that mimic aspects also seen in human PD to reveal insights into the contribution made by several mitochondrial proteins in the pathogenesis of PD. Some of the most important studies in the context of PD are described below.
The discovery that missense mutations (A30P, A53T and E46K) in SNCA associate with PD and that the SNCA protein is a major component of LBs spurred attempts at creating SNCA transgenic Drosophila, by which to study aspects of the human disease. Although Drosophila lacks endogenous SNCA and also does not possess the SNCA ortholog,[171] Feany and Bender demonstrated that flies expressing both WT or mutated (A30P/A53T) human SNCA display symptoms resembling those seen in human PD patients.[172] Features seen in these PD-like flies include gradually progressive degeneration of dopaminergic neurons, the formation of LB-like inclusions, and impaired climbing ability.
In a series of investigations led by Xun and colleagues, the proteome of the SNCA transgenic Drosophila model of PD was analyzed in an effort to address issues relating to the underlying molecular mechanisms of SNCA-mediated neurotoxicity in PD.[173,174] In the first of these studies to be discussed, LC coupled to MS and database analysis techniques were employed for analyzing the proteome of an A30P SNCA Drosophila model of PD. The proteome analysis was performed at three different disease stages (presymptomatic, early and advanced) and the results compared with changes detected in the gene expression profiles of the same animals.[173] Approximately 44% of transcriptional changes compared favorably with those seen at the proteome level, although the pattern of change in protein expression varied substantially compared with that seen in the corresponding transcripts. A total of 19 genes expressed differentially, but only at the proteome and not at the transcriptome level. Eight of these 19 proteins were mitochondrial-associated, the perturbance that were detected as early as day 1 to represent the presymptomic stage of the human disease.[173] The identified mitochondrial proteins are encoded for by CG3011, CG4685, CG6439, CG3731, CG6543, ATP synthase-γ chain, CG11015 and ATP synthase-β, while proteins related to the mitochondrial OXPHOS system, namely ATP synthase-γ chain, CG11015 and ATP synthase-β were upregulated.
Drosophila expressing the A53T SNCA point mutation develop similar human PD-like symptoms as Drosophila expressing A30P SNCA.[172] In other work performed by the same group, the proteome changes in a A30P SNCA Drosophila model of PD were compared with age-matched controls at seven different ages across the adult lifespan.[174] Data were captured using a shotgun proteomic approach, involving multidimensional LC coupled to MS. Moreover, proteins were labeled using an isotopic-labeling strategy that incorporates global internal standard technology, as previously described by Chakraborty and Regnier.[175] The study detected a total of 24 proteins that expressed differentially between the A53T transgenics and WT flies. Subsequent gene ontology analysis indicated that the dysregulated proteins primarily associate with cellular membranes, endoplasmic reticulum, actin cytoskeleton, mitochondria and ribosomes. Changes in the mitochondrial proteome were most dominant in the youngest (presymptomatic and early disease stage) flies. This indicates that future research should focus particularly on understanding molecular changes that occur at the presymptomatic stage, in efforts to address what factors cause the disease. It is anticipated that this may provide valuable insights to help develop diagnostic tools and find ways by which to intervene in the disease progression of PD.
For understanding how mutations in SNCA contribute to the pathophysiology of PD, proteomic analysis was conducted on parkinsonian transgenic mice that overexpress human mutant A30P SNCA.[176] In this regard, the A30P mutation was previously shown to accelerate SNCA aggregation.[177–180] In turn, this step could increase mitochondrial vulnerability to oxidative insult.[181] The aim of this study was to increase understanding concerning what proteins associate with impaired energy metabolism and determine whether mitochondria are particularly vulnerable to oxidative stress in mice expressing mutant A30P SNCA.
Results from the proteomic analysis, generated by 2DE followed by MS were subsequently compared with the brain proteins from WT mice. All the brain proteins that displayed oxidatively modified changes in the brains of the transgenic mice associated with mitochondria. This provides strong evidence for the notion that mitochondrial dysfunction contributes to PD pathology, while the study also implicates mitochondrial pathology in aggregated SNCA toxicity. Subsequent functional analysis of the identified proteins revealed impaired energy metabolism in the brains of these A30P transgenics compared with controls, indicating that A30P-mutant SNCA-associated proteins are particularly vulnerable to oxidative stress. The authors affirmed that oxidative stress impaired energy metabolism and found that mitochondrial dysfunction seen in these animals may be brought on by oxidative inactivation of the metabolic enzymes α-enolase, Ldh2 and Car2.
Carbonyl levels of the glycolytic enzyme enolase, which associates with the intermembrane space/outer mitochondrial membrane fraction,[182] to place it in a position to contribute to mitochondrial function, was significantly increased. Its enzyme activity was decreased in the brains of the transgenic mice compared with WT controls. This result suggests that oxidative inactivation may alter normal glycolysis and mitochondrial function in the brain, as well as contribute to altered energy metabolism in PD.
The Zn2+ metallo-enzyme Car2, which reversibly catalyzes hydration of carbon dioxide (CO2) to bicarbonate (HCO3−) was found to be significantly oxidized in the brains of A30P SNCA mutant mice. Car2 shares high (68%) amino acid sequence similarity to its mitochondrial counterpart carbonic anhydrase 5a (Car5a) and 5b (Car5b). It was proposed that they interact to maintain metabolic processes, cellular transport, gluconeogenesis and mitochondrial metabolism.[183] In the study by Poon and colleagues, mentioned previously, Car2 activity was also decreased significantly in the brains of A30P SNCA transgenic mice compared with WT controls.[176] The authors suggested that the inactivation of Car2 may underlie the resulting aggregation of synuclein and subsequent neurodegeneration seen in A30P-mediated parkinsonism. Another oxidatively modified protein was the glycolytic protein Ldh2 that catalyzes the reversible NAD-dependent interconversion of pyruvate to lactate. The study showed that Ldh2 was significantly modified and inactivated by oxidative insults in A30P SNCA transgenic mice brains. An assay to determine the enzymatic activity of Ldh2 also showed Ldh2 inactivation due to oxidative insult in the brains of A30P SNCA transgenic mice, suggesting that oxidative inactivation of Ldh may contribute to mitochondrial dysfunction seen in PD. The insights gained from this work could aid understanding of what mechanisms underlie the loss of antioxidant capacity by overexpressed A30P mutant SNCA and how this may contribute to cell death.
Parkin mainly serves as a ubiquitin ligase that is essential for the ubiquitin-proteasomal system. Subcellular fractionation has demonstrated parkin's association with the outer mitochondrial membrane.[184] Evidence was also given that parkin localizes to mitochondria in dividing SH-SY5Y neuroblastoma cells.[185] Moreover, mitochondrial export of parkin has been described, with parkin translocating to the golgi apparatus and the nucleus in dividing cells, while a similar phenomenon was described when cells were treated with METC inhibitors, such as rotenone, METC uncouplers, as well as cell cycle couplers.[186] Data suggesting that parkin is involved in mitochondrial transcription and replication also support a potential role for parkin in mitochondria.[186]
Mutations in the Parkin gene (PARK2) associate with an early-onset form of autosomal recessive parkinsonism.[187] Early clinical studies have gained support from genetic animal models, such as Drosophila and mice lacking the gene encoding for parkin in which mitochondrial defects have been described.[68,70]Parkin-deficient mice resemble the biochemical and behavioral changes observed in presymptomatic PD patients,[188] although intriguingly, neuronal degeneration is absent in these mice. The same is true for Drosophila parkin-null mutants, which show no apparent dopaminergic cell loss, but do present with a neurodegenerative phenotype, including shrinkage of the dorsomedial dopaminergic cell body, impaired flight and climbing ability, reduced longevity and male sterility.[68] Morphological and physiological mitochondrial pathology detected in such Parkin knock-out animals include swollen mitochondria, severely fragmented cristae, decreased abundance of several protein subunits of METC Co-1 (NADH dehydrogenase) and IV, as well as decrements in mitochondrial respiratory capacity.[189] Further support for a correlation between mitochondrial defect and PD was shown, such as locomotor defects and male sterility in Parkin KO flies due to a mitochondrial dysfunction, which manifests already during early development.[68]
Using 2-DE gel electrophoresis followed by MS, Palacino and colleagues aimed to determine whether mice engineered to express a Parkin loss-of-function mutation, which serves as a prevalent cause of familial PD, resulted in altered levels of abundance and/or modified protein levels in the ventral mesencephalon.[70] Although electron microscopic analysis revealed no gross morphological changes in striatal mitochondria harvested from Parkin KO mice, functional analysis of the MS-identified proteins complimented the proteomic findings. Proteomic analysis revealed decreased abundance of various proteins involved in mitochondrial function or oxidative stress, including consistent reductions in subunits of METC Co-I and IV. These findings were consistent with those derived from functional analysis, which detected reduced respiratory capacity of the striatal mitochondria in these animals. Accompanying these deficits, the animals also exhibited delayed weight-gain, suggesting for an underlying metabolic abnormality. Moreover, the animals revealed decreased serum antioxidant capacity and increased protein and lipid peroxidation, in line with proteomics results showing that the levels of proteins that fulfill a protective role against oxidative stress were significantly decreased in Parkin KO mice. Therefore, by making use of a recombinant approach, which utilized genetic, physiological and proteomic analysis techniques, evidence was provided that mitochondrial dysfunction and oxidative damage are major mechanisms underlying the deficits seen in the Parkin-KO mouse model of PD.
Periquet and colleagues conducted a study, for which the objective was to gather clues as to the pathogenic mechanisms underlying the preclinical stages of parkin-related parkinsonism, as well as the compensatory mechanisms that might be functioning at the proteome level.[188] Application of MS identified 87 proteins that differed in relative abundance between WT and Parkin KO mice (45%). Functional classification of the differentially regulated proteins revealed that a large proportion of these are involved in energy metabolism, including being prominent role-players in the METC. Several are also known for the part they play in detoxification, their role as stress-response-related chaperones and as components of the ubiquitin-proteasomal system. The authors of this study proposed that the changes in protein abundance, in the absence of a neurodegenerative phenotype might reflect the activity of adaptive mechanisms that serve to regulate cellular energy, necessary due to the induced parkin deficiency. Evidence for this was seen in the altered abundance of enzymes involved in glycolysis and changes in proteins involved in energy regeneration, such as subunits of the mitochondrial ATP synthase.[188]
Multidimensional LC, including offline strong cation exchange chromatography and reversed-phase LC was coupled with MS/MS and database searching techniques[190] for analyzing adult (1-day-old) Drosophila Parkin null mutants. The results were matched with age-matched controls. For relative protein quantification, a label-free peptide hits technique, based on extracted ion chromatogram peak area (XICPA33, with addition of 350 mM potassium chloride)[191] and an isotope-labeling strategy, based on global internal standard technology[175] was utilized. A total of 253 proteins were identified, 24 of which showed differential expression compared with WT controls. This included the mitochondrial antioxidant SOD and the mitochondrial enzymes ATP synthase subunit b and ATP synthase g chain. Similar upregulations were also reported following the proteome analysis of human PD-affected SN to possibly reflect an underlying energy deficiency.[102] Furthermore, an upregulation of cytochrome c was seen in these Parkin-null mutants, which possibly reflects an attempt at counterbalancing the energy deficiency caused by the abundance of ATP synthase.[190]
This investigation was conducted on flies within a narrow age-range. Since the Drosophila Parkin PD model may encompass more human-like PD symptoms at advanced ages, the investigators suggested that differential analysis of the proteome of aged Drosophila (e.g., at 40, 50 and 60 days postfertilization) might provide a more comprehensive view of protein expression changes that underlie Parkin-induced parkinsonism.
Genes inherent to the nematode Caenorhabditis elegans display high conservation with human genes (especially noticeable for transporter proteins[192] and the antiapoptotic chaperone 14–3–3 protein family,[193] which earns the organism a unique position in the genetic and genomic arsenal available to investigators, who work in diseases as diverse as cancer and PD.
The enzymes synthetase, protease, catalase, hydrolase, dehydrogenase, oxidase and isomerase, metabolically process amino acids, carbohydrates, lipids, nucleotides and cofactors. This class of proteins also shows a high degree of overlap between nematode and mammalian metabolic pathways. Li and colleagues recently conducted a study, where the generated information is expected to make a valuable contribution towards characterizing the role of mitochondria in nematodes, while extending our understanding of mitochondrial dynamics, mitochondrial disease, aging and life span.[194] For this study, the BeadBeater, described by Grad and colleagues, was used in an effort to overcome the technical barriers for isolating intact mitochondria in C. elegans.[195] Here, purified mitochondria were harvested from WT (N2) worms and subjected to shotgun proteomics (2D-LC-MS/MS), to compile a complete protein composition of C. elegans. A total of 1117 proteins were identified, including two unique peptides. The majority of these had middle-to-low molecular weights, consistent with the finding that most mitochondrial proteins are synthesized in the cytoplasm, from where they are transported into mitochondria. Hence their effective transmembrane transfer requires relatively light proteins.[72] By subjecting the dataset to a disease gene-identifying protein database, the Kyoto Encyclopedia of Genes and Genomes (KEGG),[196] a total of 75 proteins were assigned a PD-related role.
Representing a series of tightly related mitochondrial-regulated processes, OXPHOS is largely responsible for cell respiration and energy metabolism. The dysfunctional management of these processes has been implicated in PD pathology.[197] The importance of OXPHOS in the physiological and pathological processes of C. elegans was emphasized in this study, with over 90% of identified proteins confirmed as belonging to METC subunits.
In an effort to create an animal model of endogenous oxidative stress, Hinerfeld and colleagues generated SOD KO mice, with WT SOD that serve as the primary defense mechanism against mitochondrial superoxide.[198] In addition to having a survival-time of only 3 weeks, SOD2 KO mice develop a severe neurological phenotype presenting as degeneration of neurons in the basal ganglia and brainstem, progressive motor disturbances characterized by rapid fatigue, exaggerated circling behavior, severe spongiform encephalopathy and decreased levels of mitochondrial aconitase activity.[199,200] Proteomic analysis entailing 2-DE electrophoresis and MALDI-TOF-MS was performed on the cortices of these animals. Application of this approach led to the detection of seven proteins that displayed differences in terms of relative abundance. Moreover, a number of enzymes showed a differential display between SOD KO and WT controls, including the two tricarboxylic acid cycle enzymes ketoglutarate dehydrogenase and succinate dehydrogenase. Two enzymes with a role to play in maintaining redox balance, peroxiredoxin 5 (Prx5) and glutathione S transferase class µ1 (GST class-µ1), as well as cyanide-detoxifying enzyme 3-mercaptosulfurtransferase and glycolytic enzyme triosephosphate isomerase (TPI) were also found upregulated in the SOD KO mice, as opposed to WT mice.[198] Using this genetic animal model, the study therefore generated insight into the specific protein targets of endogenously generated mitochondrial oxidative stress. This knowledge holds implications for designing and developing anti-PD therapeutics aimed at preventing damage being incurred to proteins, through exposure to excessive mitochondrial-generated oxidative cellular stress.
Expert Rev Proteomics. 2010;7(2):205-226. © 2010 Expert Reviews Ltd.
Cite this: Mitochondrial Proteomics as a Selective Tool for Unraveling Parkinson's Disease Pathogenesis - Medscape - Apr 01, 2010.
