Biological Implications of Differential Expression of Mitochondrial-Shaping Proteins in Parkinson’s Disease

It has long been accepted that mitochondrial function and morphology is affected in Parkinson’s disease, and that mitochondrial function can be directly related to its morphology. So far, mitochondrial morphological alterations studies, in the context of this neurodegenerative disease, have been performed through microscopic methodologies. The goal of the present work is to address if the modifications in the mitochondrial-shaping proteins occurring in this disorder have implications in other cellular pathways, which might constitute important pathways for the disease progression. To do so, we conducted a novel approach through a thorough exploration of the available proteomics-based studies in the context of Parkinson’s disease. The analysis provided insight into the altered biological pathways affected by changes in the expression of mitochondrial-shaping proteins via different bioinformatic tools. Unexpectedly, we observed that the mitochondrial-shaping proteins altered in the context of Parkinson’s disease are, in the vast majority, related to the organization of the mitochondrial cristae. Conversely, in the studies that have resorted to microscopy-based techniques, the most widely reported alteration in the context of this disorder is mitochondria fragmentation. Cristae membrane organization is pivotal for mitochondrial ATP production, and changes in their morphology have a direct impact on the organization and function of the oxidative phosphorylation (OXPHOS) complexes. To understand which biological processes are affected by the alteration of these proteins we analyzed the binding partners of the mitochondrial-shaping proteins that were found altered in Parkinson’s disease. We showed that the binding partners fall into seven different cellular components, which include mitochondria, proteasome, and endoplasmic reticulum (ER), amongst others. It is noteworthy that, by evaluating the biological process in which these modified proteins are involved, we showed that they are related to the production and metabolism of ATP, immune response, cytoskeleton alteration, and oxidative stress, amongst others. In summary, with our bioinformatics approach using the data on the modified proteins in Parkinson’s disease patients, we were able to relate the alteration of mitochondrial-shaping proteins to modifications of crucial cellular pathways affected in this disease.

Mitochondrial-shaping proteins were crossed with the proteins that have been found to be altered in Parkinson's disease using the respective gene names in Venny web tool (v.2.1.0) [71]. The gene name of the common proteins (proteins that are involved in mitochondrial shape and are modified in Parkinson's disease) were further used to determine the binding partners in HIPPIE web tool [72].
Network analysis was performed using the Cytoscape software (version v3.5.1) (Cytoscape Consortium, San Diego, CA, US)) with the plugins ClueGo (version v2.3.2) and Cluepedia (version v1.3.2). We used ClueGO's default settings: merge redundant groups with >50.0% overlap; the minimum GO level used was 3 and the maximum GO level was 8; statistical test used was "Enrichment/Depletion (Two-sided hypergeometric test)"; Kappa Score Threshold was 0.4; and number of genes was set at 2 with a minimum percentage at 4.0.

Differentially Expressed Mitochondrial Proteins Associated with Parkinson's Disease
The interplay between mitochondria function and Parkinson's disease was first described as a deficiency of the mitochondrial respiratory chain Complex I [7,8]. Alterations of the mitochondrial shape have been related to their functional state [11] and, in the past few years, an increasing number of reports have shown alterations of mitochondrial morphology in the context of Parkinson's disease [73][74][75][76]. Mitochondrial morphology is tightly regulated by the combined action of proteins involved in fusion, fission, and movement along the cytoskeleton [3]. In this study we aimed to integrate the proteins related to mitochondria morphology with Parkinson's disease pathology. The flowchart followed in the present work is represented in Figure 1. The complete list of mitochondrial proteins that have been described to play a role in the control and regulation of mitochondrial morphology is depicted in Table 1. To integrate the alterations of the mitochondrial dynamics in the context of the pathobiology of Parkinson's disease, a literature search for proteomics-based studies in this disorder, that used samples from patients or cellular models (cell lines of human origin) (Supplementary Table S1), was performed. These proteins were then cross-referenced with the mitochondrial-shaping proteins listed in Table 1 ( Figure 1). To collect the information from proteomic-based studies of the differentially expressed proteins in the context of Parkinson's disease, a search on PubMed and Web of Science (version v5.24) (up to 28 July 2017) was performed using the following keywords: "Parkinson's disease mass spectrometry", "Parkinson's disease proteomics". Studies working with samples from human patients and cellular models (from cell lines of human origin) were used to build the Supplementary Table S1. Three experienced reviewers selected the list of articles relevant for data extraction, taking into consideration only the studies that match the following criteria: proteomics studies, with information on differentially expressed proteins related to control conditions, employing samples either from human patients or cellular models (using cell lines of human origin).
Mitochondrial-shaping proteins were crossed with the proteins that have been found to be altered in Parkinson's disease using the respective gene names in Venny web tool (v.2.1.0) [71]. The gene name of the common proteins (proteins that are involved in mitochondrial shape and are modified in Parkinson's disease) were further used to determine the binding partners in HIPPIE web tool [72].
Network analysis was performed using the Cytoscape software (version v3.5.1) (Cytoscape Consortium, San Diego, CA, US)) with the plugins ClueGo (version v2.3.2) and Cluepedia (version v1.3.2). We used ClueGO's default settings: merge redundant groups with >50.0% overlap; the minimum GO level used was 3 and the maximum GO level was 8; statistical test used was "Enrichment/Depletion (Two-sided hypergeometric test)"; Kappa Score Threshold was 0.4; and number of genes was set at 2 with a minimum percentage at 4.0.

Differentially Expressed Mitochondrial Proteins Associated with Parkinson's Disease
The interplay between mitochondria function and Parkinson's disease was first described as a deficiency of the mitochondrial respiratory chain Complex I [7,8]. Alterations of the mitochondrial shape have been related to their functional state [11] and, in the past few years, an increasing number of reports have shown alterations of mitochondrial morphology in the context of Parkinson's disease [73][74][75][76]. Mitochondrial morphology is tightly regulated by the combined action of proteins involved in fusion, fission, and movement along the cytoskeleton [3]. In this study we aimed to integrate the proteins related to mitochondria morphology with Parkinson's disease pathology. The flowchart followed in the present work is represented in Figure 1. The complete list of mitochondrial proteins that have been described to play a role in the control and regulation of mitochondrial morphology is depicted in Table 1. To integrate the alterations of the mitochondrial dynamics in the context of the pathobiology of Parkinson's disease, a literature search for proteomics-based studies in this disorder, that used samples from patients or cellular models (cell lines of human origin) (Supplementary Table S1), was performed. These proteins were then cross-referenced with the mitochondrial-shaping proteins listed in Table 1 ( Figure 1).  From this analysis, 32 different gene names (Figure 2), related to mitochondrial morphology, were found to be modified in the context of Parkinson's disease, which correspond to 22 different proteins ( Table 2). The vast majority of these mitochondrial proteins are related to the cristae morphology (82%), whereas only 9% are reported to be involved in the fusion and fission processes. From this analysis, 32 different gene names (Figure 2), related to mitochondrial morphology, were found to be modified in the context of Parkinson's disease, which correspond to 22 different proteins ( Table 2). The vast majority of these mitochondrial proteins are related to the cristae morphology (82%), whereas only 9% are reported to be involved in the fusion and fission processes.  Table S1) and the mitochondrial-shaping proteins described in the literature ( Table 1). The Venn diagram was constructed using the Venny 2.1 software [71]. PD-Parkinson's disease.   Table S1) and the mitochondrial-shaping proteins described in the literature ( Table 1). The Venn diagram was constructed using the Venny 2.1 software [71]. PD-Parkinson's disease. Curiously, in Parkinson's disease the most reported mitochondrial morphology alterations are associated with mitochondrial fragmentation and movement impairment [74,77]. Mechanisms proposed for these alterations include: the alteration of the interaction between mitochondria and the motor complexes, and mitophagy impairment [78][79][80][81][82][83].
In our analysis, the fusion-and fission-related proteins found to be altered were Mitofusin-2 and the synaptic vesicle membrane protein VAT-1 homolog, as well as the dynamin-like protein (Drp1) and the SH3-containing protein SH3GLB2, respectively.
Although commonly accepted as a protein involved in mitochondrial fusion, the Mitofusin-2 protein also plays a key role in Ca 2+ signaling. This function is facilitated by the physical interaction between the ER and the mitochondria for the delivery of Ca 2+ to the mitochondrial matrix, enabling mitochondrial signaling. Hence, Mitofusin-2 is involved in both mitochondrial morphology and crosstalk between the ER and the mitochondria [84]. In the process of mitochondrial fusion both Mitofusin-1 and -2 have been reported to contribute, and although they share a common function in this process; deficiency in Mitofusion-2, but not Mitofusin-1, has been linked to neurodegenerative diseases [85]. Interestingly, the synaptic vesicle membrane protein VAT-1 homolog was found to be negatively regulate mitochondrial fusion in cooperation with Mitofusin-2 [70].
Regarding the mitochondrial fission process, in the context of Parkinson's disease, the role of Drp1 has been extensively recognized in mitochondrial fragmentation in different animal and cellular models preceding neuronal death [73,86,87]. This large GTPase is a cytosolic protein that, following mitochondrial fragmentation stimuli, translocates to the outer mitochondrial membrane where it assembles into large complexes in a spiral form, enabling the constriction of the mitochondria [19]. Endophilins might be involved in membrane shaping, e.g., Endophilin B2, although they have also been described to play a role in mitophagy by promoting the degradation of the inner mitochondrial membrane [63].
Nevertheless, as reported above, most of the proteins related to mitochondrial morphology that we found to be altered in the context of Parkinson's disease are associated with the regulation of the mitochondrial cristae morphology. Interestingly, accumulating evidence shows an association between the morphology of the mitochondrial cristae and the OXPHOS complexes. This fact brings forward the idea that the formation of the supercomplexes of the respiratory chain is related to the organization of the inner mitochondrial membrane [18]. The involvement of the mitochondria in Parkinson's disease is clear, and several indications also reveal that alterations in the balance of fission and fusion processes increase the occurrence of fragmented mitochondria. However, the data collected in the present work points to a major contribution of the modification of the mitochondrial cristae. The major drawback when studying mitochondrial morphology is the fact that many studies have employed immunofluorescence using antibodies against mitochondrial membrane proteins and subsequent observation in optical microscopes. This methodology exhibits a lack of resolution needed to visualize the morphological subtleties in the mitochondrial cristae [88]. Therefore, for the study of mitochondrial dynamics, super-resolution and immunoelectron microscopy are better options since it is then possible to visualize the inner mitochondrial compartment [88,89].
The mitochondrial contact site and the cristae-organizing system (MICOS) have been described as a multiprotein complex relevant to inner membrane architecture [1,90]. In fact, it was described that some of the MICOS subunits control the morphology of the cristae in coordination with the mitochondrial Complexes III and IV from the respiratory chain [18]. In the absence of MICOS, cristae morphology is aberrant and these respiratory chain complexes are not functional [14]. Interestingly, in our study we found that two of the MICOS core subunits were altered in the context of Parkinson's disease ( Table 2): Mitofilin [18] and Mic26 [14]. The MICOS Complex interacts with proteins of the outer mitochondrial membrane, specifically with the sorting and assembly machinery component 50 (SAM50). Its depletion has been found to affect the mitochondria ultrastructure and the loss of cristae, thus affecting the assembly of the complexes of the mitochondrial respiratory chain [91]. Curiously, the SAM50 protein expression was also found to be altered in our analysis (Table 2).
Other modified proteins retrieved in our study included proteins from the mitochondrial respiratory chain such as members of the Complex III (the cytochrome b-c1 Complex subunit, the ubiquinol cytochrome c reductase iron-sulphur subunit, and the cytochrome b-c1 Complex subunit 6) and Complex IV (the cytochrome c oxidase subunit 4, the cytochrome c oxidase subunit 5A, the cytochrome c oxidase subunit 6C, the cytochrome c oxidase subunit 7C, and the cytochrome c oxidase polypeptide I).
Pivotal for the proper architecture of the mitochondrial cristae is the protein OPA1 [3]. Alterations were found to occur in the expression of the calcium-binding mitochondrial carrier protein Aralar2 (Slc25A), which acts in conjunction with OPA1 to sense modifications of the substrate levels for energy production. Following this interaction, the cristae are narrowed and the dimerization of the ATP synthase is stimulated [18].
The mitochondrial cristae structure is not solely maintained by proteins, but also by cardiolipin lipids [18], and several cardiolipin binding proteins have been described to be present at the mitochondrial membrane, amongst them the Prohibitin protein family. Prohibitin and Prohibitin-2 appeared to be altered in the context of Parkinson's disease in our analysis ( Table 2). These proteins are known to be organized in complexes within the inner mitochondrial membrane and are important for the proper organization of the cristae morphology and mitochondrial respiration [53,92]. Moreover, these proteins are involved in the turnover of the subunits of the mitochondrial respiratory chain and participate in the assembly of the Complex IV from the respiratory chain [93,94].
There is evidence that aberrant cristae morphology affects the proper assembly of the OXPHOS complexes, but also that the lack of ATP synthase subunits impacts the morphology of the cristae [95,96]. The latter has been supported by studies showing that ATP synthase dimerization forces membrane curvature [97]. Interestingly, mitochondrial morphology also relies on the cellular energetic state since, by compromising mitochondrial membrane potential, mitochondrial fragmentation is induced [98]. In addition, dimer formation of the F1F0-ATP synthase affects mitochondrial cristae structure [95].

Binding Partners of Mitochondrial Proteins Differentially Expressed in Parkinson's Disease
Mitochondria are organelles with important roles in many cellular processes, hence we next explored the binding partners described for the mitochondrial proteins differentially expressed in Parkinson's disease ( Table 2). Using the HIPPIE tool [72,[99][100][101], we determined the complete list of binding partners, which is depicted in Supplementary Table S2 ( Figure 1). This software provides information on human protein-protein interactions with high confidence scores that are due to the amount of supporting data available as well as derived from annotated information [72,99]. In total, for the 22 mitochondrial-shaping proteins altered in Parkinson's disease, we found 1683 hints of interacting proteins. Since some of the mitochondrial-shaping proteins have interacting proteins in common, these hints correspond to 1008 different proteins. When we cross-referenced these binding partners with the list of proteins found to be modified in the context of Parkinson's disease (Supplementary Table S1), 108 common hints were identified ( Figure 3, Table 3, and Supplementary  Table S3). In Supplementary Table S3, the different hints of the proteins listed in Table 3 within the different proteomics-based studies used in the present work are described.
As a first approach we assessed the cellular components, which are represented by these proteins using the plugin ClueGO in Cytoscape software ( Figure 1). In Figure 4 it is possible to see the network built from the different cellular components and their upregulation (green nodes) or downregulation (red nodes) in Parkinson's disease. The cluster of proteins that were found to be upregulated fall into diverse cellular components: mitochondrial respiratory chain Complex III, proteasome complex, muscle thin filament tropomyosin, and melanosome. On the other hand, the downregulated are represented by: inner mitochondrial membrane, integral component of the lumenal side of the endoplasmic reticulum membrane, and mitochondrial proton-transporting ATP synthase complex. Elongation factor 1-alpha 1 (EF-1-alpha-1) (Elongation factor Tu) (EF-Tu) (Eukaryotic elongation factor 1 A-1) (eEF1A-1) (Leukocyte receptor cluster member 7) EEF1B2, EEF1B, EF1B Elongation factor 1-beta (EF-1-beta)

EIF5A
Eukaryotic translation initiation factor 5A-1 (eIF-5A-1) (eIF-5A1) (Eukaryotic initiation factor 5A isoform 1) (eIF-5A) (Rev-binding factor) (eIF-4D)    interacting proteins. Since some of the mitochondrial-shaping proteins have interacting proteins in common, these hints correspond to 1008 different proteins. When we cross-referenced these binding partners with the list of proteins found to be modified in the context of Parkinson's disease (Supplementary Table S1), 108 common hints were identified ( Figure 3, Table 3, and Supplementary  Table S3). In Supplementary Table S3, the different hints of the proteins listed in Table 3 within the different proteomics-based studies used in the present work are described.  (Table 2) and the proteins found to be differentially expressed in the context of Parkinson's disease (Supplementary Table S1). The Venn diagram was constructed using the Venny 2.1 software [71]. PD-Parkinson's disease.  (Table 2) and the proteins found to be differentially expressed in the context of Parkinson's disease (Supplementary Table S1). The Venn diagram was constructed using the Venny 2.1 software [71]. PD-Parkinson's disease.
The proteins used to search for the binding partners were mitochondrial proteins, thus alterations in the mitochondrial cellular components were expected. In this regard, the downregulation of the inner mitochondrial membrane and the mitochondrial proton-transporting ATP synthase complex was anticipated. Several of the proteins found to be altered in the context of Parkinson's disease (Table 2) are proteins located at the inner mitochondrial membrane, and the alteration of these components has also previously been described in the pathology [7].
Interestingly, it has been shown that the ubiquitin-proteasome system regulates the level of proteins targeted to the mitochondrial intermembrane space, and this process depends on the mitochondrial intermembrane space import machinery [102]. Additionally, it has been shown that the ubiquitin-proteasome system acts on the regulation of the mitochondrial biogenesis [103]. In this study, we found an upregulation of the proteasome, which corroborates the evidence of its dysfunction in Parkinson's disease [103,104]. ROS levels are increased in Parkinson's disease and are responsible for the oxidative modification of lipids, DNA, and proteins [105]. These modifications might lead to misfolded proteins and aggregation [106]. Mitochondrial proteins might be dysfunctional due to the harmful effects of ROS, which not only might modify the folded proteins, but also affect the incorporation of newly synthetized mitochondrial proteins since they are translated in the cytosol and must be transported unfolded into the mitochondria [103]. In an oxidative stress scenario, as in Parkinson's disease, the risk of the alteration of unfolded proteins and consequent removal by the proteasome is higher, reducing the amount of mitochondrial proteins available. Besides, it is known that outer mitochondrial membrane proteins involved in mitochondrial fusion are regulated by ubiquitination and that this process is induced by stress [19].
Mitochondrial dynamics not only relies on mitochondrial fusion and fission proteins, but also on the contact sites between mitochondria and the ER, which are fundamental for the initial fission process [107]. It has been described that the shape-forming proteins control mitochondrial morphology by mediating the attachment of the mitochondria to the cytoskeleton and the ER [107,108], and they can also connect the inner and outer mitochondrial membranes, hence influencing the import and assembly of mitochondrial proteins [109]. Regarding the upregulation of the melanosome as a cellular component, although it is an organelle not present in neuronal cells, when we look closely at the proteins contributing to this node, we find that three of the proteins are heat shock proteins. These proteins are key components in ensuring proper protein function and are expressed in response to stress, controlling the subsequent degradation of misfolded proteins, which is also in line with the upregulation of the proteasome complex and the occurrence of the oxidative stress characteristic of the disorder.
The mitochondrial cytochrome bc1 complex from the respiratory chain (Complex III) is one of the main producers of ROS, together with the Complex I [110]. Although Complex I release superoxide into the mitochondrial matrix, Complex III does it into the intermembrane space and the cytosol [111]. In the pathobiology of Parkinson's disease, it is well accepted that there is an increase in ROS leading to oxidative stress [105], which is in agreement with the upregulation of the cellular mitochondria component of the respiratory chain Complex III found in our analysis. This complex is localized in the inner mitochondrial membrane, at the cristae, and has three transmembrane subunits in which the prosthetic groups involved in the redox reactions are located. They must be dimerized for proper functioning, which is also dependent on the mitochondrial membrane potential [110], suggesting that alterations in the organization of the inner mitochondrial membrane might affect their function.
Antioxidants 2018, 7, 1 22 of 48 but also affect the incorporation of newly synthetized mitochondrial proteins since they are translated in the cytosol and must be transported unfolded into the mitochondria [103]. In an oxidative stress scenario, as in Parkinson's disease, the risk of the alteration of unfolded proteins and consequent removal by the proteasome is higher, reducing the amount of mitochondrial proteins available. Besides, it is known that outer mitochondrial membrane proteins involved in mitochondrial fusion are regulated by ubiquitination and that this process is induced by stress [19].  A closer look at the network shows that "muscle thin filament tropomyosin" is connected to cellular components related to the actin cytoskeleton ("actin filament", "stress fiber", and "filamentous actin"). As described above, the cytoskeleton also plays a role in the dynamics and movement of the mitochondria [108]. Interestingly, in the context of Parkinson's disease some models (both genetic and drug-based) showed a negative impact on the dynamics of the actin cytoskeleton and the formation of stress fibers [108,112,113].

Biological Processes Associated with Mitochondrial-Shaping Proteins Affected in Parkinson's Disease
To obtain information on the biological processes related to the mitochondrial-shaping proteins affected in Parkinson's disease, we undertook a bioinformatic approach using the plugin ClueGo from the Cystoscope software ( Figure 5 and Table 4). This plugin allows the extraction of the biological meaning of large lists of proteins [114]. Overall, around 44% altered processes are related to energy production by the mitochondria. This contribution was expected since the dysfunction of this organelle is a hallmark of the disease.     Interestingly, other biological processes are related to the occurrence of oxidative stress and the respective alterations in proteins, which is also a known characteristic of Parkinson's disease [105]. In this regard, the positive regulation of the nitric oxide (NO) biosynthetic process have been shown to occur in this disorder, which is relevant for neuronal death. When NO synthases are ablated, animals are protected against the effect of the MPTP toxin [115]. It is also known that NO not only induces oxidative stress but also neuronal death [116,117]. The downregulation of the glutathione derivative biosynthetic process is also a characteristic of an oxidative stress scenario, which has also been described in Parkinson's disease [118]. This peptide acts as a cellular antioxidant, which is produced by neurons and glial cells, and it has been proposed as an important molecule for therapeutic purposes in the context of Parkinson's disease [118,119]. Moreover, the upregulation of the response to unfolded proteins is important in an oxidative stress scenario where proteins and peptides can be oxidatively modified with a harmful effect on their three-dimensional (3D) structure, with aggregation having a negative impact on their function [106]. Besides, protein oxidative modifications and aggregation have been also related to the decreased in glutathione levels [120].
As stated along this work, the cellular cytoskeleton is one of the mechanisms contributing to the definition of mitochondrial morphology [108]. In fact, modification of the actin cytoskeleton has been probed in Parkinson's disease [108,112,113]. Mitochondrial fusion and fission processes are affected by the interaction of the mitochondria with the cytoskeleton. It has been described that the fusion process can be delayed when actin filaments are depolymerized [121]. The actin cytoskeleton is also involved in the fission process [38]. In our network, several processes are related to the actin cytoskeleton, such as the upregulation of the process of "muscle filament sliding" and the downregulation of the "positive regulation of stress fiber assembly". Interestingly, after a closer look at the modified biological process of "binding of sperm to zona pellucida", we observed that the proteins connected to this process are molecular chaperones and, remarkably, TCP-1-epsilon is known to play a role in the folding of actin and tubulin [122].
Regarding the "auditory receptor cell morphogenesis" biological processes, it is important to note that the protein Rac1 contributes to this pathway and is involved in the regulation of secretory processes, the phagocytosis of death cells, cell polarization, and the formation of membrane ruffles. In the context of Parkinson's disease, it has been shown to contribute to a ROS generating pathway acting with Nox1, causing neuronal death [123]. Interestingly, the other component of this node is the NHERF-1 protein, which has been shown to act as a scaffold for connecting plasma membrane proteins with members of the ERM (ezrin/moesin/radixin) family, aiding in their link to the actin cytoskeleton for the regulation of their surface expression [124].
Within the highlighted process of "regulation of protein dephosphorylation" involved in the regulation of protein function, we found an interesting protein contribution to this pathway: the Peptidyl-prolyl cis-trans isomerase (Pin1). This protein has been shown to be involved in the disease, being upregulated in cellular and animal models as well as in SN in patients [125]. The alteration of this biological process might have an impact on stress responses, immune function, and neuronal survival [126]. Also, this pathway is fundamental for proper mitochondrial functioning and signaling since, in response to the metabolic state of the cell, mitochondrial proteins from the import machinery might be regulated by phosphorylation [127].
In the "regulation of exit from mitosis" process, two interesting proteins emerge: Prohibitin-2 and the NAD-dependent protein deacetylase sirtuin-2. The latter deacetylates lysines on histones, alpha-tubulin, and other proteins [128]. By acting on tubulin it has a direct impact on microtubule function. Tubulin can be subjected to different post-translational modifications with influence on the microtubule polymerization state and its function, such as acetylation [129]. This modification on the residue K40 has been reported to alter the interaction of proteins with the cytoskeleton, with subsequent impact on the intracellular transport along the microtubules [129]. As described in this work, this may affect mitochondria morphology and dynamics.
Parkinson's disease has long been linked to increased inflammatory response [130]. In our analysis we found that the processes related to the inflammatory response were upregulated: "positive regulation of neutrophil chemotaxis" and "regulation of complement activation". Interestingly, from the last process, two of the implicated proteins belong to the Prohibitin family, which have been described to be involved in the regulation of mitochondrial respiration [131]. Regarding the downregulation of the process of the "glucocorticoid receptor signaling pathway" it is important to note that a decrease in the levels of the glucocorticoid receptor in both the SN of patients and in animal models of the disease has been reported [132,133]. These receptors regulate inflammation and are dysregulated in microglia in the context of Parkinson's disease. Dysregulation has been proposed to sustain the chronic inflammatory state observed in this disorder as well as the increased permeability of the blood brain barrier, which might increase neuronal vulnerability [132,133]. Another pathway related to the inflammatory process is downregulated: "positive regulation of NF-kB signaling", in which the protein ribosomal protein S3 stands out. Interestingly, this protein has been proposed to protect the dopaminergic neurons from apoptosis [134].
Within the network, the biological processes of "midbrain development" and "substantia nigra development" share three proteins: Actin, Complex I 30 kD from the mitochondrial respiratory chain, and the 14-3-3 protein epsilon. As described previously, the dysfunction of Complex I from the mitochondrial respiratory chain was the first indication of the mitochondria involvement in the pathobiology of Parkinson's disease [7,8]. The contribution of the cytoskeleton has also been shown, in which actin has a key role in the secretion of the synaptic vesicles content that might be then translated into a decrease in the content of neurotransmitters in the synaptic cleft [135]. Although the 14-3-3 protein is ubiquitously expressed and participates in the regulation of many signaling pathways, it has also been found to be a constituent of the Lewy bodies of Parkinson's disease patients [136].
In Parkinson's disease there is a critical modification in the lipid rafts composition, and increasing evidence shows their contribution to the disorder [137][138][139]. Lipid rafts have a role in diverse cellular processes such as membrane trafficking, signal transduction, and cytoskeletal organization. Their alteration can also have a negative impact on protein-protein interactions, which are fundamental processes for the formation of protein supercomplexes [138]. Interestingly, we found the process of "membrane raft assembly" to be downregulated. Not only was the protein Flotillin-1, a well-known component of the lipid rafts, found to contribute to this node, but also the protein S100A10. The S100 family of proteins are involved in several cellular processes such as the regulation of cell proliferation and differentiation, apoptosis, calcium homeostasis, energy metabolism, and inflammation. Interestingly, they also interact with cytoskeletal and other cellular proteins [140]. Some of the membrane proteins that interact with the S100A10 are: Annexin 2, ion channels, actin binding proteins, and the serotonin receptor [140]. This protein has been proposed to function on membrane repair and was shown to be downregulated in depressive-like states in mice, with its expression being regulated by neurotrophins [141,142].
N-glycosylation is a post-translational modification that is found in membrane proteins and secreted proteins; amongst them are growth factors and their receptors [143]. In our analysis, we found that the process of "protein N-linked glycosylation via asparagine" was downregulated. This modification takes places in the ER and in the Golgi, having an effect on protein function. Evidence show that N-glycosylation is important for proper neuronal function and has a role in synaptic transmission [144], hence having a profound impact on the disease [145].
The occurrence of a role of the ER stress in the context of Parkinson's disease [146] is supported through new evidence, and the process of the "ER-nucleus signaling pathway" was found to be downregulated in our analysis. In this node, we found the LMNA. Lamins are filamentous proteins that contribute to the nucleus architecture and gene expression [147,148]. These proteins also interact with the actin cytoskeleton, which is known to be affected in Parkinson's disease [108,112,113,149]. The other proteins (calreticulin and the heat shock 70 kDa protein 5) are chaperones involved in protein folding and the formation of multimeric complexes [150,151], playing a crucial role in an oxidative stress scenario. Additionally, in the case of fission, not only actin but also the ER is involved in the process of mitochondrial preconstruction and DRP1 assembly [107].

Discussion
Mitochondria are fundamental organelles for cells, working mainly on energy production, calcium homeostasis, and apoptosis. Defects in the mitochondrial respiratory chain have received much of the attention as a key player in the pathobiology of Parkinson's disease [8,152]. However, additional modifications of the mitochondria are being increasingly reported [153,154]. Besides, it is now known that the inhibition of Complex I from the mitochondrial respiratory chain by MPP+ and rotenone does not directly trigger cytochrome c release but, instead, increases the amount of cytochrome c within the mitochondrial intermembrane space [155], indicating that there are other processes required to trigger neuronal death. Amongst them are changes in mitochondrial dynamics (i.e., alterations in the fusion and fission processes, alteration of cristae morphology) [85,156].
Increasing evidence shows that for proper mitochondrial function, processes like mitochondrial fusion, fission, and turnover are fundamental, and their dysfunction has been linked to different diseases [1,3]. In the context of Parkinson's disease, an increase in mitochondrial fission has been reported [73,157,158], suggesting that this excessive fragmentation might then enhance cytochrome c release from mitochondria and subsequently triggering apoptosis [159]. However, we found the process of cristae remodeling to be more highlighted in our analysis regarding the alteration of the mitochondrial-shaping proteins in Parkinson's disease. Notably, the alteration of the mitochondria cristae and membrane might affect the proper binding of cytochrome c, favoring its release to the cytosol and initiating the apoptotic process [155]. Some evidence of mitochondrial cristae remodeling exists in the context of Parkinson's disease. One study using a cybrid cell line constructed with mitochondria DNA isolated from cells from Parkinson's disease patients showed that there were deficiencies in both complexes of the mitochondrial respiratory chain I and IV, and cells contained a non-homogenous mitochondrial population with different morphologies ranging from enlarged to swollen and rounded in shape, which also displayed different mitochondrial membrane potential values [160]. At the ultrastructural level, some mitochondria from this cybrid displayed a decreased in the matrix density and contained a reduced number of cristae and a discontinuous outer mitochondrial membrane [160]. Similarly, in a study using transgenic mice for mutated α-synuclein, morphological alterations on the cristae were also observed, showing a disordered inner membrane and swollen matrix [34]. However, this type of study with samples from human patients is insufficient, and an increasing number of studies aiming at deciphering the ultrastructure of the mitochondria in Parkinson's disease by electron and super-resolution microscopy are required.
It is known that cristae remodeling is fundamental during apoptosis for the proper release of cytochrome c [27], and that the cristae are the sites were the OXPHOS components are located (94% of Complex II and ATP synthase [161], and 85% of cytochrome c [27]). Compelling data indicate that the shape of the cristae is crucial for the modulation of the OXPHOS function [18], and relies on the cellular state [162,163]. Disruption of the cristae junctions is a result of the release of apoptotic factors from the mitochondria [27]. Remarkably, in our analysis most of the proteins involved in mitochondrial dynamics that were found to be altered in the context of Parkinson's disease have been previously reported to play a role in the morphology of the cristae.
When imaging mitochondria, four main components can be distinguished at the ultrastructure level: the outer mitochondrial membrane (OMM), important for regulating membrane permeability and the import/export of mitochondrial proteins; the inner mitochondrial membrane (IMM), where the mitochondrial respiratory chain is placed (the invaginations of the IMM into the matrix are the so-called mitochondria cristae); the intermembrane space (IMS), which is the space between the two mitochondrial membranes; and the matrix, where the components of the tricarboxylic acid (TCA) cycle are located. The inner mitochondrial membrane is organized into three specialized zones: the inner boundary membrane, where the inner and outer membranes are associated, containing proteins of the protein import machinery; the cristae, which are the inner membrane invaginations that are enriched in proteins involved in protein translocation and synthesis as well as proteins involved in iron-sulfur biogenesis; and the cristae junctions, which are the portion of the cristae that are constricted and where the MICOS Complex is located [3].
Fundamental to cell survival is the implication of the mitochondria in the regulation of apoptosis. Within this organelle several pro-apoptotic proteins reside, triggering the apoptotic process when released into the cytosol. The permeabilization of the OMM constitutes a point of no return in the activation of this process, where the Bcl2 family of proteins participates in its regulation [164]. Interestingly, our network analysis highlighted the alteration on the permeability of membranes, supporting the apoptotic activation [165].
The OMM morphology is influenced by its interaction with the ER, ribosomes, the nucleus, and the cellular cytoskeleton [3]. Fundamental to the regulation of multiple cellular processes are the mitochondrial-ER contact sites [164]. In our network analysis, this intraorganellar interaction was highlighted as being altered in Parkinson's disease. Amongst the cellular processes are: the regulation of the intracellular calcium levels, mitochondrial fission, the endowment of membranes to phagosomes, and the formation of the inflammasome [166]. It is now clear how the ER participates in the initiation of the mitochondrial fission process. The ER enwraps the mitochondria at the constriction site where the dynamin-like protein Drp1 responsible for the fission process localizes [107,167]. Additionally, actin polymerization and the ER protein inverted formin 2 (INF2) are involved in this process [38]. Interestingly, actin filaments polymerized around the constriction sites might constitute the pulling force for the fission process [38], and several processes related to the actin cytoskeleton were found to be altered in our analysis. Additionally, the actin cytoskeleton plays a fundamental role in synaptic vesicle secretion. The alteration of this process affects synaptic transmission in the disease. Not only actin but also calcium is involved in this process.
The regulation of calcium levels is also dependent on the mitochondria-ER crosstalk, and a modification of its homeostasis has been reported in Parkinson's disease [168]. Mitochondrial calcium channels display low affinity for this ion, and for correct calcium entry into the mitochondria for the formation of the ER-mitochondrial contact sites is fundamental [169]. The relevance of this interaction has been shown by its involvement in the progression of Alzheimer's disease, where it is upregulated [170]. When calcium accumulates within the mitochondria, oxidative phosphorylation and ATP production are enhanced [171]. In addition, different chaperones are involved in the stabilization of these contact sites and could coordinate signaling between mitochondria and the ER [166]. In this direction, we also found that the expression of the chaperone grp78 was altered [172]. The ER-mitochondria contact sites are also related with the ER-stress response, which might trigger apoptosis [173]. This pathway has also been shown to be active in the context of Parkinson's disease [174]. Additionally, the calcium released by the ER at these contact sites might act as an amplifier of the apoptotic pathway [166]. Besides, the fission protein Fis1 has also been shown to facilitate the cleavage of the pro-apoptotic protein Bap31 [175]. Interestingly, these contact sites are not only relevant to the regulation of calcium levels, but also to ROS-mediating signaling [176].
Moreover, the ER contacts with the phagosomes and non-functional mitochondria degraded by mitophagy are recognized by specific OMM proteins [166]. MFN2 has also been indicated as critical to autophagosome formation, and the ER-mitochondria interaction is important for autophagosome formation [166,177]. Accumulating evidence shows that the ER might also be involved in the mitochondrial fusion process, since it was shown that mitofusin2 (MFN2) is necessary for the tethering of both organelles [178].
Relevant to proper neuronal function is the appropriate localization of the mitochondria within the synaptic terminals, where they can provide ATP for exocytosis and regulate calcium levels during synaptic transmission [179]. Mitochondria positioning within these terminals relies not only on mitochondrial fission [180], since only small mitochondria might fit into the terminals, but also in correct mitochondrial movement along microtubules and the actin cytoskeleton [164]. In this work, the network analysis of cellular components and biological pathways indicated that in the context of Parkinson's disease actin filaments are affected [108,112,113]. In addition, dysregulation of the cellular microtubules has been reported, namely in the alteration of proteins involved in tubulin acetylation. Most of the drugs used to induce Parkinson's disease modify microtubules, and specifically, acetylation has been shown to affect the interaction of proteins with the cytoskeleton [129]. Alteration of the cellular cytoskeleton might have an impact not only on mitochondrial fission but also distribution within neurons [181][182][183][184]. The modification of the actin cytoskeleton might also have an impact on the regulation of surface receptor distribution [124], which was highlighted in the present work.
The ATP produced by the mitochondria reaches the cytosol by its active transport through the adenine nucleotide translocator. To produce ATP, the processes of the TCA cycle and the respiratory chain/oxidative phosphorylation system act in conjunction. The last is located at the IMM. As a result of the mitochondrial respiratory chain deficiency, ATP production is reduced and ROS are increased, which leads to oxidative stress. This increase leads to modifications in protein, lipids, and DNA within the cells [185]. Amongst the modified lipids, the oxidation of cardiolipin from the IMM has been reported in the context of Parkinson's disease [155], and its oxidation disrupts the normal binding of cytochrome c to the membrane [155]. Furthermore, ER is fundamental for providing membrane lipids to the mitochondria [186], highlighting again the importance of this crosstalk. Phosphatidic acid is considered a fusogenic lipid required for the fusion mediated by mitofusins [187]. Cardiolipin have been reported to control mitochondrial fission [188,189]. Interestingly, in the present work we found the expression of several cardiolipin binding proteins to be modified, which might impact the proper assembly of the mitochondrial membranes. Moreover, synaptic mitochondria present lower levels of cardiolipin, which has been pointed out as a lower threshold for the release of cytochrome c in the apoptotic process [164,190]. It has also been reported that synaptic mitochondria present higher sensitivity to the inhibition of the mitochondrial respiratory chain Complex I [191]. Detachment of cytochrome c from the membrane is necessary for cytochrome c release for apoptotic activation and cristae remodeling [27]. As discussed above, these fundamental processes have been emphasized by the high percentage of mitochondrial-shaping proteins found to be altered in the context of Parkinson's disease in contrast with the proteins involved in fusion and fission.
The ER-mitochondrial connection is also important for the inflammatory response [192]. The activation of the inflammasome might occur under an oxidative stress scenario, i.e., Parkinson's disease, where there is an increase in ROS production by the mitochondria [192]. Specific receptors are translocated to the ER-mitochondrial contact sites in response to inflammation [192]. Besides, relevant to the activation of this inflammasome is the VDAC channel located at the mitochondria. Knockdown of both VDAC1 and -2 abolishes the inflammasome formation [192]. Both channels were found to be downregulated in our study. Furthermore, these channels interact with Bcl-2 proteins, therefore enabling cell survival [192,193].

Conclusions
The DA neuron loss from the SN constitutes a hallmark of Parkinson's disease. These neurons are known to be more susceptible than other DA neurons in the brain, and some of the referred sources for this vulnerability are associated with mitochondria. A more complex picture of the alterations of the mitochondria in Parkinson's disease is arising in addition to the widely known deficit in the mitochondrial Complex I dysfunction. Through the so-called "mitochondrial life cycle", these organelles can modulate their function and perform quality control. Accumulating evidence shows that there is a correlation between the morphology of these organelles and the cellular energy status. Increasing efforts have been made to associate the morphology of the mitochondria to its function. Importantly for a neurodegenerative disease such as Parkinson's disease, in which the causes have been linked to mitochondrial dysfunction, this type of analysis will aid in advancing the field, both in the pathobiology of the disease and the search for new therapies.
By network analysis we have correlated the changes in differentially expressed mitochondrialshaping proteins in the context of Parkinson's disease with the corresponding biological pathways affected in the disease. One of the most striking findings is related to the process of cristae remodeling, since most of the mitochondrial-shaping proteins found to be altered in the context of Parkinson's disease participate in the maintenance of cristae shape. Remarkably, this alteration is evident in different human disorders, including Parkinson's disease. Since these structures regulate protein and lipid distribution as well as soluble molecules (i.e., ADP and cytochrome c), their alteration might have a direct impact on neuronal physiology and survival.
In our opinion, although it is clear in the cellular and animal models of the disease that mitochondrial morphology is altered, more studies from post mortem tissue from patients are needed, aiming at unravelling the alterations of the mitochondrial morphology more specifically related to the cristae shape in the context of Parkinson's disease. These studies would provide new insights into the development of new therapies or aid in biomarkers discovery. Identification of the mitochondrial components that play a role in the process of cristae remodeling might also be fundamental for these purposes.
Supplementary Materials: The following are available online at www.mdpi.com/2076-3921/7/1/1/s1, Table S1: List of proteins found modified in different proteomics-based approaches in the context of Parkinson's disease; Table S2: List of the binding partners of the "mitochondrial-shaping proteins found modified in Parkinson's disease"; Table S3: List of the binding partners of the "mitochondrial-shaping proteins found modified in different proteomics-based approaches in the context of Parkinson's disease according to the Human Integrated Protein-Protein Interaction reference" that are modified in Parkinson's disease.
performed the literature search, collected the data meeting the criteria, performed the bioinformatics analysis and figures, and analyzed the data; Maria Gomez-Lazaro, Sara Rocha, Sofia C. Guimaraes, and Miguel Aroso wrote the paper; all authors revised the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest. O-linked N-acetylglucosamine transferase interacting protein 106 OK/SW-cl. 110 ORF name for Phosphoglycerate kinase 1 OK/SW-cl. 56 ORF name for Tubulin beta chain OK/SW-cl. 82 ORF name for 40S ribosomal protein S15a OMA1