Altered Cortical Palmitoylation Induces Widespread Molecular Disturbances in Parkinson’s Disease

Citation: Cervilla-Martínez, J.F.; Rodríguez-Gotor, J.J.; Wypijewski, K.J.; Fontán-Lozano, Á.; Wang, T.; Santamaría, E.; Fuller, W.; Mejías, R. Altered Cortical Palmitoylation Induces Widespread Molecular Disturbances in Parkinson’s Disease. Int. J. Mol. Sci. 2022,23, 14018. https://doi.org/10.3390/ijms232214018 Academic Editor: Gian Carlo Bellenchi Received: 13 October 2022 Accepted: 4 November 2022 Published: 14 November 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Article Altered Cortical Palmitoylation Induces Widespread Molecular Disturbances in Parkinson’s Disease Juan F. Cervilla-Martínez 1,†, Juan J. Rodríguez-Gotor 1,2,† , Krzysztof J. Wypijewski 3,4, Ángela Fontán-Lozano 1,5 , Tao Wang 6, Enrique Santamaría7, William Fuller 3and Rebeca Mejías 1,5,* 1Department of Physiology, School of Biology, University of Seville, Avenida de la Reina Mercedes, 6, 41012 Sevilla, Spain 2Instituto de Neurociencias CSIC-UMH, Avenida Santiago Ramón y Cajal s/n, San Juan de Alicante, 03550 Alicante, Spain 3Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow G12 8QQ, UK 4School of Life Sciences, University of Dundee, Dundee DD2 5DA, UK 5Instituto de Biomedicina de Sevilla, Campus Hospital Universitario Virgen del Rocío, Avda. Manuel Siurot, s/n, 41013 Sevilla, Spain 6McKusick—Nathans Institute of Genetic Medicine and Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA 7Clinical Neuroproteomics Unit, Navarrabiomed, Hospital Universitario de Navarra (HUN), Universidad Pública de Navarra (UPNA), IDISNA, Irunlarrea Street, 3, 31008 Pamplona, Spain *Correspondence: [email protected]; Tel.: +34-954-559-549 † These authors contributed equally to this work. Abstract: The relationship between Parkinson’s disease (PD), the second-most common neurodegenerative disease after Alzheimer’s disease, and palmitoylation, a post-translational lipid modification, is not well understood. In this study, to better understand the role of protein palmitoylation in PD and the pathways altered in this disease, we analyzed the differential palmitoyl proteome (palmitome) in the cerebral cortex of PD patients compared to controls (n= 4 per group). Data-mining of the cortical palmitome from PD patients and controls allowed us to: (i) detect a set of 150 proteins with altered palmitoylation in PD subjects in comparison with controls; (ii) describe the biological pathways and targets predicted to be altered by these palmitoylation changes; and (iii) depict the overlap between the differential palmitome identified in our study with protein interactomes of the PD-linked proteins α -synuclein, LRRK2, DJ-1, PINK1, GBA and UCHL1. In summary, we partially characterized the altered palmitome in the cortex of PD patients, which is predicted to impact cytoskeleton, mitochondrial and fibrinogen functions, as well as cell survival. Our study suggests that protein palmitoylation could have a role in the pathophysiology of PD, and that comprehensive palmitoyl-proteomics offers a powerful approach for elucidating novel cellular pathways modulated in this neurodegenerative disease. Keywords: Parkinson´s disease; proteomics; cerebral cortex; palmitoylation; interactome 1. Introduction PD is the second-most common neurodegenerative disease. Its pathological hallmarks are the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and the presence of Lewy bodies (LBs), insoluble intracytoplasmic neuronal inclusions containing misfolded α -synuclein. The loss of dopaminergic neurons in the SNc leads to a depletion of dopamine in the striatum, which in turn is responsible for the serious motor symptoms of PD [ 1 ]. As the disease evolves, neurodegeneration spreads to the rest of the central nervous system, including the cerebral cortex [ 2 ]. In addition to motor disturbances, nonmotor symptoms that profoundly affect the quality of life of PD patients—such as REM sleep disturbances, mood, constipation, and loss of smell—can appear even before the development of motor symptoms [ 3 ]. Although the exact causes of PD are unknown, in Int. J. Mol. Sci. 2022,23, 14018. https://doi.org/10.3390/ijms232214018 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022,23, 14018 2 of 18 5–15% of cases, a mutation in a Parkinson ´ s disease gene (PARK gene) has been identified, and some environmental factors, such as certain pesticides, have also been linked to the risk of PD [ 1 ]. The characterization of the protein products encoded by PD-linked genes has provided important insights into the pathophysiology of familial and sporadic PD. Thus, alterations in mitochondrial function, vesicular transport, protein degradation and oxidative balance, among other cellular functions, have been postulated as pathways involved in the neurodegeneration associated with PD [ 4 ]. At present there is no cure for PD, and treatments simply aim to improve motor and non-motor symptoms [5]. S-acylation (called palmitoylation for simplicity through the manuscript) is a posttranslational modification in which a palmitate molecule is reversibly conjugated to a cysteine residue of a protein via a thioester bond. Palmitoylation is regulated by enzymes which add palmitate (palmitoyl acyltransferases, PATs) or remove it (acyl protein thioesterases) from proteins [ 6 ]. Twenty-three PATs have been identified in mammals to date, but the knowledge about depalmitoylating enzymes remains limited [ 7 , 8 ]. Protein palmitoylation can regulate trafficking, stability and enzymatic activity of proteins, and dysregulation of this lipid modification has been implicated in several human diseases, including cancers, metabolic syndrome and infections [ 9 , 10 ]. More specifically, in the nervous system, several studies have linked alterations in palmitoylation to schizophrenia, Alzheimer ´ s disease (AD), intellectual disability, neuronal ceroid lipofuscinosis and Huntington’s disease [ 6 , 7 , 11 – 16 ]. It is well known that multiple neural signaling proteins, ion channels, cell adhesion molecules, SNARE proteins, vesicular trafficking proteins and G-protein receptors are palmitoylated [ 17 , 18 ]. Moreover, this lipid modification has been closely associated with neuronal development and synaptic plasticity [ 8 , 19 , 20 ]. However, a possible link between palmitoylation/depalmitoylation defects and PD has not been studied in vivo , and our limited understanding of this relationship comes from in-vitro and in-silico studies. Thus, palmitoylation of the dopamine transporter (DAT) and DJ-1, encoded by the PARK7 gene, have been described, and palmitoylation of LRRK2, encoded by the PARK8 gene, has been predicted [ 21 – 23 ]. In addition, it has recently been reported that upregulation of cellular palmitoylation alleviates the accumulation and neurotoxicity induced by α-synuclein [24]. In recent years, neuroproteomics has emerged as a powerful approach to characterize multiple proteotypes associated with neurodegeneration [ 25 , 26 ]. However, there is a lack of information about the impact of palmitoylation during the progression of the neurodegenerative process associated with PD. We therefore studied the cortical palmitoyl-proteome (palmitome) of PD and control subjects. After isolating palmitoylated proteins from cortical human samples using the Acyl-Rac assay, quantitative proteomics was performed to detect, identify and quantify palmitoylated proteins levels in both groups. In this pilot study, we characterized a set of 84 proteins that show altered levels of palmitoylation in the cerebral cortex of PD patients vs. controls. Interestingly, around 30% of these proteins were not previously known to be palmitoylated. These alterations in protein palmitoylation might have an impact on microtubule transport, inflammation, oxidative stress and tau pathology, among other physiological effects. Moreover, some of the proteins with altered palmitoylation levels in PD patients belong to the interactome of proteins encoded by most relevant PARK genes, suggesting that alterations in palmitoylation could contribute to pathological mechanisms causing PD. Using publicly available RNA-seq datasets, we show that the differentially palmitoylated proteins tend to map to different cell types in the cerebral cortex, but mainly in neurons. Our results demonstrate, for the first time in human brain tissue, a global deregulation of protein palmitoylation in the cortex of PD patients. We suggest that the deployment of brain palmitoyl-proteomics is an ideal approach that will support focused exploration of the imbalance in palmitoylation-dependent regulatory mechanisms associated with this disease. Int. J. Mol. Sci. 2022,23, 14018 3 of 18 2. Results and Discussion 2.1. Altered Palmitoylation Levels of Cortical Proteins in PD To characterize the palmitoyl-proteome (palmitome) signature in the brain of PD patients, we first isolated palmitoylated proteins using the Acyl-RAC assay from cortical tissue derived from PD subjects and controls with no history of neurological disorders (Supplementary Table S1), and then performed label-free LC-MS/MS-based comparative proteomics [ 27 , 28 ]. Among 2284 putatively palmitoylated proteins identified in this proteomics study (Supplementary Table S2), 84 proteins had statistically significant differences in palmitoylation when comparing PD patients and control subjects (4% of the quantified palmitome; Log2(FC): ≥ [0.6]; p-value: ≤ 0.05) (Supplementary Table S3). We will refer to these 84 proteins as differentially palmitoylated proteins (DPPs) henceforth. We compared our results (set of all palmitoylated proteins identified in human cortex and DPPs) with the database of S-palmitoylation SwissPalm [ 29 ]. SwissPalm contains information about the palmitoylation of all annotated proteins in H. sapiens, specifically whether a protein is known to be palmitoylated and the type of experiments that suggest the lipid modification (proteomics studies and/or further validation experiments). Approximately 20% of Homo sapiens proteins are palmitoylated, according to SwissPalm (Figure 1A). Focusing on the 2284 proteins identified in our cortical palmitome, more than 50% have previously been described to be palmitoylated according to SwissPalm (Figure 1B). Almost 70% of the DPPs are palmitoylated, according to the same database (Figure 1C). The increase in the percentage of palmitoylated proteins in the pie charts from Figure 1B,C compared to that of Figure 1A confirms the successful isolation of palmitoylated proteins before the proteomics analysis. In addition, these observations also suggest that our proteomics experiment identified novel palmitoylated proteins, although additional targeted studies are needed to validate this conclusion. In order to corroborate and broaden the findings of this study, future studies are needed to confirm our data in sex-balanced large cohorts and expand palmitome analysis across other brain areas relevant in the establishment and evolution of PD. In addition, comparison of palmitome data and whole proteome analysis will help to rule out differences in protein palmitoylation levels that are merely due to alterations in the total levels of proteins in PD patients. It is notable that global changes in protein palmitoylation were also found in patients with the neurodegenerative disorder neuronal ceroid lipofuscinosis [17]. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 22 Figure 1. Pie charts representing the percentage of proteins per category (palmitoylated with different levels of validation, in blue, green or orange, or non-known to be palmitoylated, in red) included in the SwissPalm database for Homo sapiens (A), for all 2284 palmitoylated proteins detected in our workflow study (B), and for the 84 differentially palmitoylated proteins in PD patients identified (C). From the 84 DPPs identified in this work, 33 proteins were more palmitoylated and 51 proteins were less palmitoylated in PD compared to the control group (Figure 2 and Supplementary Table S3). Figure 1. Pie charts representing the percentage of proteins per category (palmitoylated with different levels of validation, in blue, green or orange, or non-known to be palmitoylated, in red) included in the SwissPalm database for Homo sapiens ( A ), for all 2284 palmitoylated proteins detected in our workflow study ( B ), and for the 84 differentially palmitoylated proteins in PD patients identified ( C ). Int. J. Mol. Sci. 2022,23, 14018 4 of 18 From the 84 DPPs identified in this work, 33 proteins were more palmitoylated and 51 proteins were less palmitoylated in PD compared to the control group (Figure 2and Supplementary Table S3). Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 of 22 Figure 2. (A) Volcano plot showing proteins that are more palmitoylated (red) and less palmitoylated (blue) in cerebral cortex from PD patients. Proteins with no significant changes among groups are represented in grey; (B) Heatmap showing differential palmitoylated proteins arranged by Z-score. The UniProt accession number for each protein is shown. In order to understand the biological and functional impact of the alterations in cortical palmitoylation levels in PD subjects, we performed GO (Gene Ontology) enrichment analysis [30] (summarized in Supplementary Table S4; adjusted p-value: ≤0.05). Figure 3A shows a heatplot with the 15 most relevant GO terms obtained in this analysis. In Figure 3B, DPPs (represented by their gene symbols) included within these GO terms are shown. Figure 3C displays a cnet plot to visualize the linkages of these DPPs with the most significantly altered biological functions in PD. In summary, key palmitoylated proteins are related to coagulation (FGA, FGB and FGG), dendritic spine, postsynapse and neuron projection organization (ABHD17B, CAMK2B, CFL1, DBN1, DNM3, MAP6 and YWHAH) and cell-cell adhesion (CSRP1 and DSP). Interestingly, ABDH17 proteins have depalmitoylating activity, but their palmitoylation has not been reported to date [31]. We observed an increase in the palmitoylation levels of the three polypeptides that constitute the fibrinogen molecule (encoded by genes FGA, FGB and FGG) in our PD sample cohort. Fibrinogen has essential roles in coagulation, inflammation, extracellular matrix physiology and tissue repair, having chaperone-like activity [32–35]. Fibrinogen is undetectable in the healthy brain, but it can deposit abundantly after blood-brain barrier (BBB) disruption in a variety of neurological diseases, suggesting a potential pathological role [36–39]. Fibrinogen deposition has not been explored in the brain of PD patients, although disruption of the BBB and inflammation may be involved in disease onset [40,41]. Alterations in normal clotting of blood have not previously been described in PD patients, although Sato et al. reported a possible relationship between the use of antiparkinsonian drugs and coagulation abnormalities [42]. Studies about fibrinogen levels in plasma and cerebrospinal fluid (CSF) in PD patients have led to conflicting results. Chen et al. reported no relationship between plasma levels of fibrinogen and the risk of developing PD [43], while other reports found high fibrinogen levels in plasma and CSF associated with PD [44–46]. Our data suggest that PD induces an up-regulation in the levels of palmitoylated fibrinogen polypeptides, at least at cortical level in the brain. Interestingly, palmitoylation of Figure 2. ( A ) Volcano plot showing proteins that are more palmitoylated (red) and less palmitoylated (blue) in cerebral cortex from PD patients. Proteins with no significant changes among groups are represented in grey; ( B ) Heatmap showing differential palmitoylated proteins arranged by Z-score. The UniProt accession number for each protein is shown. In order to understand the biological and functional impact of the alterations in cortical palmitoylation levels in PD subjects, we performed GO (Gene Ontology) enrichment analysis [ 30 ] (summarized in Supplementary Table S4; adjusted p-value: ≤ 0.05). Figure 3A shows a heatplot with the 15 most relevant GO terms obtained in this analysis. In Figure 3B, DPPs (represented by their gene symbols) included within these GO terms are shown. Figure 3C displays a cnet plot to visualize the linkages of these DPPs with the most significantly altered biological functions in PD. In summary, key palmitoylated proteins are related to coagulation (FGA,FGB and FGG), dendritic spine, postsynapse and neuron projection organization (ABHD17B,CAMK2B,CFL1,DBN1,DNM3,MAP6 and YWHAH) and cell-cell adhesion (CSRP1 and DSP). Interestingly, ABDH17 proteins have depalmitoylating activity, but their palmitoylation has not been reported to date [ 31 ]. We observed an increase in the palmitoylation levels of the three polypeptides that constitute the fibrinogen molecule (encoded by genes FGA,FGB and FGG) in our PD sample cohort. Fibrinogen has essential roles in coagulation, inflammation, extracellular matrix physiology and tissue repair, having chaperone-like activity [ 32 – 35 ]. Fibrinogen is undetectable in the healthy brain, but it can deposit abundantly after blood-brain barrier (BBB) disruption in a variety of neurological diseases, suggesting a potential pathological role [ 36 – 39 ]. Fibrinogen deposition has not been explored in the brain of PD patients, although disruption of the BBB and inflammation may be involved in disease onset [ 40 , 41 ]. Alterations in normal clotting of blood have not previously been described in PD patients, although Sato et al. reported a possible relationship between the use of antiparkinsonian drugs and coagulation abnormalities [ 42 ]. Studies about fibrinogen levels in plasma and cerebrospinal fluid (CSF) in PD patients have Int. J. Mol. Sci. 2022,23, 14018 5 of 18 led to conflicting results. Chen et al. reported no relationship between plasma levels of fibrinogen and the risk of developing PD [ 43 ], while other reports found high fibrinogen levels in plasma and CSF associated with PD [ 44 – 46 ]. Our data suggest that PD induces an up-regulation in the levels of palmitoylated fibrinogen polypeptides, at least at cortical level in the brain. Interestingly, palmitoylation of fibrinogen subunits has not been described to date, and consequences of this lipid modification in the functioning of the fibrinogen complex are unknown. Therefore, further experiments are needed to validate fibrinogen palmitoylation, and characterize its role in the onset and progression of PD. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 of 22 fibrinogen subunits has not been described to date, and consequences of this lipid modification in the functioning of the fibrinogen complex are unknown. Therefore, further experiments are needed to validate fibrinogen palmitoylation, and characterize its role in the onset and progression of PD. Figure 3. (A) Heatplot showing the 15 most significant terms obtained by performing GO enrichment analysis. (B) Barplot showing the DPPs (represented by their gene symbols) and their relationship with the 15 most relevant GO terms. (C) Cnet plot of significantly enriched GO terms. To further study pathways affected by the alterations in the DPPs in PD patients, and complementing the results obtained by the GO enrichment analysis, we performed Metascape analysis (results summarized in Supplementary Table S5; p-values are provided in logarithmic based ten: “LogP”; p-value: ≤0.05). Metascape is a resource that incorporates a core set of default ontologies for enrichment analysis, including GO processes (GO), KEGG pathways (hsa), Reactome gene sets (R-HSA) and WikiPathways (WP) [47]. Interestingly, as shown in Figure 4, the Parkinson´s disease pathway (hsa05012 term) was the most affected route, according to KEGG Pathway Database. More specifically, nine of the DPPs are involved in PD-related molecular pathways, according to KEGG: CAMK2B- Calcium/Calmodulin Dependent Protein Kinase II Beta, GPR37-G Protein-Coupled Receptor 37, MAPT-Microtubule Associated Protein Tau, NDUFV1- NADH: Ubiquinone Oxidoreductase Core Subunit V1 (in mitochondrial Complex I), TUBB2A-Tubulin Beta 2A Class IIa, TXN-Thioredoxin, UQCRC1- Ubiquinol-Cytochrome C Reductase Core Protein 1 (in mitochondrial complex III), TUBB3- Tubulin Beta 3 Class III, and TUBB4A-Tubulin Beta 4A Class IVa (Gene Symbols- Protein coding). A visualization of the PD pathway can be seen at https://www.kegg.jp/kegg-bin/show_path- way?hsa05012/816/2861/4137/4723/7280/7295/7384/10381/10382 (accessed on 7 November 2022) with altered DPPs proteins (in a red rectangle and in red font color) and their interactions with proteins encoded by PD-associated genes DJ-1, PaelR, Parkin, PINK1, SNCA and UCHL1 (in a black rectangle and in red font color). It is notable that dysfunction of mitochondrial complex I has been recently linked to the loss of dopaminergic neurons and metabolic changes in the substantia nigra of mice, causing a human-like parkinsonism [48], and that low activity of mitochondrial complexes I, II and III has been reported in Figure 3. ( A ) Heatplot showing the 15 most significant terms obtained by performing GO enrichment analysis. ( B ) Barplot showing the DPPs (represented by their gene symbols) and their relationship with the 15 most relevant GO terms. (C) Cnet plot of significantly enriched GO terms. To further study pathways affected by the alterations in the DPPs in PD patients, and complementing the results obtained by the GO enrichment analysis, we performed Metascape analysis (results summarized in Supplementary Table S5; p-values are provided in logarithmic based ten: “LogP”; p-value: ≤ 0.05). Metascape is a resource that incorporates a core set of default ontologies for enrichment analysis, including GO processes (GO), KEGG pathways (hsa), Reactome gene sets (R-HSA) and WikiPathways (WP) [ 47 ]. Interestingly, as shown in Figure 4, the Parkinson ´ s disease pathway (hsa05012 term) was the most affected route, according to KEGG Pathway Database. More specifically, nine of the DPPs are involved in PD-related molecular pathways, according to KEGG: CAMK2B- Calcium/Calmodulin Dependent Protein Kinase II Beta, GPR37-G Protein-Coupled Receptor 37, MAPT-Microtubule Associated Protein Tau, NDUFV1- NADH: Ubiquinone Oxidoreductase Core Subunit V1 (in mitochondrial Complex I), TUBB2A-Tubulin Beta 2A Class IIa, TXN-Thioredoxin, UQCRC1- Ubiquinol-Cytochrome C Reductase Core Protein 1 (in mitochondrial complex III), TUBB3- Tubulin Beta 3 Class III, and TUBB4A-Tubulin Beta 4A Class IVa (Gene Symbols- Protein coding). A visualization of the PD pathway can be seen at https://www.kegg.jp/kegg-bin/show_pathway?hsa05012/816/2861/41 37/4723/7280/7295/7384/10381/10382 (accessed on 9 October 2022) with altered DPPs proteins (in a red rectangle and in red font color) and their interactions with proteins encoded by PD-associated genes DJ-1, PaelR, Parkin, PINK1, SNCA and UCHL1 (in a black rectangle and in red font color). It is notable that dysfunction of mitochondrial complex I has been recently linked to the loss of dopaminergic neurons and metabolic changes in the Int. J. Mol. Sci. 2022,23, 14018 6 of 18 substantia nigra of mice, causing a human-like parkinsonism [ 48 ], and that low activity of mitochondrial complexes I, II and III has been reported in lymphocytes and platelets of PD patients [ 49 , 50 ]. Thioredoxin has antioxidant activity that protects dopaminergic neurons in animal models of PD [ 51 ], and its palmitoylation has been shown to modulate its activity [ 52 , 53 ]. Although the role of Tubulin Beta in PD is not well defined, its degradation seems to be regulated by Parkin [ 54 ], and its palmitoylation has been suggested [ 55 ]. Metascape analysis also predicted changes in MAPK signaling for integrins (R-HSA-354194; FGA,FGB,FGG and RAP1B) and cellular responses to stimuli (R-HSA-8953897; ATP6V1A, CAMK2B,CSRP1,EIF2S3,HSPA2,RPL7A,TKT,TUBB2A,TXN,KHSRP,TUBB3,TUBB4A and PRDX3), among other pathways. Taken together, the analyses of our palmitome study in PD cortex highlights both well-known and novel molecular pathways that could be affected in this disease. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 of 22 lymphocytes and platelets of PD patients [49,50]. Thioredoxin has antioxidant activity that protects dopaminergic neurons in animal models of PD [51], and its palmitoylation has been shown to modulate its activity [52,53]. Although the role of Tubulin Beta in PD is not well defined, its degradation seems to be regulated by Parkin [54], and its palmitoylation has been suggested [55]. Metascape analysis also predicted changes in MAPK signaling for integrins (R-HSA-354194; FGA, FGB, FGG and RAP1B) and cellular responses to stimuli (R-HSA-8953897; ATP6V1A, CAMK2B, CSRP1, EIF2S3, HSPA2, RPL7A, TKT, TUBB2A, TXN, KHSRP, TUBB3, TUBB4A and PRDX3), among other pathways. Taken together, the analyses of our palmitome study in PD cortex highlights both well-known and novel molecular pathways that could be affected in this disease. Figure 4. Barplot from Metascape analysis showing the 13 most relevant and non-redundant biofunctions affected by the palmitoylation changes in the DPPs detected at cortical level in PD. 2.2. Network-Driven Palmitoyl-Proteomics Reveals an Imbalance in Cytoskeletal Architecture and Mitochondrial Homeostasis in PD To enhance the biological outcome of our palmitome approach, proteome-scale interaction networks were constructed using STRING (p ≤ 0.05) [56] and QIAGEN Ingenuity Pathway Analysis (IPA). The outcome obtained by the STRING analysis of DPPs can be seen at https://version-11-5.string-db.org/cgi/network?taskId=bx7tBdv4gtLt&sessionId=bAXrCBiw5ZOI (accessed on 7 November 2022). Figure 5A shows a functional and physical protein-protein interaction (PPI) network of DPPs, and Figure 5B displays just the physical PPI network of DPPs obtained using the STRING tool. In this analysis, one of the affected molecular functions involved constituents of the cytoskeleton. Tubulin beta polypeptides (encoded by TUBB2A, TUBB3 and TUBB4A), Microtubule Associated Protein Tau (encoded by MAPT) and Calcium/Calmodulin Dependent Protein Kinase II Beta (encoded by CAMK2B) are found in the same PPI network, with lower levels of palmitoylation in PD patients compared to the controls (Figure 5A). Tubulin alpha and beta heterodimers are structural components of the microtubules and have key roles on multiple cellular functions, such as the intracellular molecular trafficking [57]. Microtubule Associated Protein Tau, usually named Tau for simplicity, is responsible for stabilizing the microtubules, and its pathological deposition is known as tauopathy. In tauopathies, the soluble tau protein separates from microtubules, and creates abnormal, fibrillar structures of aggregated, hyperphosphorylated and acetylated tau [58,59]. Some tauopathies are characterized by parkinsonism, but PD was not initially considered to be a typical tauopathy. However, recent studies have demonstrated increasing evidence of tau pathology in PD. Interestingly, hyperphosphorylated tau proteins have been shown to interact with αsynuclein promoting aggregation and fibrilization to each other, accelerating the formation of Lewy bodies and axonal transport dysfunction [60–63]. Our study supports the existence of tau pathology in the cortex of PD patients, and suggests that tau could be palmitoylated in vivo. Calcium/Calmodulin Dependent Protein Kinase II Beta (CAMKIIβ), whose possible palmitoylation has not been studied, can associate with the Figure 4. Barplot from Metascape analysis showing the 13 most relevant and non-redundant biofunctions affected by the palmitoylation changes in the DPPs detected at cortical level in PD. 2.2. Network-Driven Palmitoyl-Proteomics Reveals an Imbalance in Cytoskeletal Architecture and Mitochondrial Homeostasis in PD To enhance the biological outcome of our palmitome approach, proteome-scale interaction networks were constructed using STRING (p ≤ 0.05) [ 56 ] and QIAGEN Ingenuity Pathway Analysis (IPA). The outcome obtained by the STRING analysis of DPPs can be seen at https://version-11-5.string-db.org/cgi/network?taskId=bx7tBdv4gtLt&sessionId= bAXrCBiw5ZOI (accessed on 9 October 2022). Figure 5A shows a functional and physical protein-protein interaction (PPI) network of DPPs, and Figure 5B displays just the physical PPI network of DPPs obtained using the STRING tool. In this analysis, one of the affected molecular functions involved constituents of the cytoskeleton. Tubulin beta polypeptides (encoded by TUBB2A,TUBB3 and TUBB4A), Microtubule Associated Protein Tau (encoded by MAPT) and Calcium/Calmodulin Dependent Protein Kinase II Beta (encoded by CAMK2B) are found in the same PPI network, with lower levels of palmitoylation in PD patients compared to the controls (Figure 5A). Tubulin alpha and beta heterodimers are structural components of the microtubules and have key roles on multiple cellular functions, such as the intracellular molecular trafficking [ 57 ]. Microtubule Associated Protein Tau, usually named Tau for simplicity, is responsible for stabilizing the microtubules, and its pathological deposition is known as tauopathy. In tauopathies, the soluble tau protein separates from microtubules, and creates abnormal, fibrillar structures of aggregated, hyperphosphorylated and acetylated tau [ 58 , 59 ]. Some tauopathies are characterized by parkinsonism, but PD was not initially considered to be a typical tauopathy. However, recent studies have demonstrated increasing evidence of tau pathology in PD. Interestingly, hyperphosphorylated tau proteins have been shown to interact with α -synuclein promoting aggregation and fibrilization to each other, accelerating the formation of Lewy bodies and axonal transport dysfunction [ 60 – 63 ]. Our study supports the existence of tau pathology in the cortex of PD patients, and suggests that tau could be palmitoylated in vivo . Calcium/Calmodulin Dependent Protein Kinase II Beta (CAMKII β ), whose possible palmi- Int. J. Mol. Sci. 2022,23, 14018 7 of 18 toylation has not been studied, can associate with the actin cytoskeleton (microfilaments), and has a role in regulating dendritic arborization and synapse density [ 64 , 65 ]. Interestingly, cytoskeleton dysfunctions in PD have been suggested [ 66 – 68 ], and it would be valuable to unravel the consequences of altered palmitoylation on the function of this crucial cellular structure. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 8 of 22 actin cytoskeleton (microfilaments), and has a role in regulating dendritic arborization and synapse density [64,65]. Interestingly, cytoskeleton dysfunctions in PD have been suggested [66–68], and it would be valuable to unravel the consequences of altered palmitoylation on the function of this crucial cellular structure. In accordance with the data obtained from Metascape (MAPK signaling for integrins, R-HSA-354194; Figure 4 and Supplementary Table S5), STRING analysis also predicted a functional and physical PPI network, including the fibrinogen components and Ras-Re- lated Protein Rap-1b (encoded by RAP1B gene) (Figure 5A,B). Modulation of the binding of fibrinogen to integrins by Rap-1b has been studied in platelet aggregation and megakaryocytes [69,70], but no information is available related to the interactions of these proteins in the brain. Figure 5. (A) Functional and physical protein-protein interaction (PPI) network of DPPs (represented by their gene symbols) obtained with STRING analysis. (B) Only physical PPI network of DPPs (represented by their gene symbols) obtained with the STRING tool are shown. Green and red halos represent down- or up-regulation of the palmitoylation levels, respectively. Color intensity of the halo is in accordance with the fold-change. To further study the functional connection of our proteomics data with signaling hubs, we used IPA. To enrich our proteomic dataset, we included the qualitative palmitoylated proteins (QPPs, 66 proteins listed in Supplementary Table S6) to the 84 DPPs in this analysis (total input dataset of 150 proteins). QPPs are proteins whose palmitoylation is almost undetectable in the cortex of one of the groups, either the control or the PD group, but detectable in the other one, and therefore the differences in palmitoylation levels are quite striking, but cannot be quantified. As shown in Figure 6, protein network analysis revealed that the deregulated cortical palmitome of PD patients is functionally associated to survival mediators such as Protein Kinase B (Akt) and Protein Phosphatase 2 (PP2A). Both Akt and PP2 have been implicated in a variety of neurodegenerative disorders, including PD. Specifically, Akt has been reported to promote neuronal survival [71] and PP2A to modulate mitochondrial function [72,73]. Although palmitome changes were predicted to inhibit AKT (Figure 6), additional research is needed to Figure 5. ( A ) Functional and physical protein-protein interaction (PPI) network of DPPs (represented by their gene symbols) obtained with STRING analysis. ( B ) Only physical PPI network of DPPs (represented by their gene symbols) obtained with the STRING tool are shown. Green and red halos represent down- or up-regulation of the palmitoylation levels, respectively. Color intensity of the halo is in accordance with the fold-change. In accordance with the data obtained from Metascape (MAPK signaling for integrins, R-HSA-354194; Figure 4and Supplementary Table S5), STRING analysis also predicted a functional and physical PPI network, including the fibrinogen components and Ras-Related Protein Rap-1b (encoded by RAP1B gene) (Figure 5A,B). Modulation of the binding of fibrinogen to integrins by Rap-1b has been studied in platelet aggregation and megakaryocytes [ 69 , 70 ], but no information is available related to the interactions of these proteins in the brain. To further study the functional connection of our proteomics data with signaling hubs, we used IPA. To enrich our proteomic dataset, we included the qualitative palmitoylated proteins (QPPs, 66 proteins listed in Supplementary Table S6) to the 84 DPPs in this analysis (total input dataset of 150 proteins). QPPs are proteins whose palmitoylation is almost undetectable in the cortex of one of the groups, either the control or the PD group, but detectable in the other one, and therefore the differences in palmitoylation levels are quite striking, but cannot be quantified. As shown in Figure 6, protein network analysis revealed that the deregulated cortical palmitome of PD patients is functionally associated to survival mediators such as Protein Kinase B (Akt) and Protein Phosphatase 2 (PP2A). Both Akt and PP2 have been implicated in a variety of neurodegenerative disorders, including PD. Specifically, Akt has been reported to promote neuronal survival [ 71 ] and PP2A to modulate mitochondrial function [ 72 , 73 ]. Although palmitome changes were predicted to inhibit AKT (Figure 6), additional research is needed to characterize the potential crosstalk between cellular palmitoylation and survival mediators in the context of PD. Int. J. Mol. Sci. 2022,23, 14018 8 of 18 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 9 of 22 characterize the potential crosstalk between cellular palmitoylation and survival mediators in the context of PD. Figure 6. High-scoring protein interactome map focused on cell survival (Score 38). Visual representation of the relationships between DPPs and QPPs with functional interactors. DPPs are highlighted in red when palmitoylation is up-regulated, and in green when palmitoylation is downregulated in PD cortex. Dataset molecules that did not pass the analyses cutoffs are shown with a grey background. Continuous and discontinuous lines represent direct and indirect interactions, respectively. Predicted interactors of DPPs and QPPs, not part of the dataset, are shown in white. See complete QIAGEN IPA legend at https://qiagen.my.salesforce-sites.com/KnowledgeBase/articles/Knowledge/Legend (accessed on 7 November 2022). Our functional interactome data also recognized a protein set and their PPI interactions that affect mitochondrial homeostasis, specifically the mitochondrial complex I functionality in the cortex of PD subjects (Figure 7). In accordance with this information, Metascape analyses also identified a mitochondrial disruption linked to dyshomeostatic palmitoylation (Parkinson´s disease pathway, hsa05012 term, Ubiquinone Oxidoreductase Core Subunit V1 and Ubiquinol-Cytochrome C Reductase Core Protein 1). Peroxiredoxin 3 (PRDX3) is associated with the mitochondria, where it exerts its antioxidant role Figure 6. High-scoring protein interactome map focused on cell survival (Score 38). Visual representation of the relationships between DPPs and QPPs with functional interactors. DPPs are highlighted in red when palmitoylation is up-regulated, and in green when palmitoylation is down-regulated in PD cortex. Dataset molecules that did not pass the analyses cutoffs are shown with a grey background. Continuous and discontinuous lines represent direct and indirect interactions, respectively. Predicted interactors of DPPs and QPPs, not part of the dataset, are shown in white. See complete QIAGEN IPA legend at https://qiagen.my.salesforce-sites.com/KnowledgeBase/articles/Knowledge/Legend (accessed on 9 October 2022). Our functional interactome data also recognized a protein set and their PPI interactions that affect mitochondrial homeostasis, specifically the mitochondrial complex I functionality in the cortex of PD subjects (Figure 7). In accordance with this information, Metascape analyses also identified a mitochondrial disruption linked to dyshomeostatic palmitoylation (Parkinson ´ s disease pathway, hsa05012 term, Ubiquinone Oxidoreductase Core Subunit V1 and Ubiquinol-Cytochrome C Reductase Core Protein 1). Peroxiredoxin 3 (PRDX3) is associated with the mitochondria, where it exerts its antioxidant role [ 74 ]. Both altered mitochondrial complexes activity and oxidative stress have been described as molecular mechanisms underlying the pathophysiology of PD [ 48 – 50 , 75 ]. On the other hand, O- Linked N-Acetylglucosamine (GlcNAc) Transferase (OGT), whose palmitoylation levels are decreased in the cortex of PD patients (see Figure 7), catalyzes the post-translational modification of proteins named O-GlcNAcylation. O-GlcNAcylation modulates protein function; for instance, the ability of the palmitoyl acyltransferase zDHHC5 to recruit a substrate [ 76 ]. Inhibition of OGT might be neuroprotective against AD, and tau protein can be O-GlcNAcylated, at least in animal models [ 77 – 79 ]. GlcNAcylation on α -synuclein has also been established, which has an inhibitory effect on fibril formation, neurotoxicity, Int. J. Mol. Sci. 2022,23, 14018 9 of 18 and aggregation [ 80 , 81 ]. Although there is no information about palmitoylation of OGT in the literature, palmitoylation of PRDX3 has been described in mice [ 82 ]. According to IPA knowledgebase, PRDX3 and OGT are functionally linked to NF- κβ , a key regulator of inflammation and apoptosis, whose dysregulations could be involved in the onset of PD [83,84]. Thus, NF-κβ activation in the brain of PD patients could contribute to chronic neuroinflammation, and its inhibition has been proposed to slow down the progression of this disease [ 85 , 86 ]. Accordingly, IPA predicts a potential activation of NF- κβ in the cortex of PD patients. NF- κβ is not known to be palmitoylated. In summary, network data from STRING and IPA analyses indicate that cytoskeletal-mitochondrial axis could be modulated, at least in part, by levels of protein palmitoylation in the cortex of PD patients. Some studies have pointed to a relationship between cytoskeleton alterations and mitochondrial dysfunction in PD [87,88]. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 11 of 22 Figure 7. High-scoring protein interactome map unveiling a mitochondrial imbalance (Score 57). Visual representation of the relationships between DPPs and QPPs with functional interactors. DPPs are highlighted in red when palmitoylation is up-regulated, and in green when palmitoylation is down-regulated in the cortex of PD patients. Dataset molecules that did not pass the analyses cutoffs are shown with a grey background. Continuous and discontinuous lines represent direct and indirect interactions, respectively. Predicted interactors of DPPs and QPPs, not part of the dataset, are shown in white. See complete QIAGEN IPA legend at https://qiagen.my.salesforcesites.com/KnowledgeBase/articles/Knowledge/Legend (accessed on 7 November 2022). 2.3. Differential Palmitoyl-Proteins Are Components of Interactomes Associated to PD Genes and Are Located in Multiple Cell Types Discovery of unexpected relationships between apparently unrelated and PD-linked proteins may be a powerful strategy for the characterization of novel PD targets and pathways. We explored whether the interactome of well-established PD-related proteins were Figure 7. High-scoring protein interactome map unveiling a mitochondrial imbalance (Score 57). Visual representation of the relationships between DPPs and QPPs with functional interactors. DPPs are highlighted in red when palmitoylation is up-regulated, and in green when palmitoylation is down-regulated in the cortex of PD patients. Dataset molecules that did not pass the analyses cutoffs are shown with a grey background. Continuous and discontinuous lines represent direct and indirect interactions, respectively. Predicted interactors of DPPs and QPPs, not part of the dataset, are shown in white. 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