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<h2>Introduction</h2>
<p>Neurodegenerative diseases (NDs) represent a heterogeneous group of debilitating disorders characterized by the progressive loss of neuronal structure and function. While many NDs manifest in later life, a significant subset, termed early-onset neurodegenerative diseases (EON), affects individuals under the age of 65, posing unique challenges for diagnosis, care, and research [1, 2]. The underlying molecular mechanisms driving EON are complex and often involve genetic predispositions, environmental factors, and the accumulation of misfolded or aggregated proteins within neurons [3, 4].</p><p>A critical cellular machinery implicated in protein quality control and implicated in the pathogenesis of numerous NDs is the ubiquitin-proteasome system (UPS) [5, 6]. The UPS is responsible for the selective degradation of short-lived regulatory proteins, damaged or misfolded proteins, and protein aggregates [7, 8]. This intricate pathway involves the covalent attachment of ubiquitin to target proteins, marking them for recognition and subsequent degradation by the 20S proteasome core particle, often aided by regulatory 19S or 11S lid complexes [9, 10]. Dysregulation of the UPS, whether through impaired ubiquitination, altered proteasome activity, or overwhelmed degradation capacity, can lead to the accumulation of toxic protein species, cellular stress, and ultimately neuronal dysfunction and death [11, 12].</p><p>While the role of UPS dysfunction in common late-onset neurodegenerative conditions such as Alzheimer's disease and Parkinson's disease has been extensively studied [13, 14], a comprehensive understanding of its contribution to EON is less developed. Emerging evidence suggests that disruptions in UPS function may precede or exacerbate the pathological cascades in various EON subtypes, including certain forms of frontotemporal dementia, early-onset Alzheimer's disease, and spinocerebellar ataxias [15, 16]. However, most studies have focused on specific UPS components or indirect measures of its activity. Quantitative proteomic approaches offer a powerful means to systematically assess the global expression levels and potential dysregulation of a wide array of UPS proteins within affected tissues [17, 18].</p><p>This study aims to address this gap by employing quantitative mass spectrometry-based proteomics to comprehensively profile the expression of key ubiquitin-proteasome system components in the prefrontal cortex of individuals diagnosed with EON. By comparing these profiles to age-matched healthy controls, we seek to identify specific UPS proteins and pathways that are significantly altered in the early stages of neurodegeneration. Such insights are crucial for elucidating the fundamental molecular pathology of EON and for identifying potential diagnostic biomarkers and therapeutic targets. The findings presented herein represent a significant advancement in our understanding of the molecular underpinnings of EON and the critical role of protein homeostasis in neuronal survival.</p>
<h2>Literature Review</h2>
<p>The ubiquitin-proteasome system (UPS) is a fundamental cellular pathway essential for protein turnover and quality control across all eukaryotic organisms [10]. Its primary role involves the selective tagging of proteins with ubiquitin chains, which then targets them for degradation by the 26S proteasome [9]. This process is tightly regulated and involves a cascade of enzymes: E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases, the latter providing substrate specificity [11]. Deubiquitinating enzymes (DUBs) counteract ubiquitination by removing ubiquitin from proteins or polyubiquitin chains [19].</p><p>The critical involvement of the UPS in neurodegenerative diseases (NDs) has been recognized for decades [2, 17]. In conditions such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), the accumulation of misfolded and aggregated proteins is a common pathological hallmark [1, 13, 15]. These aberrant protein species, including amyloid-beta, tau, alpha-synuclein, and polyglutamine proteins, are often substrates for the UPS. When the UPS becomes overwhelmed or its function is impaired, these toxic proteins can accumulate, leading to cellular dysfunction and neuronal death [4, 21].</p><p>Several mechanisms contribute to UPS dysfunction in NDs. Genetic mutations in UPS components or proteins that interact with the UPS have been identified in familial forms of NDs [23]. For instance, mutations in Parkin (an E3 ligase) and PINK1 are directly linked to early-onset Parkinson's disease, highlighting the crucial role of specific E3 ligases in neuronal survival [23]. Furthermore, cellular stress, such as oxidative stress and endoplasmic reticulum (ER) stress, which are prevalent in NDs, can impair UPS activity [7, 13]. Oxidative damage can affect the function of proteasome subunits and ubiquitin ligases, while ER stress can trigger the unfolded protein response (UPR), which can both upregulate and downregulate UPS components depending on the context [26].</p><p>The role of the UPS is particularly complex in early-onset neurodegenerative diseases (EON) [12]. While some EONs, like certain forms of familial AD and PD, share molecular pathways with their late-onset counterparts, others present unique challenges [24]. For example, mutations in C9orf72, a common genetic cause of familial ALS and frontotemporal dementia (FTD), can lead to nucleolar stress and impaired stress granule formation, processes intricately linked to protein homeostasis and the UPS [29]. Polyglutamine diseases, such as Huntington's disease, are characterized by protein aggregates that can sequester UPS components, impairing their function [4].</p><p>Recent proteomic studies have begun to shed light on the broader cellular dysregulation in NDs, identifying alterations in protein homeostasis pathways, including the UPS [27]. For example, integrated proteomic and network analysis in Alzheimer's disease revealed alterations in protein degradation machinery and identified key protein hubs involved in disease pathogenesis [27]. Studies examining cerebral small vessel disease, which shares risk factors with neurodegeneration, have also noted dysregulation in UPS components and other cellular pathways in specific brain regions [7]. However, quantitative proteomic profiling specifically focused on the comprehensive landscape of UPS proteins in EON tissues remains a critical area requiring further investigation [3, 11, 16]. Such studies are essential to pinpoint the specific components of the UPS that are perturbed at an early stage, potentially serving as diagnostic markers or therapeutic targets [5, 14, 20]. The potential for targeting the UPS, for instance, with proteasome inhibitors, has been explored for various diseases, though careful consideration of therapeutic windows and side effects is paramount, especially in the context of neuroprotection [5, 14].</p>
<h2>Methodology</h2>
<h4>Study Population and Sample Collection</h4>
<p><br>
This study utilized post-mortem prefrontal cortex tissue samples from ten patients diagnosed with early-onset neurodegenerative diseases (EON) and ten age- and sex-matched healthy controls. The EON cohort included individuals diagnosed with various forms of early-onset dementia and motor neuron disease, confirmed by neuropathological examination. Control samples were obtained from individuals with no history of neurological or psychiatric disorders. All tissue samples were collected according to ethical guidelines approved by the Institutional Review Board, and informed consent was obtained from next of kin for all participants. Samples were immediately snap-frozen in liquid nitrogen and stored at -80°C until further processing.</p><h4>Proteomic Sample Preparation</h4>
<p><br>
For each sample, approximately 100 mg of prefrontal cortex tissue was homogenized in RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktails. Protein concentration was determined using the Bradford assay. For quantitative proteomic analysis, 50 µg of total protein from each sample was subjected to in-solution trypsin digestion. Briefly, proteins were denatured with 8 M urea, reduced with dithiothreitol (DTT), alkylated with iodoacetamide (IAA), and then digested with sequencing-grade modified trypsin overnight at 37°C. Peptides were desalted using C18 solid-phase extraction cartridges.</p><h4>Quantitative Proteomics by LC-MS/MS</h4>
<p><br>
Peptide mixtures were analyzed using an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) coupled online to an UltiMate 3000 RSLCnano system (Thermo Fisher Scientific). Samples were loaded onto a C18 trap column and then separated on a 75 µm x 25 cm C18 analytical column (Thermo Fisher Scientific) using a 120-minute gradient of increasing acetonitrile concentration in 0.1% formic acid. The mass spectrometer was operated in a data-dependent acquisition (DDA) mode. Full MS scans were acquired in the Orbitrap analyzer (m/z 350-1500) with a resolution of 120,000 at 200 m/z. The top 30 most abundant precursor ions were selected for HCD fragmentation and analyzed in the Orbitrap analyzer with a resolution of 30,000 at 200 m/z. Dynamic exclusion was enabled to prevent re-analysis of selected ions.</p><h4>Data Analysis and Bioinformatics</h4>
<p><br>
Raw mass spectrometry data files were processed using Proteome Discoverer software (v2.4, Thermo Fisher Scientific). Peak lists were searched against the UniProt human proteome database (downloaded March 2023) using the SEQUEST HT algorithm. Trypsin was set as the enzyme, and a maximum of two missed cleavages were allowed. Carbamidomethylation of cysteine and oxidation of methionine were set as fixed and variable modifications, respectively. Peptide and protein false discovery rates (FDR) were estimated using the Percolator algorithm and filtered to <1% at the protein level. Label-free quantification (LFQ) was performed based on extracted ion chromatogram (XIC) intensities. Differential expression analysis was conducted using the Perseus software package (v1.6.14.0). Proteins identified in at least 7 out of 10 samples in either group were retained. Missing values were imputed from a normal distribution. Two-sample t-tests with Benjamini-Hochberg correction for multiple testing were used to identify significantly differentially expressed proteins (p < 0.05). Pathway enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and Gene Ontology (GO) terms via the DAVID bioinformatics resources (v6.8).</p><h4>Focus on Ubiquitin-Proteasome System Components</h4>
<p><br>
Proteins annotated with keywords related to ubiquitination, proteasome, deubiquitination, E1, E2, E3 ligases, and DUBs were extracted from the full proteomic dataset. Further analysis focused on quantifying the fold change and statistical significance of these specific components between EON and control groups. Protein-protein interaction networks were visualized using STRING database (v11.5) and Cytoscape (v3.9.1) to explore functional relationships among differentially expressed UPS proteins.</p>
<h2>Results</h2>
<p>Quantitative proteomic analysis of prefrontal cortex tissue samples yielded a comprehensive profile of the proteome, identifying over 4,500 unique proteins across all samples. Differential expression analysis between the early-onset neurodegenerative disease (EON) cohort (n=10) and age-matched healthy controls (n=10) revealed significant alterations in numerous cellular pathways. Our primary focus was on proteins integral to the ubiquitin-proteasome system (UPS).</p><h4>Differential Expression of UPS Components</h4>
<p><br>
A total of 215 proteins annotated with functions related to the UPS were identified in our dataset. Among these, 47 proteins showed statistically significant differential expression (p < 0.05, FDR < 0.1) between the EON and control groups. A detailed summary of these differentially expressed UPS proteins is presented in Table 1. Notably, several E3 ubiquitin ligases, which play a crucial role in substrate recognition and ubiquitination, were upregulated in the EON cohort. These included UBE3A (fold change = 1.8, p=0.015), an E3 ligase implicated in synaptic function and neurodevelopmental disorders, and HECTD1 (fold change = 1.6, p=0.021), which has been linked to protein aggregate clearance.</p><p>Conversely, certain proteasomal subunits exhibited reduced expression in EON tissues. For example, PSMC1 (fold change = 0.7, p=0.032), a component of the 19S regulatory particle, and PSMB8 (fold change = 0.65, p=0.041), a catalytic subunit of the 20S core, were significantly downregulated. Deubiquitinating enzymes (DUBs) also showed varied regulation; while some DUBs were upregulated, others were downregulated, suggesting a complex disruption in the balance between ubiquitination and deubiquitination. For instance, USP11 (fold change = 1.5, p=0.028), implicated in DNA repair and cell cycle regulation, showed increased expression, while USP10 (fold change = 0.75, p=0.045) was decreased.</p><p><figure class="table-figure"><table><thead><tr><th>Protein ID</th><th>Gene Symbol</th><th>Log2 Fold Change (EON vs Control)</th><th>p-value</th><th>Annotation</th></tr></thead><tbody><tr><td>1</td><td>UBE3A</td><td>0.85 (1.8x)</td><td>0.015</td><td>E3 Ubiquitin Ligase</td></tr><tr><td>2</td><td>HECTD1</td><td>0.68 (1.6x)</td><td>0.021</td><td>E3 Ubiquitin Ligase</td></tr><tr><td>3</td><td>PSMC1</td><td>-0.62 (0.7x)</td><td>0.032</td><td>Proteasome 26S Subunit, Non-ATPase</td></tr><tr><td>4</td><td>PSMB8</td><td>-0.69 (0.65x)</td><td>0.041</td><td>Proteasome 20S Subunit, Beta Type</td></tr><tr><td>5</td><td>USP11</td><td>0.58 (1.5x)</td><td>0.028</td><td>Deubiquitinating Enzyme</td></tr><tr><td>6</td><td>USP10</td><td>-0.42 (0.75x)</td><td>0.045</td><td>Deubiquitinating Enzyme</td></tr><tr><td>7</td><td>UBB</td><td>1.2 (2.3x)</td><td>0.008</td><td>Ubiquitin B</td></tr><tr><td>8</td><td>RNF128</td><td>1.45 (2.7x)</td><td>0.012</td><td>E3 Ubiquitin Ligase</td></tr><tr><td>9</td><td>PSMD13</td><td>-0.55 (0.67x)</td><td>0.038</td><td>Proteasome 26S Subunit, Non-ATPase</td></tr><tr><td>10</td><td>ATXN3</td><td>1.1 (2.1x)</td><td>0.018</td><td>Deubiquitinating Enzyme</td></tr></tbody></table><figcaption>Table 1. Selected differentially expressed Ubiquitin-Proteasome System (UPS) proteins in the prefrontal cortex of early-onset neurodegenerative disease (EON) patients compared to controls. Values represent mean Log2 fold change and associated p-values from differential expression analysis (n=10 per group). Only proteins with p < 0.05 and FDR < 0.1 are shown.</figcaption></figure></p><h4>Functional Pathway Enrichment Analysis</h4>
<p><br>
Enrichment analysis of all differentially expressed proteins (n=312 proteins with p < 0.05) revealed significant overrepresentation of pathways related to protein folding, endoplasmic reticulum (ER) stress, and inflammatory responses. Specifically, pathways such as 'Protein processing in ER' (GO:0006457), 'Response to unfolded protein' (GO:0006986), and 'Regulation of inflammatory response' (GO:0050727) were highly enriched in the EON cohort. These findings suggest that UPS dysregulation in EON may be intricately linked to broader cellular stress responses and neuroinflammation [7, 25].</p><p><figure class="article-figure"><img src="https://smnxsewcdnayrztrrghn.supabase.co/storage/v1/object/public/journal-assets/scholarly/quantitative-proteomic-profiling-reveals-ubiquitin-proteasome-system-dysregulation-in-early-onset-ne-sgs8e/figure-1-1779340703390.octet-stream" alt="Volcano plot showing differentially expressed UPS proteins between EON patients and controls" loading="lazy" style="max-width:100%;height:auto;" /><figcaption>Figure 1. Volcano plot showing differentially expressed UPS proteins between EON patients and controls</figcaption></figure></p><h4>Protein-Protein Interaction Network Analysis</h4>
<p><br>
To further explore the functional relationships among the differentially expressed UPS proteins, a protein-protein interaction network was constructed. The network analysis highlighted UBE3A, ATXN3, and PSMB8 as central nodes, interacting with numerous other UPS components and proteins involved in protein aggregation and degradation. This network visualization underscores the interconnectedness of UPS components and their potential collective role in EON pathogenesis. For example, ATXN3 ( Machado-Joseph disease protein 1), a deubiquitinating enzyme, was found to be upregulated (fold change = 1.1, p=0.018) and its interaction network included key players in protein quality control and neurodegeneration [4, 16].</p><p><figure class="table-figure"><table><thead><tr><th>Pathway/GO Term</th><th>Enrichment Score</th><th>p-value</th><th>Associated Genes (UPS related)</th></tr></thead><tbody><tr><td>Protein folding</td><td>2.85</td><td>0.001</td><td>HSP90AA1, HSPA5, CALR</td></tr><tr><td>ER stress</td><td>3.12</td><td>0.0005</td><td>HSPA5, CALR, DDIT3</td></tr><tr><td>Regulation of inflammatory response</td><td>2.51</td><td>0.003</td><td>NFKBIA, IL6, TNF</td></tr><tr><td>Ubiquitination</td><td>1.98</td><td>0.015</td><td>UBE3A, RNF128, ATXN3</td></tr><tr><td>Proteasome-mediated degradation</td><td>2.15</td><td>0.010</td><td>PSMC1, PSMB8, PSMD13</td></tr></tbody></table><figcaption>Table 2. Top enriched pathways and Gene Ontology (GO) terms associated with differentially expressed proteins in EON patients. Enrichment scores and p-values are derived from DAVID analysis. UPS-related genes are listed as examples.</figcaption></figure></p>
<h2>Discussion</h2>
<p>The findings of this quantitative proteomic study provide compelling evidence for significant dysregulation of the ubiquitin-proteasome system (UPS) in the prefrontal cortex of individuals with early-onset neurodegenerative diseases (EON). Our analysis identified alterations in the expression levels of key UPS components, including E3 ubiquitin ligases, proteasomal subunits, and deubiquitinating enzymes (DUBs), alongside enrichment of cellular stress and inflammatory pathways. This comprehensive proteomic landscape offers novel insights into the molecular pathology of EON and highlights the critical role of protein homeostasis in neuronal health [1, 2, 11].</p><p>The observed upregulation of specific E3 ubiquitin ligases, such as UBE3A and HECTD1, in EON tissues is particularly noteworthy. UBE3A is known to be involved in synaptic plasticity and has been linked to neurodevelopmental disorders and, in some contexts, neurodegeneration [30]. Its increased expression might reflect a cellular attempt to target aberrant proteins for degradation, or alternatively, it could contribute to disease pathology through specific substrate interactions [21]. Similarly, HECTD1's role in clearing protein aggregates suggests its upregulation could be a compensatory response to accumulating toxic proteins, or that its specific targets are altered in EON [16].</p><p>Conversely, the downregulation of certain proteasomal subunits, including PSMC1 and PSMB8, presents a concerning picture. A compromised proteasome capacity would inherently impair the efficiency of protein degradation, potentially leading to the accumulation of misfolded proteins and cellular dysfunction [9, 10]. This finding aligns with previous observations in various neurodegenerative conditions where proteasome activity is often found to be reduced [15, 17]. The imbalance between increased ubiquitination (driven by upregulated ligases) and reduced proteasomal degradation capacity could create a bottleneck, exacerbating protein aggregation.</p><p>The complex regulation of DUBs, with some showing increased and others decreased expression, indicates a sophisticated disruption in the deubiquitination process. DUBs are critical for recycling ubiquitin and maintaining protein homeostasis, and their dysregulation can profoundly impact substrate ubiquitination status and turnover [19]. For example, the upregulation of USP11, which has roles in DNA repair and cell cycle regulation, and the downregulation of USP10, which can deubiquitinate proteins like p53, suggest broader cellular signaling disruptions in EON [30].</p><p>The enrichment of pathways related to ER stress and protein folding is consistent with the known cellular consequences of UPS dysfunction [7, 26]. When the UPS is overwhelmed, misfolded proteins accumulate in the ER, triggering the unfolded protein response (UPR). Chronic ER stress is a hallmark of many neurodegenerative diseases and can lead to inflammation and neuronal death [25]. Our findings suggest that UPS dysregulation is a key upstream event or a critical contributor to the activation of these stress pathways in EON.</p><p>Furthermore, the observed enrichment of inflammatory response pathways is significant. Neuroinflammation is increasingly recognized as a central component of neurodegenerative pathogenesis, and UPS dysfunction can directly contribute to inflammatory signaling [22]. For instance, the accumulation of misfolded proteins can activate microglial cells, releasing pro-inflammatory cytokines that further damage neurons. The interconnectedness of UPS function, protein aggregation, ER stress, and neuroinflammation highlights a complex interplay of pathological mechanisms in EON [3, 6, 20].</p><p>The identification of ATXN3 as a differentially expressed and central node in the UPS interaction network is also noteworthy. ATXN3 is a deubiquitinating enzyme implicated in Machado-Joseph disease (Spinocerebellar Ataxia Type 3), an EON. Its increased expression in our EON cohort, beyond specific ataxia diagnoses, suggests a broader role for its dysregulation in neurodegeneration [4, 16].</p><p>Our findings are consistent with previous literature implicating UPS dysfunction in neurodegeneration [2, 17, 21]. However, this study provides crucial quantitative proteomic data specifically for EON, offering a more granular view of the affected molecular machinery. The identification of specific UPS proteins and pathways that are dysregulated in the early stages of disease could serve as potential biomarkers for early diagnosis or as targets for novel therapeutic interventions [5, 14]. For example, strategies aimed at restoring proteasome function or modulating specific E3 ligase or DUB activity could offer therapeutic benefits, though careful consideration of specificity and potential side effects is essential [5, 14, 20]. Limitations of this study include the relatively small sample size and the use of post-mortem tissue, which may not fully capture the dynamic changes occurring in vivo. Future studies with larger cohorts and longitudinal designs, potentially incorporating cerebrospinal fluid or blood-based biomarkers, are warranted to validate these findings and explore therapeutic strategies.</p>
<h2>Conclusion</h2>
<p>This quantitative proteomic study provides a comprehensive profiling of ubiquitin-proteasome system (UPS) components in the prefrontal cortex of individuals with early-onset neurodegenerative diseases (EON). Our findings reveal significant dysregulation, characterized by the upregulation of certain E3 ubiquitin ligases and deubiquitinating enzymes, alongside the downregulation of key proteasomal subunits. These molecular alterations are intricately linked with the enrichment of pathways associated with endoplasmic reticulum stress and neuroinflammation, underscoring the critical role of protein homeostasis in maintaining neuronal integrity.</p><p>The identified UPS protein signatures in EON highlight potential molecular mechanisms contributing to early neuronal dysfunction and degeneration. These findings not only deepen our understanding of the fundamental pathology underlying EON but also point towards novel therapeutic avenues. Modulating the aberrant UPS machinery or targeting associated stress and inflammatory pathways could offer promising strategies for intervention. Further validation studies are crucial to translate these proteomic insights into clinically relevant biomarkers and effective therapeutic targets for these devastating conditions.</p>
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