Eleni Zamba-Papanicolaou

There is accumulating evidence that distinct mitochondrial DNA (mtDNA) methylation and hydroxymethylation patterns exist in Parkinson’s disease (PD). However, most studies have been limited to the investigation of specific target regions, rather than the entire mtDNA, and have been further hindered by other methodological discrepancies and the lack of non-CpG context investigation. Here, we provide a comprehensive profile of methylation and hydroxymethylation levels across the mitochondrial genome, at global and single-base resolution, in CpG and non-CpG (CHG, CHH) contexts in blood samples from early-onset PD (EOPD) patients (n = 39) and age- and sex-matched controls (n = 63). Bisulfite (BS) and oxidative-bisulfite (oxBS) conversions in parallel workflows followed by next-generation sequencing (NGS) using Illumina’s Novaseq 6000 sequencing system identified mitochondrial 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in all contexts. Global mtDNA methylation was significantly higher in EOPD patients vs. matched controls in the CpG context (p = 5.63 × 10⁻³) in the BS status, and in all contexts [CpG (p = 2.67 × 10⁻⁴), CHG (p = 0.015), CHH (p = 0.012)] in the oxBS status, i.e., “true methylation”. At single-base resolution, the most statistically significant sites across the mitogenome, in the D-loop region, and CpG context, were primarily hypomethylated in EOPD patients compared to matched controls. Upon further validation, both global and base resolution mtDNA (hydroxy)methylation results could act as blood-based biomarkers for EOPD.

Over the years, there has been extensive research conducted on Parkinson’s Disease (PD), a neurodegenerative disorder known for causing motor impairment and behavioral changes. In more recent years, the roles of dysregulated microRNAs (miRNAs) in PD pathology have been studied in the hopes of developing new diagnostic methods or even treatments. This systematic review pinpoints and examines studies between 2010 and 2024 that have identified significant dysregulation of miRNAs in patients with PD. Upon filtering out the search results by a series of exclusion criteria, this review was conducted using 56 relevant studies. These studies revealed a vast array of significantly dysregulated miRNAs identified in the samples of patients with PD, when compared to healthy controls. A number of these miRNAs, such as miR-29c-3p, are likely biomarkers for more accurate PD diagnosis, and many, such as miR-485-3p, were found to be involved in PD pathogenesis. With further research, miRNAs could become a helpful diagnostic and prognostic tool for PD, with some of them even being candidate therapeutic targets for future treatments.

Myasthenia gravis (MG) is a chronic antibody-mediated autoimmune neuromuscular disorder causing fluctuating muscle weakness due to autoantibodies that target the acetylcholine receptor (AChR) in the neuromuscular junction (NMJ). Despite extensive research, MG remains unpredictable and heterogeneous, underscoring the need for better insight into disease pathogenesis and clinical course. The objective of this study was to identify clinical and immunologic disease-related parameters that correlate with disease severity. This large-scale, multicenter, cross-sectional study included prospectively recruited patients with MG and MG serum samples procured from reputable biobanks. Inclusion criteria required an MG clinical presentation and a positive anti-AChR immunoglobulin G (IgG) titer (≥0.5 nM), confirmed by 2 independent tests. Enrolled patients underwent profiling of subunit-specific anti-AChR IgG titers. Disease severity patterns and their relation to age, sex, disease onset, thymic involvement, anti-AChR titer, and anti-AChR subunit immunodominance were investigated. The study included 513 patients with MG (50.5% female) with a median age of 64 years (range 14-98.5). Extended clinical data were available for 232 patients. Anti-AChR titers increased monotonically with disease severity (ρ = 0.31, 95% CI 0.17-0.44, p < 0.0001) and were higher in female patients (median 14.6 nM vs 7.7 nM, 95% CI for Δ 2.1-7.3 nM, p < 0.0001) in whom higher antibody titers decreased monotonically with age. Male patients' titers were predominantly alpha-immunodominant (male 23.2% vs female 16.6%, 95% CI for Δ 0%-7.6%, p = 0.031), while female patients' titers were gamma-immunodominant (female 30% vs male 24.5%, 95% CI for Δ 2%-11%, p = 0.0027). Gamma immunodominance correlated with higher anti-AChR titer (ρ = 0.25, 95% CI 0.16-0.33, p < 0.0001) and severe (Myasthenia Gravis Foundation of America IV-V) disease (44% vs 27%, 95% CI for Δ 4%-34%, p = 0.0141). Unsupervised principal component analysis disclosed 2 distinct disease endotypes: female patients with moderate to severe early-onset MG, gamma subunit immunodominance, high anti-AChR titers, and frequent thymic hyperplasia (endotype A) and the remaining patients, mostly patients with late-onset MG, alpha subunit immunodominance, and low anti-AChR titers (endotype B). Two distinct MG endotypes emerge based on sex, age, thymic involvement, autoantibody titer, and immunoglobulin AChR subunit immunodominance-suggesting different underlying etiologies with potential implications for sex-dependent precision medicine, women's health, and the development of new therapeutic modalities for MG.

Background: Huntington’s Disease (HD) is a monogenic neurodegenerative disease resulting in a CAG repeat expansion in the HTT gene. Despite this genetic simplicity, its molecular mechanisms remain highly complex. Methods: In this study, untargeted serum proteomics, bioinformatics analysis, biomarker filtering and ELISA validation were implemented to characterize the proteomic landscape across the three HD stages—asymptomatic, early symptomatic and symptomatic advanced—alongside gender/age-matched controls. Results: We identified 84 over-expressed and 118 under-expressed differentially expressed proteins. Enrichment analysis revealed dysregulation in pathways including the complement cascade, LXR/RXR activation and RHOGDI signaling. Biomarker analysis highlighted key proteins with diagnostic potential, including CAP1 (AUC = 0.809), CAPZB (AUC = 0.861), TAGLN2 (AUC = 0.886), THBS1 (AUC = 0.883) and CFH (AUC = 0.948). CAP1 and CAPZB demonstrated robust diagnostic potential in linear mixed-effects models. CAP1 decreased in the asymptomatic stage, suggesting early cytoskeletal disruption, while CAPZB was consistently increased across HD stages. Conclusions: Our findings illuminate the dynamic proteomic and molecular landscape of HD. Future studies should validate these candidates in larger, more diverse cohorts and explore their mechanistic roles in HD pathology and progression.

Valosin Containing Protein-associated multisystem proteinopathy (VCP-MSP) is a progressive, autosomal dominant disorder caused by pathogenic variants in the VCP gene, resulting in a heterogeneous clinical presentation. Muscle biopsy findings are characteristic but not pathognomonic. This study aimed to comprehensively analyse VCP-related myopathology and explore correlations with clinical phenotypes, genetic variants, and disease progression. Muscle biopsy images and data were collected retrospectively from adults (≥18 years) with pathogenic or likely pathogenic VCP variants enrolled in the VCP Multicentre International Study. Biopsy data were standardized using the “Common Data Elements for Muscle Biopsy Reporting.” Variations in biopsy findings were analysed by biopsy site, time from disease onset, the four most common VCP variants, and clinical phenotypes. A total of 112 muscle biopsies were included. Most individuals were male (66.0%). The mean age at biopsy was 53.3 years (SD 10.0), with a mean disease duration of 6.5 years (SD 4.5). The most frequent VCP variant was c.464G>A (p.Arg155His) (18.8%). The top clinical phenotypes were isolated myopathy (37.5%), myopathy with Paget disease of bone (17.9%), and myopathy with motor neuron involvement (13.4%). The vastus lateralis was the most common biopsy site (34.8%), and 91% were open biopsies. Histopathologic findings included atrophic fibres (87.5%), rimmed vacuoles (72.3%), endomysial fibrosis (58.0%), and protein aggregates (51.8%), primarily p62 (60.3%) and VCP (36.2%). Degeneration niches with fibrofatty replacement and atrophic fibres were seen in 33.3% of biopsies without frequency differences by clinical phenotypes. There were no differences in biopsy findings among the 4 most common VCP gene variants, except for the absence of degeneration niches in muscle biopsies of 12 patients with c.277C>T (p.Arg93Cys). MRI data from 30 patients showed fat pockets corresponding to these niches and STIR hyperintensity correlated with inflammatory infiltrates in 42.9%. Concordance between clinical phenotype, biopsy, and neurophysiology was observed in only 49.4% of cases, indicating significant heterogeneity. VCP-MSP muscle biopsies consistently show myopathic or mixed patterns with rimmed vacuoles and p62/VCP-positive inclusions, regardless of clinical phenotype, age, or progression. Some lack vacuoles, challenging diagnosis. Discrepancies between clinical, neurophysiology, and biopsy findings should prompt consideration of VCP-MSP to improve detection and management.

Background/Objectives: Dementia is not a single disease but an umbrella term that encompasses a range of symptoms, such as memory loss and cognitive impairments, which are severe enough to disrupt daily life. One of the most common forms of dementia is Alzheimer’s Disease (AD), a complex neurodegenerative condition influenced by both genetic and environmental factors. Recent research has highlighted diet as a potential modifiable risk factor for AD. Decades of research have explored the role of dietary patterns, including the Mediterranean Diet (MD) and its components, in neuroprotection and cognitive health. Systematic review examines studies investigating the impact of the Mediterranean Diet, Mediterranean-like diets, the Nordic Diet (ND), dietary intake patterns, and specific components such as extra virgin olive oil and rapeseed oil on cognitive function, disease onset, and progression in AD and dementia. Methods: A comprehensive search of PubMed, the Directory of Open Access Journals, and the Social Science Research Network was conducted independently by two reviewers using predefined search terms. The search period included studies from 2006 to 2024. Eligible studies meeting the inclusion criteria were systematically reviewed, yielding 88 studies: 85 focused on the MD and its relationship to AD and dementia, while only 3 investigated the ND. Results: The findings suggest that adherence to the Mediterranean and Nordic diets is generally associated with improved cognitive function and delayed cognitive decline and that adherence to both these diets can improve cognitive function. Some studies identified that higher legume consumption decreased dementia incidence, while fruits and vegetables, carbohydrates, and eggs lowered dementia prevalence. Most studies demonstrated that high MD or ND adherence was associated with better cognitive function and a lower risk of poor cognition in comparison to individuals with lower MD or ND adherence. However, some studies reported no significant benefits of the MD on cognitive outcomes, while two studies indicated that higher red meat consumption was linked to better cognitive function. Conclusion: Despite promising trends, the evidence remains varying across studies, underscoring the need for further research to establish definitive associations between diet and cognitive function. These findings highlight the essential role of dietary interventions in the prevention and management of dementia and AD, therefore offering critical insights into the underlying mechanisms by which the diet may impact brain health.

Over the last years, an increased interest in pharmacological and non-pharmacological therapeutic approaches have increased in HD research, at the moment HD remains a non-curable disease and current approaches involve symptomatic disease treatment. The systematic review identifies studies that have investigated pharmacological and non-pharmacological interventions, in HD individuals and mouse models to identify the clinical impact, regarding i) silencing and reduction of mHTT, ii) safety, efficiency and toxicity of antisense nucleotides, zinc finger proteins, CRISPR-Cas9 and transcription activator-like effector nuclease, iii) genetic modifiers contributing to early or delayed HD onset and, iv) importance of physical activity on motor and cognitive function. A systematic search of PubMed and Directory of Open Access Journal was performed by two independent reviewers using specific search term criteria for studies. The search period included studies from 2000 until 2024. The identified abstracts were screened and studies within the review fulfilled predetermined inclusion criteria. Furthermore, reference screening of included studies was also conducted. A total of forty-two studies were included. Most pharmacological studies identified that oligonucleotides, zinc finger proteins and transcription activator-like effector nuclease, reduced mHTT and HD mRNA, decreased toxicity and allele-specific targeting to prevent wild-type HTT targeting. In addition, studies involving medication such as Tetrabenazine showed to suppress HD-related chorea symptoms, while Atomoxetine did not suppress symptoms and Pridopidine showed an efficiency regarding motor symptoms. The safety and efficacy of these drugs are similar to that of other studies. Physical activity studies demonstrated that HD patients showed improvement in their gait and motor functions, an increase in cognitive function and quality of life, and a decrease in anxiety and depression, this indicates the beneficial health effects of physical activity in HD. However, further research is required to assess the full impact of pharmacological and non-pharmacological approaches in larger clinical trials assessing safety, feasibility and efficacy within HD mouse models and the HD population.

Huntington’s Disease (HD) is a rare, progressive neurodegenerative disease caused by CAG repeat expansion in the Huntingtin gene. HD is an incurable disease; therefore, there is a growing need for effective therapeutic treatments and candidate biomarkers for prognosis and diagnosis of HD. Technological advancements over the past couple of years, have led to high-throughput experiments and omics data. The use of System Bioinformatics (SB) approaches, allows for the integration of information across different -omics, this can clarify synergistic relationships across biological molecules, resulting in complex biological networks. SB and network-based approaches, are able to shed light on the potential interactions of genes, proteins, metabolites and pathways participating in HD pathogenesis and how dysregulation of these biological entities, can affect age on onset, disease severity and progression. Moreover, −omics data analysis and network-based approaches can provide better understanding how these biological molecules interact with each other and provides potential drug targets and biomarkers that can be used to treat HD or delay symptom onset; therefore, opening the door towards precision medicine. The aim of the following chapter, is to discuss the most popular -omics related to HD research, and the growing popularity of single cell analysis, repositories and software available for bulk and single cell analysis. In addition, network-based approaches regarding HD will also be mentioned.

Introduction: Amyotrophic lateral sclerosis (ALS) is a devastating, uniformly lethal degenerative disease of motor neurons, presenting with relentlessly progressive muscle atrophy and weakness. The etiology of ALS remains unexplained for over 85% of all cases, suggesting that besides the genetic basis of the disease, various environmental factors are implicated in the pathogenesis of ALS. This study aimed to investigate the contribution of known environmental risk factors of ALS in the Cypriot population.Methods: We conducted a case–control study with a total of 56 ALS cases and 56 healthy gender/age-matched controls of Cypriot nationality. Demographic, lifestyle characteristics, medical conditions, and environmental exposures were collected through the use of a detailed questionnaire. Statistical analyses using the R programming language examined the association between the above environmental factors and ALS.Results: A chi-square test analysis revealed a statistically significant (p = 0.000461) difference in smoking status between the two groups. In addition, univariate logistic regression analysis showed a statistically significant association between ALS cases for head trauma/injury (p = 0.0398) and exposure to chemicals (p = 0.00128), compared to controls.Conclusion: This case–control investigation has shed some light on the epidemiological data of ALS in Cyprus, by identifying environmental determinants of ALS, such as smoking, head trauma, and chemical exposure, in the Cypriot population.

Background: Motor symptoms are well-characterized in Parkinson’s disease (PD). However, non-motor symptoms, such as depression, are commonly observed and can appear up to 10 years before motor features, resulting in one-third of individuals being misdiagnosed with a neuropsychiatric disorder. Thus, identifying diagnostic biomarkers is crucial for accurate PD diagnosis during its prodromal or early stages.Methods: We employed an integrative approach, combining single nucleus RNA and bulk mRNA transcriptomics to perform comparative molecular signatures analysis between PD and major depressive disorder (MDD). We examined 39,834 nuclei from PD (GSE202210) and 32,707 nuclei from MDD (GSE144136) in the dorsolateral prefrontal cortex (dlPFC) of Brodmann area 9. Additionally, we analyzed bulk mRNA peripheral blood samples from PD compared to controls (GSE49126, GSE72267), as well as MDD compared to controls (GSE39653).Results: Our findings show a higher proportion of astrocytes, and oligodendrocyte cells in the dlPFC of individuals with PD vs. MDD. The excitatory to inhibitory neurons (E/I) ratio analysis indicates that MDD has a ratio close to normal 80/20, while PD has a ratio of 62/38, indicating increased inhibition in the dlPFC. Microglia displayed the most pronounced differences in gene expression profiles between the two conditions. In PD, microglia display a pro-inflammatory phenotype, while in MDD, they regulate synaptic transmission through oligodendrocyte-microglia crosstalk. Analysis of bulk mRNA blood samples revealed that the COL5A, MID1, ZNF148, and CD22 genes were highly expressed in PD, whereas the DENR and RNU1G2 genes were highly expressed in MDD. CD22 is involved in B-cell activation and the negative regulation of B-cell receptor signaling. Additionally, CD86, which provides co-stimulatory signals for T-cell activation and survival, was found to be a commonly differentially expressed gene in both conditions. Pathway analysis revealed several immune-related pathways common in both conditions, including the complement and coagulation cascade, and B-cell receptor signaling.Discussion: This study demonstrates that bulk peripheral immune cells play a role in both conditions, but neuroinflammation in the dlPFC specifically manifests in PD as evidenced by the analysis of single nucleus dlPFC datasets. Integrating these two omics levels offers a better understanding of the shared and distinct molecular pathophysiology of PD and MDD in both the periphery and the brain. These findings could lead to potential diagnostic biomarkers, improving accuracy and guiding pharmacological treatments.