The study of the deubiquitinase USP8 in Parkinson’s disease Pathogenesis

Parkinson's disease is the second commonest neurodegenerative disease currently treated symptomatically. It is a multifactorial disease involving mechanisms ranging from protein aggregation to mitochondrial dysfunction, oxidative stress and dopamine dysregulation. The levels of α-synuclein have been causatively linked to the development and progression of Parkinson's disease. Therefore α-synuclein lowering strategies are valid approaches in Parkinson's disease. Neuropathologically, Lewy Bodies in the vulnerable substantia nigra of Parkinson's disease patients are less ubiquitinated and specifically less K-63 ubiquitinated than Lewy bodies in the cortex, suggesting differential activation or regulation of ubiquitin interactors. A targeted screen for such interactors revealed that the Deubiquitinating enzyme Usp8 is upregulated in the substantia nigra of Parkinson’s disease brains and is inversely correlated with the degree of total and K-63 ubiquitination. Using genetic knockdown and overexpression techniques, Usp8 was found to colocalize and directly interact with α-synuclein. It was found to de-ubiquitinate α-synuclein and increase its half-life. Its knockdown increased the total and K-63 α-synuclein ubiquitination and decreased its levels by 35&amp;percnt; at least partly by increasing its degradation via the lysosome. In vivo in the Drosophila melanogaster, Usp8 knockdown demonstrated protection against α-synuclein toxicity. It rescued in a specific manner the rough eye phenotype, the age-dependent locomotive defect and the loss of dopaminergic neurons caused by the expression of α-synuclein. Specific and effective pharmacological Usp8 inhibition also has the potential to lower α-synuclein levels. Collectively, the evidence produced in my thesis suggests that Usp8 could be a potential target for the future disease-modifying therapies in Parkinson's disease.</p

The study of the deubiquitinase USP8 in Parkinsonâs disease Pathogenesis

Parkinson's disease is the second commonest neurodegenerative disease currently treated symptomatically. It is a multifactorial disease involving mechanisms ranging from protein aggregation to mitochondrial dysfunction, oxidative stress and dopamine dysregulation. The levels of &alpha;-synuclein have been causatively linked to the development and progression of Parkinson's disease. Therefore &alpha;-synuclein lowering strategies are valid approaches in Parkinson's disease. Neuropathologically, Lewy Bodies in the vulnerable substantia nigra of Parkinson's disease patients are less ubiquitinated and specifically less K-63 ubiquitinated than Lewy bodies in the cortex, suggesting differential activation or regulation of ubiquitin interactors. A targeted screen for such interactors revealed that the Deubiquitinating enzyme Usp8 is upregulated in the substantia nigra of Parkinsonâs disease brains and is inversely correlated with the degree of total and K-63 ubiquitination. Using genetic knockdown and overexpression techniques, Usp8 was found to colocalize and directly interact with &alpha;-synuclein. It was found to de-ubiquitinate &alpha;-synuclein and increase its half-life. Its knockdown increased the total and K-63 &alpha;-synuclein ubiquitination and decreased its levels by 35&percnt; at least partly by increasing its degradation via the lysosome. In vivo in the Drosophila melanogaster, Usp8 knockdown demonstrated protection against &alpha;-synuclein toxicity. It rescued in a specific manner the rough eye phenotype, the age-dependent locomotive defect and the loss of dopaminergic neurons caused by the expression of &alpha;-synuclein. Specific and effective pharmacological Usp8 inhibition also has the potential to lower &alpha;-synuclein levels. Collectively, the evidence produced in my thesis suggests that Usp8 could be a potential target for the future disease-modifying therapies in Parkinson's disease.</p

Cognitive decline and diabetes: a systematic review of the neuropathological correlates accounting for cognition at death

Given conflicting findings in epidemiologic studies, we determined the relative contributions of different neuropathologies to the excess risk of cognitive decline in diabetes mellitus (DM) through a systematic review of the literature. Included studies compared subjects with and without DM and reported neuropathological outcomes accounting for cognition at death. Data on Alzheimer’s disease (AD) pathology, cerebrovascular disease and non-vascular, non-AD pathology were extracted from each study. Eleven studies (n=6 prospective cohorts, n=5 retrospective post-mortem series, total n=6330) met inclusion criteria. All 11 studies quantified AD changes and 10/11 measured cerebrovascular disease: macroscopic lesions (n=9), microinfarcts (n=8), cerebral amyloid angiopathy (CAA, n=7), lacunes (n=6), white matter disease (n=5), haemorrhages (n=4), microbleeds (n=1), hippocampal microvasculature (n=1). Other pathology was infrequently examined. No study reported increased AD pathology in DM, three studies showed a decrease (n=872) and four (n= 4018) showed no difference, after adjustment for cognition at death. No study reported reduced cerebrovascular pathology in DM. Three studies (n=2345) reported an increase in large infarcts, lacunes and microinfarcts. One study found lower cognitive scores in DM compared to non-DM subjects despite similar cerebrovascular and AD-pathology load suggesting contributions from other neuropathological processes. In conclusion, lack of an association between DM and AD-related neuropathology was consistent across studies, irrespective of methodology. In contrast to AD, DM was associated with increased large and small vessel disease. Data on other pathologies such as non-AD neurodegeneration, and blood-brain-barrier breakdown were lacking. Further studies evaluating relative contributions of different neuropathologies to the excess risk of DM are needed

The Greek Neuropathic Pain Registry: The structure and objectives of the sole NPR in Greece

Objectives Neuropathic pain (NP) is a complex condition that impairs the patients’ quality of life. Registries are useful tools, increasingly used as they provide high-quality data. This article aims to describe the Greek Neuropathic Pain Registry (Gr.NP.R.) design, the patients’ baseline data, and real-world treatment outcomes. Methods The Gr.NP.R. collects electronically, stores, and shares real-world clinical data from Pain and Palliative Care centers in Greece. It is a web-based application, which ensures security, simplicity, and transparency. VAS, DN4, and Pain Detect were used for pain and NP assessment. Results From 2016 to 2020, 5980 patients with chronic pain, of cancer or non-cancer origin, were examined and 2334 fulfilled the NP inclusion criteria (VAS &gt; 5, DN4 &gt; 4, and Pain Detect &gt;= 19). At the first visit, the mean age was 64.8 years, 65.5% were female patients, and 97.9% were Greek. The mean (SD) time from pain initiation to visiting the pain clinics was 1.5 (3.8) years. Most patients were undertreated. Following the patients’ registration, the national guidelines were implemented. The majority of the prescribed medications were gabapentinoids (70.2%), especially pregabalin (62.6%), and opioids (tramadol, 55.3%). At visits 1 and 6, mean VAS was 7.1 and 5, and mean DN4 score was 5.6 and 3.5, respectively. Conclusions The Gr.NP.R. provides information on the demographics, clinical progress, treatment history, treatment responses, and the drugs of choice for patients with cancer and non-cancer NP. The collected data may help physicians plan the management of their patients

Evaluation of Two Highly-Multiplexed Custom Panels for Massively Parallel Semiconductor Sequencing on Paraffin DNA

<div><p>Background—Aim</p><p>Massively parallel sequencing (MPS) holds promise for expanding cancer translational research and diagnostics. As yet, it has been applied on paraffin DNA (FFPE) with commercially available highly multiplexed gene panels (100s of DNA targets), while custom panels of low multiplexing are used for re-sequencing. Here, we evaluated the performance of two highly multiplexed custom panels on FFPE DNA.</p><p>Methods</p><p>Two custom multiplex amplification panels (B, 373 amplicons; T, 286 amplicons) were coupled with semiconductor sequencing on DNA samples from FFPE breast tumors and matched peripheral blood samples (n samples: 316; n libraries: 332). The two panels shared 37% DNA targets (common or shifted amplicons). Panel performance was evaluated in paired sample groups and quartets of libraries, where possible.</p><p>Results</p><p>Amplicon read ratios yielded similar patterns per gene with the same panel in FFPE and blood samples; however, performance of common amplicons differed between panels (p<0.001). FFPE genotypes were compared for 1267 coding and non-coding variant replicates, 999 out of which (78.8%) were concordant in different paired sample combinations. Variant frequency was highly reproducible (Spearman’s rho 0.959). Repeatedly discordant variants were of high coverage / low frequency (p<0.001). Genotype concordance was (a) high, for intra-run duplicates with the same panel (mean±SD: 97.2±4.7, 95%CI: 94.8–99.7, p<0.001); (b) modest, when the same DNA was analyzed with different panels (mean±SD: 81.1±20.3, 95%CI: 66.1–95.1, p = 0.004); and (c) low, when different DNA samples from the same tumor were compared with the same panel (mean±SD: 59.9±24.0; 95%CI: 43.3–76.5; p = 0.282). Low coverage / low frequency variants were validated with Sanger sequencing even in samples with unfavourable DNA quality.</p><p>Conclusions</p><p>Custom MPS may yield novel information on genomic alterations, provided that data evaluation is adjusted to tumor tissue FFPE DNA. To this scope, eligibility of all amplicons along with variant coverage and frequency need to be assessed.</p></div

Performance of individual amplicons in blood and FFPE DNA.

<p><b>A.</b> Amplicon performance grading did not significantly differ between the two panels. Columns: combined evaluation of each panel in blood and FFPE. Numbers within boxes: actual amplicon number per category, as indicated. <b>B.</b> Performance of the 83 common amplicons was significantly different in the two panels (p<0.0001). The most unstable amplicons were of performance grade 1 and 2. <b>C.</b> A non-linear distribution of mean read ratios according to amplicon GC content was observed. Read ratios of amplicons with >75% or <25% GC content were almost uniformly below the 10^th percentile cut off (dotted line in both graphs) (Kruskal-Wallis test p<0.0001 for each panel and sample group). <b>D.</b> Very high and very low GC content was significantly associated with failed amplicons (gr 0). This pattern was also present for the B-panel in blood samples, despite the absence of statistical significance. Except for these extreme cases, however, all other amplicon categories did not significantly differ with regards to GC%.</p

Performance of the B and T panels in DNA from matched blood and tumor FFPE samples.

<p><b>A:</b> B panel; <b>B:</b> T panel. Read ratios for all amplicons for matched samples sorted per gene are shown, corresponding to 70192 observations with the B and 26128 observations with the T panel. Lanes and dots therein represent mean read ratios per amplicon for all samples tested in the respective group. Amplicon order is the same in all graphs. Solid and dotted horizontal lines within graphs: mean values + 3xSD per panel per sample type, respectively. Amplicon reading efficiency was overall constant between blood—FFPE samples with the same panel. For some genes with frequent gains in breast cancer, e.g., <i>CCND1</i>, <i>EGFR</i>, <i>ERBB2</i>, <i>PIK3CA</i> (B panel) and <i>AKT1</i>, <i>EGFR</i> (T panel) outliers with maximal amplicon read ratios outside the Y-axis were observed (A & B, red stars in tumor graphs). By contrast, for genes frequently lost in breast cancer, e.g., TP53 with both panels, mean read ratios in tumor DNA were lower than in blood (A & B, turquoise stars). FFPE-specific over-representation of individual amplicons was occasionally observed (A & B, diagonal arrows). Importantly, patterns of read ratios occasionally differed for genes targeted with the same amplicons in panels B and T, e.g., for <i>ARID1B</i>, <i>MAP3K1</i>, <i>TP53</i> (A & B, black stars with coloured outlines in blood graphs).</p