Computer-based evaluation of α-synuclein pathology in multiple system atrophy as a novel tool to recognize disease-subtypes.

Multiple system atrophy (MSA) is a neurodegenerative disorder with variable disease course and distinct constellations of clinical (cerebellar (MSA-C) or parkinsonism (MSA-P)) and pathological phenotypes, suggestive of distinct α-synuclein (αSyn) strains. Neuropathologically, MSA is characterized by the accumulation of αSyn in oligodendrocytic glial cytoplasmic inclusions (GCI). Using a novel computer-based method, this study quantified the size of GCIs, density of all αSyn pathology, density of only the GCIs, and number of GCIs in MSA cases (n = 20). The putamen and cerebellar white matter (WM) were immuno-stained with the disease-associated 5G4 anti-αSyn antibody. Following digital scanning and image processing, total 5G4-immunoreactive pathology (i.e., neuronal, neuritic, and glial) and GCIs were optically dissected for inclusion size and density measurement then evaluated applying a novel computer-based method using ImageJ. GCI size varied between cases and brain regions (p < 0.0001) and heterogeneity in the density of all αSyn pathology including the density and number of GCIs were observed between regions and across cases, where MSA-C cases had significantly higher density of all αSyn pathology in the cerebellar WM (p = 0.049). Some region-specific morphological variables inversely correlated with the age of onset and death, suggestive of an underlying aging-related cellular mechanism. Unsupervised K-means cluster analysis classified MSA cases into three distinct groups based on region-specific morphological variables. In conclusion, we developed a novel computer-based method that is easily accessible, providing a first step to developing artificial-intelligence-based evaluation strategies for large scale comparative studies. Our observations on the variability of morphological variables between brain regions and cases highlight i) the importance of computer-based approaches to detect features not considered in the routine diagnostic practice, and ii) novel aspects for the identification of previously unrecognized MSA subtypes that do not necessarily reflect the current clinical classification of MSA-C or MSA-P.

Multiple system atrophy with amyloid-ß-predominant Alzheimer's disease neuropathologic change.

Multiple system atrophy is a neurodegenerative disease with α-synuclein pathology predominating in the striatonigral and olivopontocerebellar systems. Mixed pathologies are considered to be of low frequency and mostly comprise primary age-related tauopathy or low levels of Alzheimer's disease-related neuropathologic change. Therefore, the concomitant presence of different misfolded proteins in the same brain region is less likely in multiple system atrophy. During the neuropathological evaluation of 21 consecutive multiple system atrophy cases, we identified four cases exhibiting an unusual discrepancy between high Thal amyloid-ß phase and low transentorhinal Braak neurofibrillary tangle stage. We mapped α-synuclein pathology, measured the size and number of glial cytoplasmic inclusions and compared the amyloid-ß peptides between multiple system atrophy and Alzheimer's disease. In addition, we performed α-synuclein seeding assay from the affected putamen samples. We performed genetic testing for APOE, MAPT, PSEN1, PSEN2 and APP. We refer to the four multiple system atrophy cases with discrepancy between amyloid-ß and tau pathology as 'amyloid-ß-predominant Alzheimer's disease neuropathologic change-multiple system atrophy' to distinguish these from multiple system atrophy with primary age-related tauopathy or multiple system atrophy with typical Alzheimer's disease neuropathologic change. As most multiple system atrophy cases with mixed pathologies reported in the literature, these cases did not show a peculiar clinical or MRI profile. Three amyloid-ß-predominant Alzheimer's disease neuropathologic change-multiple system atrophy cases were available for genetic testing, and all carried the APOE ɛ4 allele. The extent and severity of neuronal loss and α-synuclein pathology were not different compared with typical multiple system atrophy cases. Analysis of amyloid-ß peptides revealed more premature amyloid-ß plaques in amyloid-ß-predominant Alzheimer's disease neuropathologic change-multiple system atrophy compared with Alzheimer's disease. α-Synuclein seeding amplification assay showed differences in the kinetics in two cases. This study highlights a rare mixed pathology variant of multiple system atrophy in which there is an anatomical meeting point of amyloid-ß and α-synuclein, i.e. the striatum or cerebellum. Since biomarkers are entering clinical practice, these cases will be recognized, and the clinicians have to be informed that the prognosis is not necessarily different than in pure multiple system atrophy cases but that the effect of potential α-synuclein-based therapies might be influenced by the co-presence of amyloid-ß in regions where α-synuclein also aggregates. We propose that mixed pathologies should be interpreted not only based on differences in the clinical phenotype but also on whether protein depositions regionally overlap, potentially leading to a different response to α-synuclein-targeted therapies.

QSOX2 Upregulated in triple-negative breast cancer exacerbates patient prognosis by stabilizing integrin ß1.

Breast cancer (BC) remains a significant global health threat, with triple-negative breast cancer (TNBC) standing out as a particularly aggressive subtype lacking targeted therapies. Addressing this gap, we propose Quiescin Q6 sulfhydryl oxidase 2 (QSOX2) as a potential therapeutic target, a disulfide bond-forming enzyme implicated in cancer progression. Using publicly available datasets, we conducted a comprehensive analysis of QSOX2 expression in BC tumor and non-tumor tissues, assessing its specificity across different molecular subtypes. We further explored correlations between QSOX2 expression and patient outcomes, utilizing datasets like TCGA and METABRIC. In addition, we performed in vitro experiments to evaluate QSOX2 expression in BC cell lines and investigate the effects of QSOX2 knockdown on various TNBC cellular processes, including cell proliferation, apoptosis resistance, migration, and the epithelial-to-mesenchymal transition (EMT). Our results reveal significantly elevated QSOX2 expression in BC tumor tissues, particularly in TNBC, and establish an association between high QSOX2 expression and increased patient mortality, cancer progression, and recurrence across various BC subtypes. Notably, QSOX2 knockdown in TNBC cell lines reduces cell proliferation, enhances apoptosis, and suppresses migration, potentially mediated through its influence on the EMT process. Furthermore, we identify a significant link between QSOX2 and integrin ß1 (ITGB1), suggesting that QSOX2 enhances ITGB1 stability, subsequently exacerbating the malignancy of TNBC. In conclusion, elevated QSOX2 expression emerges as a key factor associated with adverse patient outcomes in BC, particularly in TNBC, contributing to disease progression through various mechanisms, including the modulation of ITGB1 stability. Our findings underscore the potential of targeting QSOX2 as a therapeutic strategy for improving patient prognoses not only in TNBC but also in other BC subtypes.

4R-Tau seeding activity unravels molecular subtypes in patients with Progressive Supranuclear Palsy.

Progressive Supranuclear palsy (PSP) is a 4-repeat (4-R) tauopathy. We hypothesized that the molecular diversity of tau could explain the heterogeneity seen in PSP disease progression. To test this hypothesis, we performed an extensive biochemical characterisation of the high molecular weight tau species (HMW-Tau) in 20 different brain regions of 25 PSP patients. We found a correlation between the HMW-Tau species and tau seeding capacity in the primary motor cortex, where we confirmed that an elevated 4R-Tau seeding activity correlates with a shorter disease duration. To identify factors that contribute to these differences, we performed proteomic and spatial transcriptomic analysis that revealed key mechanistic pathways, in particular those involving the immune system, that defined patients demonstrating high and low tau seeding capacity. These observations suggest that differences in the tau seeding activity may contribute to the considerable heterogeneity seen in disease progression of patients suffering from PSP.

Advances in Recombinant Adeno-Associated Virus Vectors for Neurodegenerative Diseases.

Recombinant adeno-associated virus (rAAV) vectors are gene therapy delivery tools that offer a promising platform for the treatment of neurodegenerative diseases. Keeping up with developments in this fast-moving area of research is a challenge. This review was thus written with the intention to introduce this field of study to those who are new to it and direct others who are struggling to stay abreast of the literature towards notable recent studies. In ten sections, we briefly highlight early milestones within this field and its first clinical success stories. We showcase current clinical trials, which focus on gene replacement, gene augmentation, or gene suppression strategies. Next, we discuss ongoing efforts to improve the tropism of rAAV vectors for brain applications and introduce pre-clinical research directed toward harnessing rAAV vectors for gene editing applications. Subsequently, we present common genetic elements coded by the single-stranded DNA of rAAV vectors, their so-called payloads. Our focus is on recent advances that are bound to increase treatment efficacies. As needed, we included studies outside the neurodegenerative disease field that showcased improved pre-clinical designs of all-in-one rAAV vectors for gene editing applications. Finally, we discuss risks associated with off-target effects and inadvertent immunogenicity that these technologies harbor as well as the mitigation strategies available to date to make their application safer.

Disease-Specific α-Synuclein Seeding in Lewy Body Disease and Multiple System Atrophy Are Preserved in Formaldehyde-Fixed Paraffin-Embedded Human Brain.

Recent studies have been able to detect α-synuclein (αSyn) seeding in formaldehyde-fixed paraffin-embedded (FFPE) tissues from patients with synucleinopathies using seed amplification assays (SAAs), but with relatively low sensitivity due to limited protein extraction efficiency. With the aim of introducing an alternative option to frozen tissues, we developed a streamlined protein extraction protocol for evaluating disease-specific seeding in FFPE human brain. We evaluated the protein extraction efficiency of different tissue preparations, deparaffinizations, and protein extraction buffers using formaldehyde-fixed and FFPE tissue of a single Lewy body disease (LBD) subject. Alternatively, we incorporated heat-induced antigen retrieval and dissociation using a commercially available kit. Our novel protein extraction protocol has been optimized to work with 10 sections of 4.5-µm-thickness or 2-mm-diameter micro-punch of FFPE tissue that can be used to seed SAAs. We demonstrated that extracted proteins from FFPE still preserve seeding potential and further show disease-specific seeding in LBD and multiple system atrophy. To the best of our knowledge, our study is the first to recapitulate disease-specific αSyn seeding behaviour in FFPE human brain. Our findings open new perspectives in re-evaluating archived human brain tissue, extending the disease-specific seeding assays to larger cohorts to facilitate molecular subtyping of synucleinopathies.

Lack of difference between amyloid-beta burden at gyral crests and sulcal depths in diverse neurodegenerative diseases.

AIMS: The aim of this study is to clarify whether there is a difference in amyloid-beta burden between gyral crests (GCs) and sulcal depths (SDs) in different neurodegenerative proteinopathies. METHODS: We analysed the burden and distribution of amyloid-beta deposition in post-mortem brain samples from 138 autopsies, including Alzheimer's disease (n = 30), Down's syndrome (n = 11), Lewy body disease (LBD; n = 53), multiple system atrophy (n = 8) and progressive supranuclear palsy (n = 36). We applied quantitative amyloid-beta burden analysis to compare amyloid-beta deposition in both GCs and SDs. We also evaluated the prevalence of amyloid-beta plaques in both regions in samples exhibiting high or low amounts of amyloid-beta pathology. RESULTS: Amyloid-beta burden was evaluated in 67 and 84 samples of the frontal and temporal cortices, respectively. We did not find significant differences in the amyloid-beta burden between GCs and SDs in these regions in any examined disease. In addition, amyloid-beta plaques were almost evenly distributed in both regions in cases with low amounts of amyloid-beta pathology. Females in the LBD group showed significantly higher amyloid-beta burden than males (temporal cortex, p < 0.01). Furthermore, only one LBD case showed SD-predominant deposition associated with the coarse-grained plaques. CONCLUSIONS: We have shown that amyloid-beta is almost evenly distributed in both GCs and SDs in the frontal and temporal lobes from the early stage, in diverse neurodegenerative diseases. Sex may contribute to differences in the amyloid-beta burden. The coarse-grained plaque may show SD-predominant neuritic tau deposition that must be carefully distinguished from chronic traumatic encephalopathy-related SD tau pathology.

Progressive Supranuclear Palsy Syndrome Associated With a Novel Tauopathy: Case Study.

OBJECTIVES: To report a novel tauopathy in a patient with protracted coursed progressive supranuclear palsy (PC-PSP). METHODS: Clinical follow-up, gene analysis, neuropathological study. RESULTS: A 73-year-old man presented with diplopia, slowness, shuffling gait and falls. Neurological examination revealed slowed saccades, restricted up-gaze and mild parkinsonism. Three years after onset he developed personality changes. Slowly progressive parkinsonism was associated with memory and executive deficits. MRI showed subtle bilateral hippocampal and midbrain tegmentum atrophy and hyper-intensity in the brainstem tegmentum and white matter of the medial temporal lobe. Duration of illness was 11 years. There were no pathogenic mutations in 80 genes known to be involved in neurodegeneration, including MAPT (H1/H1 haplotype) and APOE (ε3/ε3 genotype). Neuropathology revealed PSP type pathology together with the pathology described in the novel limbic-predominant neuronal inclusion body 4-repeat tauopathy (LNT) correlating well with the signal alterations seen in MRI. DISCUSSION: Our observation broadens the spectrum of tau pathology associated with PC-PSP and suggests that memory deficit and hippocampal atrophy may be suggestive of non-Alzheimer's disease pathology, including LNT. Understanding the diverse range of tau morphologies may help explain phenotypic heterogeneity seen in PSP.

Association of glial tau pathology and LATE-NC in the ageing brain.

Ageing-related pathologies of the brain include neurofibrillary tangles, argyrophilic grains, ageing-related tau astrogliopathy (ARTAG), limbic-predominant age-related TDP-43 encephalopathy-neuropathological change (LATE-NC), vascular pathology and corpora amylacea. This study used an unbiased approach to evaluate a broad range of pathologies in an unselected European community-dwelling ageing cohort of 101 individuals (77-90 years). Pathological alterations observed included neurofibrillary tangles and corpora amylacea in all cases, ARTAG (79%), Thal amyloid-ß phase >1 (60%), cerebral amyloid angiopathy (39%), Lewy bodies (22%), LATE-NC (21%), oligodendroglial tau-positive coiled bodies (33%), and argyrophilic grains (15%). We demonstrate association of LATE-NC with the previously unappreciated age-related tau oligodendrogliopathy (ARTOG) and highlight the association of LATE-NC with various ARTAG types pointing toward common pathogenic aspects. Only neurofibrillary tangles and LATE-NC were associated with cognitive decline. This study broadens the spectrum of age-related brain pathologies and highlights a novel ageing-related tau pathology in oligodendroglia. Results from this study suggest overlapping pathogenic mechanisms between LATE-NC and glial tau pathologies in the medial temporal lobe.

α-Synuclein molecular behavior and nigral proteomic profiling distinguish subtypes of Lewy body disorders.

Lewy body disorders (LBD), characterized by the deposition of misfolded α-synuclein (α-Syn), are clinically heterogeneous. Although the distribution of α-Syn correlates with the predominant clinical features, the burden of pathology does not fully explain the observed variability in clinical presentation and rate of disease progression. We hypothesized that this heterogeneity might reflect α-Syn molecular diversity, between both patients and different brain regions. Using an ultra-sensitive assay, we evaluated α-Syn seeding in 8 brain regions from 30 LBD patients with different clinical phenotypes and disease durations. Comparing seeding across the clinical phenotypes revealed that hippocampal α-Syn from patients with a cognitive-predominant phenotype had significantly higher seeding capacity than that derived from patients with a motor-predominant phenotype, whose nigral-derived α-Syn in turn had higher seeding capacity than that from cognitive-predominant patients. Interestingly, α-Syn from patients with rapid disease progression (< 3 years to development of advanced disease) had the highest nigral seeding capacity of all the patients included. To validate these findings and explore factors underlying seeding heterogeneity, we performed in vitro toxicity assays, and detailed neuropathological and biochemical examinations. Furthermore, and for the first time, we performed a proteomic-wide profiling of the substantia nigra from 5 high seeder and 5 low seeder patients. The proteomic data suggests a significant disruption in mitochondrial function and lipid metabolism in high seeder cases compared to the low seeders. These observations suggest that distinct molecular populations of α-Syn may contribute to heterogeneity in phenotypes and progression rates in LBD and imply that effective therapeutic strategies might need to be directed at an ensemble of differently misfolded α-Syn species, with the relative contribution of their differing impacts accounting for heterogeneity in the neurodegenerative process.

Protracted course progressive supranuclear palsy.

BACKGROUND AND PURPOSE: Progressive supranuclear palsy (PSP) encompasses a broader range of disease courses than previously appreciated. The most frequent clinical presentations of PSP are Richardson syndrome (RS) and PSP with a predominant Parkinsonism phenotype (PSP-P). Time to reach gait dependence and cognitive impairment have been proposed as prognostic disease milestones. Genetic polymorphisms in TRIM11 and SLC2A13 genes have been associated with longer disease duration (DD). METHODS: Methods used include retrospective chart review, genetic single nucleotide polymorphism analyses (in three cases), and neuropathology. RESULTS: We identified four cases with long (>10-15 years) or very long (>15 years) DD. Stage 1 PSP tau pathology was present in two cases (one PSP-P and one undifferentiated phenotype), whereas pallidonigroluysian atrophy (PSP-RS) and Stage 4/6 (PSP-P) PSP pathology were found in the other two cases. Three cases were homozygous for the rs564309-C allele of the TRIM11 gene and the H1 MAPT haplotype. Two were heterozygous for rs2242367 (G/A) in SLC2A13, whereas the third was homozygous for the G-allele. CONCLUSIONS: We propose a protracted course subtype of PSP (PC-PSP) based on clinical or neuropathological criteria in two cases with anatomically restricted PSP pathology, and very long DD and slower clinical progression in the other two cases. The presence of the rs564309-C allele may influence the protracted disease course. Crystallizing the concept of PC-PSP is important to further understand the pathobiology of tauopathies in line with current hypotheses of protein misfolding, seeding activity, and propagation.

Alpha-synuclein seeding shows a wide heterogeneity in multiple system atrophy.

BACKGROUND: Multiple system atrophy (MSA) is a neurodegenerative condition characterized by variable combinations of parkinsonism, autonomic failure, cerebellar ataxia and pyramidal features. Although the distribution of synucleinopathy correlates with the predominant clinical features, the burden of pathology does not fully explain observed differences in clinical presentation and rate of disease progression. We hypothesized that the clinical heterogeneity in MSA is a consequence of variability in the seeding activity of α-synuclein both between different patients and between different brain regions. METHODS: The reliable detection of α-synuclein seeding activity derived from MSA using cell-free amplification assays remains challenging. Therefore, we conducted a systematic evaluation of 168 different reaction buffers, using an array of pH and salts, seeded with fully characterized brain homogenates from one MSA and one PD patient. We then validated the two conditions that conferred the optimal ability to discriminate between PD- and MSA-derived samples in a larger cohort of 40 neuropathologically confirmed cases, including 15 MSA. Finally, in a subset of brains, we conducted the first multi-region analysis of seeding behaviour in MSA. RESULTS: Using our novel buffer conditions, we show that the physicochemical factors that govern the in vitro amplification of α-synuclein can be tailored to generate strain-specific reaction buffers that can be used to reliably study the seeding capacity from MSA-derived α-synuclein. Using this novel approach, we were able to sub-categorize the 15 MSA brains into 3 groups: high, intermediate and low seeders. To further demonstrate heterogeneity in α-synuclein seeding in MSA, we conducted a comprehensive multi-regional evaluation of α-synuclein seeding in 13 different regions from 2 high seeders, 2 intermediate seeders and 2 low seeders. CONCLUSIONS: We have identified unexpected differences in seed-competent α-synuclein across a cohort of neuropathologically comparable MSA brains. Furthermore, our work has revealed a substantial heterogeneity in seeding activity, driven by the PBS-soluble α-synuclein, between different brain regions of a given individual that goes beyond immunohistochemical observations. Our observations pave the way for future subclassification of MSA, which exceeds conventional clinical and neuropathological phenotyping and considers the structural and biochemical heterogeneity of α-synuclein present. Finally, our methods provide an experimental framework for the development of vitally needed, rapid and sensitive diagnostic assays for MSA.

Compatibility of endoclips in the gastrointestinal tract with magnetic resonance imaging.

There are no clear guidelines on the compatibility between endoclips that remain in the gastrointestinal (GI) tract and magnetic resonance imaging (MRI). The purpose of this study was to investigate the effect of 3T (T) MRI on endoclips placed in excised pig tissues. Two types of endoclips were assessed: Olympus EZ (HX-610-135L) and QuickClip Pro (HZ-202LR). We assessed tissue damage or perforation and detachment of endoclips under 3T MRI magnetic field. We also evaluated the magnitude of force required to detach the endoclips from the porcine tissue. We measured the magnetic force acting on the Olympus EZ clips. QuickClip Pro clips were used as a control in this study. There was no tissue damage and no detachment of the endoclips (Olympus EZ and QuickClip Pro) during 3T MRI. The force required to detach the Olympus EZ clips ranged from 0.9 to 3.0 N. The translational magnetic force acting on the endoclips was 3.18 × 10-3 N. Ex vivo experiments showed that the magnetic field generated by 3 MRI did not cause tissue damage or perforation and did not detach the endoclips. Olympus EZ clips and QuickClip Pro clips in the GI tract appear to be safe during 3T MRI.

Efficacy and safety of endoscopic submucosal dissection for gastric epithelial neoplasia in elderly patients aged 80 years and older.

BACKGROUND: Endoscopic submucosal dissection (ESD) has been widely used in gastric tumor as a minimally invasive treatment. The efficacy and safety of ESD is still unclear in the elderly who have high frequency comorbidities. The aim of this study is to evaluate the efficacy and safety of ESD for gastric epithelial neoplasia in patients aged 80 years and older. METHODS: Between March 2013 and July 2017, a total of 438 gastric epithelial neoplasia patients treated with ESD were analyzed. Clinical outcomes including en bloc and complete resection rates, adverse events (AE) related procedure and sedation were compared between the elderly group and the non-elderly group. RESULTS: Sex, Body Mass Index, medication history and American Society of Anesthesiologists physical status did not differ between the two groups. Tumor characteristics except size of resected specimen (elderly vs. non-elderly; 36.5 ± 10.5 vs. 32.3 ± 8.7 mm, p < 0.011) did not differ. There were no significant differences in AE-related sedation. En bloc resection (elderly vs. non-elderly; 100% vs. 98.3%, p = 0.454), and complete resection rate (elderly vs. non-elderly; 93.8% vs. 96.3%, p = 0.471) did not differ significantly between the two groups. Procedure time, hospital stay, AE-related procedure and delayed bleeding were also similar between the two groups. However, procedure time of preventive hemostasis (elderly vs. non-elderly; 10.4 ± 7.7 vs. 7.4 ± 5.2 min, p = 0.040) was significantly higher in the elderly group. CONCLUSIONS: ESD for gastric epithelial neoplasia is effective and safe in elderly patients ≥ 80 years as in non-elderly patients.