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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.

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.

Progressive supranuclear palsy (PSP) is a neurodegenerative disease characterized by abnormal accumulation of the protein tau in the brain, leading to motor and cognitive symptoms that vary between individuals. The reasons for this clinical heterogeneity are unknown. Here we show that distinct molecular forms of tau, particularly high molecular weight (HMW) assemblies, differ in abundance and biological activity across PSP brains. By combining biochemical examination, seed amplification assays, proteomic profiling, and spatial transcriptomics, we identify that HMW tau species drive the strongest aggregation activity in the primary motor cortex. Cases with high tau seeding activity display molecular signatures of altered immune and metabolic pathways. These findings reveal that tau seeding activity reflects underlying molecular heterogeneity in PSP and suggest that measuring 4R-tau seeding capacity could help stratify patients and guide the development of targeted therapeutic approaches. This study shows that high-molecular weight tau has enhanced seeding capacity, and seeding differences can define molecular subtypes of progressive supranuclear palsy, underpinning the disease’s heterogeneity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

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.

Neurodegenerative diseases are progressive and debilitating conditions affecting millions of people worldwide. Since misfolded proteins are associated with neuronal degeneration and clinical symptoms, neurodegenerative diseases are also called proteinopathies. Synucleinopathies belong to the group of neurodegenerative diseases that are characterized by the loss of neurons and deposition of misfolded α-synuclein (αSyn) protein in various cell types of the nervous system. Synucleinopathies comprise Lewy Body Disorders (LBD) and multiple system atrophy (MSA). Based on the theory that misfolded αSyn spreads cell-to-cell, the seeding potential, which is the ability of misfolded proteins to act as a template to convert normal proteins into the pathological form, can be evaluated using seed amplification assay (SAA). This is a biochemical amplification technique developed to detect minute amounts of pathological protein in real-time. Recent evidence shows that in addition to morphological differences under the microscope, seeding behavior differs between Synucleinopathies, individuals, and even brain regions within the same individual. We hypothesized that the cell-type specific accumulation of αSyn might be an important aspect contributing to the different seeding behaviors. Therefore, our aim was to: i) establish a protein extraction method using formalin-fixed paraffin-embedded (FFPE) brain tissue to confirm disease-specific αSyn seeding as observed in frozen tissue; ii) link various cytopathology (i.e., cell-type specific accumulation of αSyn) in LBD and MSA with αSyn seeding using the established FFPE-SAA; and iii) evaluate whether Synucleinopathies can be further subclassified by morphological features using artificial intelligence (AI)-based machine learning algorithms.Upon confirming the preservation of disease-specific αSyn seeding in FFPE brain tissue, small regions with mostly neuronal or glial αSyn inclusions were identified on FFPE sections under the microscope. Proteins from these precise areas were extracted using our established method for SAA-mediated evaluation of αSyn seeding. AI-based analysis provided insight into the different seeding behaviors of cell-specific pathological αSyn. Moreover, further stratification and aging-related difference within MSA were found by AI-based measurement of morphological features. Our results will open new perspectives for understanding whether cellular pathological differences seen in histological evaluations contribute to seeding molecular behavior observed through SAA. Moreover, novel sub-classification of Synucleinopathies will provide additional insight into the biological relevance - potential difference in response to therapeutics between patients with different subtypes of the disease.

Synucleinopathies are a group of neurodegenerative diseases characterized by the deposition of misfolded α‐synuclein (αSyn), predominantly in oligodendrocytes in multiple system atrophy (MSA) and in neurons in Lewy body diseases (LBD). The contribution of αSyn cytopathologies to the pathogenesis of these diseases is underappreciated. Seed amplification assays of MSA and LBD brains have revealed striking differences in αSyn seeding between regions and cases. Therefore, our aim was to evaluate whether different brain regions containing distinct αSyn cytopathologies contribute to different seeding characteristics. We collected 2‐mm micro‐punches of regions in MSA (n = 10) and LBD (n = 15) cases from formalin‐fixed paraffin‐embedded tissues. We performed double immuno‐labeling for disease‐associated αSyn and cellular markers on tissue microarrays, evaluated co‐deposition of other neurodegenerative disease‐related proteins and, from the same micro‐punched samples, we analyzed αSyn seeding. Based on these variables, machine learning algorithms were used to reduce dimensionality of the dataset and cluster the regions in MSA and LBD cases, revealing that different compositions of αSyn cytopathologies influence αSyn seeding patterns. Our results support the notion of different cellular processing of αSyn and its contribution to the variability in seeding. This has implications for understanding disease progression, interpretation of seed amplification assays, and opens avenues for the development of cell type‐specific antibodies against αSyn. This study investigated whether brain regions with distinct predominance of α‐synuclein (αSyn) cytopathologies show different αSyn seeding patterns in multiple system atrophy (MSA) and Lewy body disease (LBD), using an interdisciplinary approach. High seeding activity is observed in regions with oligodendrocytic‐predominant αSyn pathology in MSA. In regions with combined neuronal cytoplasmic‐ and astrocytic‐αSyn pathology, the increased involvement of astrocytes shows higher seeding activity in LBD. Lower seeding activity is observed in regions with predominant neuronal cytoplasmic‐αSyn in LBD.

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.

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 3T 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.