Assessment of technical and clinical utility of a bead-based flow cytometry platform for multiparametric phenotyping of CNS-derived extracellular vesicles.

BACKGROUND: Extracellular vesicles (EVs) originating from the central nervous system (CNS) can enter the blood stream and carry molecules characteristic of disease states. Therefore, circulating CNS-derived EVs have the potential to serve as liquid-biopsy markers for early diagnosis and follow-up of neurodegenerative diseases and brain tumors. Monitoring and profiling of CNS-derived EVs using multiparametric analysis would be a major advance for biomarker as well as basic research. Here, we explored the performance of a multiplex bead-based flow-cytometry assay (EV Neuro) for semi-quantitative detection of CNS-derived EVs in body fluids. METHODS: EVs were separated from culture of glioblastoma cell lines (LN18, LN229, NCH82) and primary human astrocytes and measured at different input amounts in the MACSPlex EV Kit Neuro, human. In addition, EVs were separated from blood samples of small cohorts of glioblastoma (GB), multiple sclerosis (MS) and Alzheimer's disease patients as well as healthy controls (HC) and subjected to the EV Neuro assay. To determine statistically significant differences between relative marker signal intensities, an unpaired samples t-test or Wilcoxon rank sum test were computed. Data were subjected to tSNE, heatmap clustering, and correlation analysis to further explore the relationships between disease state and EV Neuro data. RESULTS: Glioblastoma cell lines and primary human astrocytes showed distinct EV profiles. Signal intensities were increasing with higher EV input. Data normalization improved identification of markers that deviate from a common profile. Overall, patient blood-derived EV marker profiles were constant, but individual EV populations were significantly increased in disease compared to healthy controls, e.g. CD36+EVs in glioblastoma and GALC+EVs in multiple sclerosis. tSNE and heatmap clustering analysis separated GB patients from HC, but not MS patients from HC. Correlation analysis revealed a potential association of CD107a+EVs with neurofilament levels in blood of MS patients and HC. CONCLUSIONS: The semi-quantitative EV Neuro assay demonstrated its utility for EV profiling in complex samples. However, reliable statistical results in biomarker studies require large sample cohorts and high effect sizes. Nonetheless, this exploratory trial confirmed the feasibility of discovering EV-associated biomarkers and monitoring circulating EV profiles in CNS diseases using the EV Neuro assay. Video Abstract.

Extracellular vesicles (EVs) are tiny particles released by cells, carrying unique biomolecules specific to their cell of origin. EVs from the central nervous system (CNS) can reach the blood, where they could serve as liquid-biopsy markers for diagnosing brain diseases like neurodegenerative disorders and tumors. This study evaluated a flow cytometry platform (here termed EV Neuro assay), which can detect multiple EV-associated markers simultaneously, to assess its potential for identifying CNS-derived EVs and disease-specific markers in complex samples including the blood. The study compared different sample materials and methods for isolating EVs. We found distinct EV profiles in EVs derived from glioblastoma and human astrocytes, with signal intensities increasing as more EVs were present. Analyzing serum or plasma from patients with brain diseases and healthy individuals, we observed that EV marker intensities were varying between individuals. Importantly, data normalization improved the identification of disease-specific markers, such as CD36⁺EVs in glioblastoma and GALC⁺EVs in multiple sclerosis, which were significantly higher in disease compared to healthy controls. Advanced clustering analysis techniques effectively distinguished glioblastoma patients from controls. Furthermore, a potential correlation between CD107a⁺EVs and neurofilament levels in multiple sclerosis patients was discovered. Overall, the semi-quantitative EV Neuro assay proved useful for profiling EVs in complex samples. However, for more reliable results in biomarker studies, larger sample cohorts and higher effect sizes are necessary. Nonetheless, this initial trial confirmed the potential of the EV Neuro assay for discovering disease-associated EV markers and monitoring circulating EV profiles in CNS diseases.

The Aging Enteric Nervous System.

The gut and the brain communicate via the nervous system, hormones, microbiota-mediated substances, and the immune system. These intricate interactions have led to the term "gut-brain axis". Unlike the brain-which is somewhat protected-the gut is exposed to a variety of factors throughout life and, consequently, might be either more vulnerable or better adapted to respond to these challenges. Alterations in gut function are common in the elder population and associated with many human pathologies, including neurodegenerative diseases. Different studies suggest that changes in the nervous system of the gut, the enteric nervous system (ENS), during aging may result in gastrointestinal dysfunction and initiate human pathologies of the brain via its interconnection with the gut. This review aims at summarizing the contribution of normal cellular aging to the age-associated physiological changes of the ENS. Morphological alterations and degeneration of the aging ENS are observed in different animal models and humans, albeit with considerable variability. The aging phenotypes and pathophysiological mechanisms of the aging ENS have highlighted the involvement of enteric neurons in age-related diseases of the central nervous system such as Alzheimer's or Parkinson's disease. To further elucidate such mechanisms, the ENS constitutes a promising source of material for diagnosis and therapeutic predictions, as it is more accessible than the brain.

Role of NLRP3 Inflammasome in Parkinson's Disease and Therapeutic Considerations.

Parkinson's disease (PD) is the second most common neurodegenerative disease, with two main pathological features: misfolded α-synuclein protein accumulation and neurodegeneration. Inflammation has recently been identified as a contributor to a cascade of events that may aggravate PD pathology. Inflammasomes, a group of intracellular protein complexes, play an important role in innate immune responses to various diseases, including infection. In PD research, accumulating evidence suggests that α-synuclein aggregations may activate inflammasomes, particularly the nucleotide-binding oligomerization domain-leucine-rich repeat-pyrin domain-containing 3 (NLRP3) type, which exacerbates inflammation in the central nervous system by secreting proinflammatory cytokines like interleukin (IL)-18 and IL-1ß. Afterward, activated NLRP3 triggers local microglia and astrocytes to release additional IL-1ß. In turn, the activated inflammatory process may contribute to additional α-synuclein aggregation and cell loss. This review summarizes current research evidence on how the NLRP3 inflammasome contributes to PD pathogenesis, as well as potential therapeutic strategies targeting the NLRP3 inflammasome in PD.

PTEN-Induced Putative Kinase 1 Dysfunction Accelerates Synucleinopathy.

BACKGROUND: Mutations in PTEN-induced putative kinase 1 (PINK1) cause autosomal recessive Parkinson's disease (PD) and contribute to the risk of sporadic PD. However, the relationship between PD-related PINK1 mutations and alpha-synuclein (α-syn) aggregation-a main pathological component of PD-remains unexplored. OBJECTIVE: To investigate whether α-syn pathology is exacerbated in the absence of PINK1 after α-syn preformed fibril (PFF) injection in a PD mouse model and its effects on neurodegeneration. METHODS: In this study, 10-week-old Pink1 knockout (KO) and wildtype (WT) mice received stereotaxic unilateral striatal injection of recombinant mouse α-syn PFF. Then, α-syn pathology progression, inflammatory responses, and neurodegeneration were analyzed via immunohistochemistry, western blot analysis, and behavioral testing. RESULTS: After PFF injection, the total α-syn levels significantly increased, and pathological α-syn was markedly aggregated in Pink1 KO mice compared with Pink1 WT mice. Then, earlier and more severe neuronal loss and motor deficits occurred. Moreover, compared with WT mice, Pink1 KO mice had evident microglial/astrocytic immunoreactivity and prolonged astrocytic activation, and a higher rate of protein phosphatase 2A phosphorylation, which might explain the greater α-syn aggravation and neuronal death. CONCLUSION: The loss of Pink1 function accelerated α-syn aggregation, accumulation and glial activation, thereby leading to early and significant neurodegeneration and behavioral impairment in the PD mouse model. Therefore, our findings support the notion that PINK1 dysfunction increases the risk of synucleinopathy.

Temporal Evolution of Inflammation and Neurodegeneration With Alpha-Synuclein Propagation in Parkinson's Disease Mouse Model.

According to a few studies, α-synuclein (αSyn) propagation has been suggested to play a key role in the pathomechanism of Parkinson's disease (PD), but neurodegeneration and the involvement of inflammation in its pathologic progression are not well understood with regard to temporal relationship. In this study, with the help of the PD mouse model injected with intrastriatal αSyn preformed fibril (PFF), the temporal evolution of αSyn propagation, inflammation, and neurodegeneration was explored in the perspective of the striatum and the whole brain. In the PFF-injected striatum, inflammatory response cells, including microglia and astrocytes, were activated at the earliest stage and reduced with time, and the phosphorylated form of αSyn accumulation increased behind it. Afterward, the degeneration of striatal dopaminergic neurons became significant with the conspicuity of behavioral phenotype. Similar patterns of forefront eruption of inflammation and then followed by αSyn propagation were noted in the opposite striatum, which were not injured by PFF injection. In analyzing the whole brain, inflammatory responses were activated at the earliest stage, and the soluble αSyn expression increased concurrently. The inflammatory response decreased afterward, and the accumulation of the insoluble form of αSyn increased behind it. Our results suggested that the inflammatory response may precede the accumulation of the pathologic form of αSyn; thereafter, the neurodegeneration and motor dysfunction followed αSyn proliferation in the PD mouse model. From this model, recognizing the temporal relationship between inflammation, αSyn propagation, and neurodegeneration may be helpful in establishing the PD animal model and monitoring the effect of interventional therapy.