Lewy body (LB) disorders, such as dementia with LBs and Parkinson’s disease with dementia (PDD) are characterized by their pathological hallmark, i.e., the gradual appearance of LBs. Therefore, a deep understanding of the molecular and cellular mechanism, favoring the formation of LBs is required.These proteinaceous inclusions are found in different cell types of the nervous system. The key constituent protein-component of such inclusions is the protein alpha-synuclein (α-Syn). α-Syn along with other bio-active material gets secreted as content of extracellular vesicles (EVs). These vesicles not only excrete bioactive material but are also able to transport it to distant sites, and therefore have implications for disease pathology seeding and spreading.Using patch-clamp electrophysiology techniques along with simultaneous real-time molecular imaging, we determined the kinetics of EV-release in hippocampal neurons. We designed a pH-sensitive molecular tag, targeted specifically to membranes of EVs and applied this method to study stimulus-responsive MVB-PM fusion, in order to determine the kinetics, specific stimuli and regulators of such events. In addition, we analyzed the EV-content by performing mass spectrometry (MS) analysis and identifyed LBP-related molecular interactions.
Contents
1. Introduction
1.1. Lewy bodies and Parkinson’s disease
1.2. Lewy bodies
1.3. Lewy body pathology and hippocampus
1.4. Lewy body pathology propagation and extra cellular vesicles
1.5. Extracellular vesicle
1.6. Neurotrophic factors and Lewy body pathology
1.7. Functional periphery of basic fibroblast growth factor
1.8. Aim and scope of the thesis
2. Methodology, methods, and materials
2.1. Experimental design
2.1.1. Patch clamp electrophysiology
2.1.2. Cell cultures
2.1.3. Exosome labelling
2.1.4. Working model of pHluorin tag
2.1.5. A suitable bioelectric stimulus
2.1.5.1.100 Hz
2.1.6. Nano particle tracking analysis
2.1.7. Weighted protein co-expression network analysis
2.2. Material and methods
2.2.1. Chemicals used for electrophysiology
2.2.2. Miscellaneous
2.2.3. LUHMES cell culture
2.2.4. Calcium imaging fura-2 loading experiments
2.2.5. Immunocytochemistry for imaging
2.2.6. Other materials and resources
3. Results
3.1. Abundant α-Syn expression and exosomal release
3.1.1. α-Syn overexpression results an elongated morphology in LUHMES cells
3.1.2. α-Syn overexpression resulted in enhanced secretion of extracellular vesicles in LUHMES cells
3.1.3. Exosome release in LUHMES cells is calcium dependent
3.1.4. α-Syn overexpression enhances extracellular vesicles in primary hippocampal neurons
3.2. Calcium level and exosomal release
3.2.1. A relative slower rate of MVB-PM fusion
3.2.2. HFS enhanced MVB-PM fusion events with subsided success rate
3.3. Growth factors and exosomal release
3.3.1. The effects of growth factors are restricted largely to intraneuronal physiological environment
3.3.2. bFGF regulates exosomal release
3.3.3. SNARE proteins mediate bFGF regulated exosomal release
3.3.4. VAMP3 regulates bFGF enhanced EV release
3.3.5. A spectrum of EV biomarkers is affected by the bFGF treatment
3.4. Growth factors and Lewy body pathophysiology
3.4.1. bFGF induces changes to cell lysate and extracellular vesicle proteome
3.4.2. Development of protein-protein interaction networks among co-expressing proteins and pruning their LBP associations
3.4.3. High bFGF level triggers linearized up/down-regulation of protein expression
3.4.4. High bFGF levels can manipulate the neurophysiological milieu in LBP
3.4.5. bFGF induced LB pathology associated molecular interactions and possible offshoot in central nervous system
3.4.6. bFGF affected LBP-proteins/interactions are predominantly associated to the neuronal protein metabolism
4. Discussion
4.1. Complex calcium-homeostatic mechanisms govern the slower exosome release
4.2. Neurotrophic factors are the boosters of MVB-PM fusion
4.3. bFGF mediated enhanced EV-release is depends on tyrosine kinase activity and is calcium dependent
4.4. “e-SNAREsâ€
4.5. bFGF has a broad effect on protein expression and their linear exodus as EV-content
4.6. The expression levels of many Lewy body pathology associated proteins are changed by bFGF treatment
4.7. Stimulus-responsive enhanced EV release has system level implications
5. Concluding remarks and future recommendations
Research Objectives and Themes
This thesis aims to elucidate the mechanisms underlying the stimulus-responsive release of extracellular vesicles (EVs), specifically exosomes, in neurons and their role in the propagation of Lewy body pathology. By integrating electrophysiological techniques with real-time molecular imaging and proteomic network analysis, the research seeks to resolve the kinetic and regulatory factors of exosome secretion and assess the modifying influence of neurotrophic factors on these processes.
- Temporal kinetics of MVB-PM fusion events in single neurons.
- Role of bioelectric and neurotrophic stimuli (specifically bFGF) in regulating EV release.
- Identification of molecular machinery (e.g., SNARE proteins) controlling exosomal secretion.
- Impact of enhanced EV secretion on the propagation of α-Synuclein-related pathology.
- System-level proteomic changes and molecular interactions associated with bFGF-induced LBP.
Excerpt from the Book
3.1. Abundant α-Syn expression and exosomal release
A shift in somatic volume is observed for α-Syn over-expressing LUHMES cells (Figure 16A and B) as compared to WT LUHMES cells (Figure 16A). α-Syn over-expression resulted in a significant increase in the surface area (≈ mean area = 2.8 x 10^-8 a.u.: n=15) as compared to WT cells (≈ mean area = 1.2 x 10^-8 a.u.: n=15) (Figure 16C). The elongated morphological appearance of α-Syn-overexpressing cells is also evident from decreased somatic circularity (≈ to 1/2) as compared to the WT cells (Figure 16D). No change was found in the somatic solidity between WT and SNCA overexpressing LUHMES cells, thus suggesting that the overall quality of plasma membrane was uncompromised since the solidity is a ratiomeric measure between surface area and convex hull of the surface (data not included).
Using patch-clamp electrophysiology, it has been long established that the plasma membrane (PM) of a cell behaves closely like a parallel plate capacitor and increases in the surface area of plates corresponds to an increase in membrane capacitance (Cm) and vice versa [130]. Therefore, a higher Cm would correspond to a fusion of vesicular bodies.
Summary of Chapters
1. Introduction: Provides a comprehensive background on Lewy body pathology, the role of α-Synuclein, and the current understanding of extracellular vesicles in disease propagation.
2. Methodology, methods, and materials: Details the experimental setup, including patch-clamp electrophysiology, cell culture models, imaging techniques, and proteomic network analysis methods.
3. Results: Presents the primary findings on how α-Synuclein overexpression influences exosome release and the mechanisms through which bFGF and SNARE proteins regulate this process.
4. Discussion: Interconnects the experimental results to propose a model of EV-mediated pathology propagation and emphasizes the role of bFGF in modulating these events.
5. Concluding remarks and future recommendations: Summarizes key findings and suggests future directions for leveraging EV-based insights for therapeutic or diagnostic strategies.
Keywords
Parkinson's disease, Lewy bodies, α-Synuclein, Extracellular vesicles, Exosomes, MVB-PM fusion, Patch-clamp, bFGF, Neurotrophic factors, Proteomics, WPCNA, SNARE proteins, VAMP3, Neurodegeneration, LBP.
Frequently Asked Questions
What is the core focus of this doctoral thesis?
The research focuses on understanding the molecular mechanisms and the temporal dynamics of exosome release in neurons, specifically investigating how bioelectric and neurotrophic stimuli affect this process in the context of Lewy body pathology.
What are the central themes discussed in the work?
The central themes include the kinetics of multivesicular body fusion to the plasma membrane (MVB-PM fusion), the influence of α-Synuclein aggregation, the regulatory role of neurotrophic factors like bFGF, and the protein-level interaction networks that drive disease propagation.
What is the primary goal of this research?
The primary goal is to resolve the physiological factors and stimuli that modulate the release of extracellular vesicles and to identify if these pathways contribute significantly to the seeding and spreading of pathological protein aggregates in neurodegenerative diseases.
Which scientific methods are employed throughout the study?
The study utilizes a multi-disciplinary approach, combining patch-clamp electrophysiology for real-time membrane capacitance measurements, live-cell molecular imaging, nanoparticle tracking analysis (NTA), and weighted protein co-expression network analysis (WPCNA) of mass spectrometry-derived proteomics data.
What is addressed in the results section?
The results demonstrate that α-Synuclein overexpression enhances EV release, that these fusion events are calcium-dependent, and that specific growth factors like bFGF modulate this pathway through tyrosine kinase activity and the enrichment of v-SNARE proteins like VAMP3.
Which keywords best describe this research?
Key terms include Parkinson's disease, α-Synuclein, extracellular vesicles, bFGF, MVB-PM fusion, and proteomic network analysis.
How does bFGF manipulate the neurophysiological milieu of LBP?
The study suggests that high bFGF levels influence protein metabolism and interact with specific α-Synuclein binding proteins (LRBPs and receptors), potentially creating molecular conditions that favor the sequestration of pathological aggregates into exosomes.
What is the clinical implication of the "slow-release" kinetics observed?
The slow kinetics of exosome release, compared to fast synaptic neurotransmission, suggest that exosomes facilitate a distinct mode of intercellular communication that is well-suited for the slow, chronic progression of disease spreading over several years.
- Arbeit zitieren
- Rohit Kumar (Autor:in), 2021, How Stimulus-responsive Extracellular Vesicle Release is Regulated and Associated to Lewy body disease, München, GRIN Verlag, https://www.grin.com/document/1351407
