Neurodegenerative diseases are a heterogeneous group of maladies, characterized by progressive loss of neurons. These diseases involve an intricate pattern of cross-talk between different types of cells to maintain specific signaling pathways. A component of such intercellular cross-talk is the exchange of various types of extracellular vesicles (EVs). Exosomes are a subset of EVs, which are increasingly being known for the role they play in the pathogenesis and progression of neurodegenerative diseases, e.g., synucleinopathies and tauopathies. The ability of the central nervous system exosomes to cross the blood–brain barrier into blood has generated enthusiasm in their study as potential biomarkers. However, the lack of standardized, efficient, and ultra-sensitive methods for the isolation and detection of brain-derived exosomes has hampered the development of effective biomarkers. Exosomes mirror heterogeneous biological changes that occur during the progression of these incurable illnesses, potentially offering a more comprehensive outlook of neurodegenerative disease diagnosis, progression and treatment. In this review, we aim to discuss the challenges and opportunities of peripheral biofluid-based brain-exosomes in the diagnosis and biomarker discovery of Alzheimer’s and Parkinson’s diseases. In the later part, we discuss the traditional and emerging methods used for the isolation of exosomes and compare their advantages and disadvantages in clinical settings.

Tau (Tubulin associated unit) protein is a major hallmark of Alzheimer’s disease (AD) and tauopathies. Tau is predominantly an axonal protein with a crucial role in the stabilization and dynamics of the microtubules. Since the discovery of Tau protein in 1975, research efforts were concentrated on the pathophysiological role of Tau protein in the context of the microtubules. Although, for more than three decades, different localizations of Tau protein have been discovered e.g., in the nuclear compartments. Discovery of the role of Tau protein in various cellular compartments especially in the nucleus opens up a new fold of complexity in tauopathies. Data from cellular models, animal models, and the human brain indicate that nuclear Tau is crucial for genome stability and to cope with cellular distress. Moreover, it’s nature of nuclear translocation, its interactions with the nuclear DNA/RNA and proteins suggest it could play multiple roles in the nucleus. To comprehend Tau pathophysiology and efficient Tau-based therapies, there is an urgent need to understand whole repertoire of Tau species (nuclear and cytoplasmic) and their functional relevance. To complete the map of Tau repertoire, understanding of various species of Tau in the nucleus and cytoplasm, identification if specific transcripts of Tau, isoforms and post-translational modifications could foretell Tau’s localizations and functions, and how they are modified in neurodegenerative diseases like AD, is urgently required. In this review, we explore the nuclear face of Tau protein, its nuclear localizations and functions and its linkage with Alzheimer’s disease.

Very solid evidence suggests that the core of full length PrP^Sc is a 4-rung β-solenoid, and that individual PrP^Sc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the β-strands and connecting loops that make up the PrP^Sc solenoid. Using high resolution SDS-PAGE followed by epitope analysis, and mass spectrometry, we identified positions ~116/118, 133–134, 141, 152–153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrP^Sc. Such sites likely define loops and/or borders of β-strands, helping us to predict the threading of the β-solenoid. We have now extended this approach to recombinant PrP^Sc (recPrP^Sc). The term recPrP^Sc refers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrP^Sc species yielded N-terminally truncated PK-resistant fragments similar to those seen in brain-derived PrP^Sc, albeit with varying relative yields. Along with these fragments, doubly N- and C-terminally truncated fragments, in particular ~89/97-152, were detected in some recPrP^Sc preparations; similar fragments are characteristic of atypical strains of brain-derived PrP^Sc. Our results suggest a shared architecture of recPrP^Sc and brain PrP^Sc prions. The observed differences, in particular the distinct yields of specific PK-resistant fragments, are likely due to differences in threading which result in the specific biochemical characteristics of recPrP^Sc. Furthermore, recombinant PrP^Sc offers exciting opportunities for structural studies unachievable with brain-derived PrP^Sc. PrP^Sc Prions propagate by inducing the refolding of the natively folded normal cellular prion protein (PrP^C) into the prion conformation in brain and other mammalian tissues (wild-type). Understanding the structure of PrP^Sc is essential to understanding how PrP^Sc prions propagate. The secondary structure of PrP^C is composed of four α-helical regions, two very small β-sheets and random coil, while PrP^Sc is composed entirely of β-sheets and random coil. The β-sheets of PrP^Sc wind four times to form a spring-like 4-rung β-solenoid. The rungs are connected by stretches of random coil. We have used proteinase K (PK), to cleave these random coil connecters, allowing us to identify the location of the more PK-resistant β-strand stretches within wild-type PrP^Sc. In this work, we use recombinant PrP^Sc, (in vitro generated infectious prions) to show that patterns of PK cleavage for recombinant and wild-type PrP^Sc are very similar, indicating that they share a common architecture. This also means that recombinant PrP^Sc is a true surrogate for wild-type PrP^Sc. Since recombinant PrP^Sc is derived from recombinant PrP, future structural studies employing specific amino acid or stable-isotope labeled amino acid substitutions are easily achievable. Such substitutions are essential for NMR studies.

Aims: Tau is a key player in Alzheimer's disease (AD) and other Tauopathies. Tau pathology in the brain directly correlates with neurodegeneration in AD. The recent identification of a rapid variant of AD demands an urgent need to uncover underlying mechanisms leading to differential progression in AD. Accordingly, we aimed to dissect the underlying differential mechanisms of toxicity associated with the Tau protein in AD subtypes and to find out subtype‐dependent biomarkers and therapeutic targets.Methods: To identify and characterise subtype‐specific Tau‐associated mechanisms of pathology, we performed comparative interactome mapping of Tau protein in classical AD (cAD) and rapidly progressive AD (rpAD) cases using co‐immunoprecipitation coupled with quantitative mass spectrometry. The mass spectrometry data were extensively analysed using several bioinformatics approaches.Results: The comparative interactome mapping of Tau protein revealed distinct and unique interactors (DPYSL4, ARHGEF2, TUBA4A and UQCRC2) in subtypes of AD. Interestingly, an analysis of the Tau‐interacting proteins indicated enrichment of mitochondrial organisation processes, including negative regulation of mitochondrion organisation, mitochondrial outer membrane permeabilisation involved in programmed cell death, regulation of autophagy of mitochondrion and necroptotic processes, specifically in the rpAD interactome. While, in cAD, the top enriched processes were related to oxidation–reduction process, transport and monocarboxylic acid metabolism.Conclusions: Overall, our results provide a comprehensive map of Tau‐interacting protein networks in a subtype‐dependent manner and shed light on differential functions/pathways in AD subtypes. This comprehensive map of the Tau‐interactome has provided subsets of disease‐related proteins that can serve as novel biomarkers/biomarker panels and new drug targets.

Background: Aberrant stress granules (SGs) are emerging as prime suspects in the nucleation of toxic protein aggregates. Understanding the molecular networks linked with aggregation-prone proteins (prion protein, synuclein, and tau) under stressful environments is crucial to understand pathophysiological cascades associated with these proteins. Methods: We characterized and validated oxidative stress-induced molecular network changes of endogenous aggregation-prone proteins (prion protein, synuclein, and tau) by employing immunoprecipitation coupled with mass spectrometry analysis under basal and oxidative stress conditions. We used two different cell models (SH-SY5Y: human neuroblastoma and HeLa cell line) to induce oxidative stress using a well-known inducer (sodium arsenite) of oxidative stress. Results: Overall, we identified 597 proteins as potential interaction partners. Our comparative interactome mapping provides comprehensive network reorganizations of three aggregation-prone hallmark proteins, establish novel interacting partners and their dysregulation, and validates that prion protein and synuclein localize in cytoplasmic SGs. Localization of prion protein and synuclein in TIA1-positive SGs provides an important link between SG pathobiology and aggregation-prone proteins. In addition, dysregulation (downregulation) of prion protein and exportin-5 protein, and translocation of exportin-5 into the nucleus under oxidative stress shed light on nucleocytoplasmic transport defects during the stress response. Conclusions: The current study contributes to our understanding of stress-mediated network rearrangements and posttranslational modifications of prion/prion-like proteins. Localization of prion protein and synuclein in the cytoplasmic SGs provides an important link between stress granule pathobiology and aggregation-prone proteins. In addition, our findings demonstrate nucleocytoplasmic transport defects after oxidative stress via dysregulation and nuclear accumulation of exportin-5.

Intracellular aggregation of proteins such as Tau, TDP43, FUS, prion protein, and α-synuclein is a major hallmark of many major neurodegenerative diseases. Aberrant stress granules (SGs) are emerging as key contributors to the nucleation of toxic protein aggregates in these disorders. SGs are dynamic, membrane less cytoplasmic assemblies that form transiently through liquid–liquid phase separation (LLPS) of RNA binding proteins (RBPs) containing low complexity domains, together with stalled mRNAs, to help cells cope with stress. While physiological SGs facilitate cellular resilience to acute stress and undergo rapid disassembly, chronic or excessive stress leads to persistent SGs, driving pathological protein aggregation characteristic of age related neurodegeneration. The inherent reversible aggregation of RBPs crucial for cellular function paradoxically exposes them to misfolding disorders. Notably, recent findings expand this paradigm by demonstrating that Tau itself participates in SG formation, with Tau–SG interactions potentiating Tau aggregation and disease progression in tauopathies. Despite these insights, the precise cellular stressors and posttranslational modifications (PTMs) governing the shift from physiological granules to pathological aggregates remain poorly defined. Emerging evidence highlights oxidative stress as a central upstream mediator of this transition. In this perspective, we synthesize current understanding of how SG dynamics intersect with oxidative stress to potentiate protein aggregation, proposing molecular mechanisms that bridge SG biology and neurodegenerative disease. Elucidating these pathways is essential for the development of targeted therapeutic interventions for disorders such as Alzheimer’s disease and related tauopathies.