Dysfunctional RNA-binding proteins (RBPs) have been implicated in several neurodegenerative disorders. Recently, this paradigm of RBPs has been extended to pathophysiology of Alzheimer's disease (AD). Here, we identified disease subtype specific variations in the RNA-binding proteome (RBPome) of sporadic AD (spAD), rapidly progressive AD (rpAD), and sporadic Creutzfeldt Jakob disease (sCJD), as well as control cases using RNA pull-down assay in combination with proteomics. We show that one of these identified proteins, splicing factor proline and glutamine rich (SFPQ), is downregulated in the post-mortem brains of rapidly progressive AD patients, sCJD patients and 3xTg mice brain at terminal stage of the disease. In contrast, the expression of SFPQ was elevated at early stage of the disease in the 3xTg mice, and in vitro after oxidative stress stimuli. Strikingly, in rpAD patients' brains SFPQ showed a significant dislocation from the nucleus and cytoplasmic colocalization with TIA-1. Furthermore, in rpAD brain lesions, SFPQ and p-tau showed extranuclear colocalization. Of note, association between SFPQ and tau-oligomers in rpAD brains suggests a possible role of SFPQ in oligomerization and subsequent misfolding of tau protein. In line with the findings from the human brain, our in vitro study showed that SFPQ is recruited into TIA-1-positive stress granules (SGs) after oxidative stress induction, and colocalizes with tau/p-tau in these granules, providing a possible mechanism of SFPQ dislocation through pathological SGs. Furthermore, the expression of human tau in vitro induced significant downregulation of SFPQ, suggesting a causal role of tau in the downregulation of SFPQ. The findings from the current study indicate that the dysregulation and dislocation of SFPQ, the subsequent DNArelated anomalies and aberrant dynamics of SGs in association with pathological tau represents a critical pathway which contributes to rapid progression of AD.

There is an increasing demand for the understanding of pathophysiology on neurodegeneration diseases at early stages. Changes in endocytic machinery and the cytoskeleton-associated response are the first alterations observed in Creutzfeldt-Jakob disease (CJD) and Alzheimer's disease AD brain. In this study, we performed a targeted search for endocytic pathway proteins in the different regions of the brain. We found late endosome marker Rab7a which was significantly upregulated in the frontal cortex region in the rapid progressive CJD form (MM1) and rapid progressive AD (rpAD) forms. However, Rab9 expression was significantly downregulated only in CJD-MM1 brain frontal cortex region. In the cerebellum, Rab7a expression showed significant upregulation in both subtype MM1 and VV2 CJD forms, in contrast to Rab9 which showed significant downregulation in both subtype MM1 and VV2 CJD forms at terminal stage of the disease. To check regulatory response at pre-symptomatic stage of the disease, we checked the regulatory interactive response of Rab7a, Rab9, and known biomarkers PrP and tau forms in frontal cortex at pre-symptomatic stage of the disease in tg340 mice expressing about fourfold of human PrP-M129 with PrP-null background that had been inoculated with human sCJD MM1 brain tissue homogenates (sCJD MM1 mice). In addition, we analyzed 5XFAD mice, exhibiting five mutations in the APP and presenilin genes related to familial Alzheimer's disease (FAD), to validate specific regulatory response of Rab7a, Rab9, tau, and phosphorylated form of tau by immunostaining 5XFAD mice in comparison with the wild-type age-matched mice brain. The cortical region of 5XFAD mice brain showed accumulated form of Rab7a in puncta that co-label for p-Tau, indicating colocalization by using confocal laser-scanning microscopy and was confirmed by using reverse co-immunoprecipitation. Furthermore, synthetic RNA (siRNA) against the Rab7a gene decreased expression of Rab7a protein, in cortical primary neuronal cultures of PrP wild type. This depleted expression of Rab7a led to the increased accumulation of PrP in Rab9-positive endosomal compartments and consequently an increased co-localization between PrP/Rab9; however, total tau level decreased. Interestingly, siRNA against tau gene in cortical primary neuronal cultures of PrP wild-type mice showed enhanced Rab7a and Rab9 expression and increase formation of dendritic spines. The work described highlighted the selective involvement of late endosomal compartment marker Rab7a in CJD, slow and rapid progressive forms of AD pathogenesis.

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.

Very solid evidence suggests that the core of full length PrPSc is a 4-rung β-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the β-strands and connecting loops that make up the PrPSc 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 PrPSc. 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 PrPSc (recPrPSc). The term recPrPSc refers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrPSc species yielded N-terminally truncated PK-resistant fragments similar to those seen in brain-derived PrPSc, 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 recPrPSc preparations; similar fragments are characteristic of atypical strains of brain-derived PrPSc. Our results suggest a shared architecture of recPrPSc and brain PrPSc 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 recPrPSc. Furthermore, recombinant PrPSc offers exciting opportunities for structural studies unachievable with brain-derived PrPSc.

Rapidly progressive Alzheimer's disease (rpAD) is a variant of AD distinguished by a rapid decline in cognition and short disease duration from onset to death. While attempts to identify rpAD based on biomarker profile classifications have been initiated, the mechanisms which contribute to the rapid decline and prion mimicking heterogeneity in clinical signs are still largely unknown. In this study, we characterized prion protein (PrP) expression, localization, and interactome in rpAD, slow progressive AD, and in non-dementia controls. PrP along with its interacting proteins were affinity purified with magnetic Dynabeads Protein-G, and were identified using Q-TOF-ESI/MS analysis. Our data demonstrated a significant 1.2-fold decrease in di-glycosylated PrP isoforms specifically in rpAD patients. Fifteen proteins appeared to interact with PrP and only two proteins3/4histone H2B-type1-B and zinc alpha-2 protein3/4were specifically bound with PrP isoform isolated from rpAD cases. Our data suggest distinct PrP involvement in association with the altered PrP interacting protein in rpAD, though the pathophysiological significance of these interactions remains to be established.

A high priority in the prion field is to identify pre-symptomatic events and associated profile of molecular changes. In this study, we demonstrate the pre-symptomatic dysregulation of cytoskeleton assembly and its associated cofilin-1 pathway in strain and brain region-specific manners in MM1 and VV2 subtype-specific Creutzfeldt-Jakob disease at clinical and pre-clinical stage. At physiological level, PrP interaction with cofilin-1 and phosphorylated form of cofilin (p-cofilin(Ser3)) was investigated in primary cultures of mouse cortex neurons (PCNs) of PrP wild-type and knockout mice (PrP). Short-interfering RNA downregulation of active form of cofilin-1 resulted in the redistribution/downregulation of PrP, increase of activated form of microglia, accumulation of dense form of F-actin, and upregulation of p-cofilin(Ser3). This upregulated p-cofilin(Ser3) showed redistribution of expression predominantly in the activated form of microglia in PCNs. At pathological level, cofilin-1 expression was significantly altered in cortex and cerebellum in both humans and mice at pre-clinical stage and at early symptomatic clinical stage of the disease. Further, to better understand the possible mechanism of dysregulation of cofilin-1, we also demonstrated alterations in upstream regulators; LIM kinase isoform 1 (LIMK1), slingshot phosphatase isoform 1 (SSH1), RhoA-associated kinase (Rock2), and amyloid precursor protein (APP) in sporadic Creutzfeldt-Jakob disease MM1 mice and in human MM1 and VV2 frontal cortex and cerebellum samples. In conclusion, our findings demonstrated for the first time a key pre-clinical response of cofilin-1 and the associated pathway in prion disease.

Background High-density oligomers of the prion protein (HDPs) have previously been identified in brain tissues of patients with rapidly progressive Alzheimer’s disease (rpAD). The current investigation aims at identifying interacting partners of HDPs in the rpAD brains to unravel the pathological involvement of HDPs in the rapid progression. Methods HDPs from the frontal cortex tissues of rpAD brains were isolated using sucrose density gradient centrifugation. Proteins interacting with HDPs were identified by co-immunoprecipitation coupled with mass spectrometry. Further verifications were carried out using proteomic tools, immunoblotting, and confocal laser scanning microscopy. Results We identified rpAD-specific HDP-interactors, including the growth arrest specific 2-like 2 protein (G2L2). Intriguingly, rpAD-specific disturbances were found in the localization of G2L2 and its associated proteins i.e., the end binding protein 1, α-tubulin, and β-actin. Discussion The results show the involvement of HDPs in the destabilization of the neuronal actin/tubulin infrastructure. We consider this disturbance to be a contributing factor for the rapid progression in rpAD.

The molecular determinants of atypical clinical variants of Alzheimer’s disease, including the recently discovered rapidly progressive Alzheimer’s disease (rpAD), are unknown to date. Fibrilization of the amyloid-β (Aβ) peptide is the most frequently studied candidate in this context. The Aβ peptide can exist as multiple proteoforms that vary in their post-translational processing, amyloidogenesis, and toxicity. The current study was designed to identify these variations in Alzheimer’s disease patients exhibiting classical (sAD) and rapid progression, with the primary aim of establishing if these variants may constitute strains that underlie the phenotypic variability of Alzheimer’s disease. We employed two-dimensional polyacrylamide gel electrophoresis and MALDI-ToF mass spectrometry to validate and identify the Aβ proteoforms extracted from targeted brain tissues. The biophysical analysis was conducted using RT-QuIC assay, confocal microscopy, and atomic force microscopy. Interactome analysis was performed by co-immunoprecipitation. We present a signature of 33 distinct pathophysiological proteoforms, including the commonly targeted Aβ40, Aβ42, Aβ4-42, Aβ11-42, and provide insight into their synthesis and quantities. Furthermore, we have validated the presence of highly hydrophobic Aβ seeds in rpAD brains that seeded reactions at a slower pace in comparison to typical Alzheimer’s disease. In vitro and in vivo analyses also verified variations in the molecular pathways modulated by brain-derived Aβ. These variations in the presence, synthesis, folding, and interactions of Aβ among sAD and rpAD brains constitute important points of intervention. Further validation of reported targets and mechanisms will aid in the diagnosis of and therapy for Alzheimer’s disease.