Comparative interactome mapping of Tau-protein in classical and rapidly progressive Alzheimer's disease identifies subtype-specific pathways.

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.

SWATH Mass Spectrometry-Based CSF Proteome Profile of GBA-Linked Parkinson's Disease Patients.

ß-glucocerebrosidase (GBA)-associated mutations are a significant risk factor for Parkinson's disease (PD) that aggravate the disease pathology by upregulating the deposition of α-Synuclein (α-Syn). The resultant clinical profile varies for PD patients without GBA mutations. The current study aimed to identify the proteomic targets involved in the pathogenic pathways leading to the differential clinical presentation of GBA-associated PD. CSF samples (n = 32) were obtained from PD patients with GBA mutations (n = 22), PD patients without GBA mutations (n = 7), and healthy controls that were carriers of GBA mutations (n = 3). All samples were subjected to in-gel tryptic digestion followed by the construction of the spectral library and quantitative SWATH-based analysis. CSF α-Syn levels were reduced in both PDIdiopathic and PDGBA cases. Our SWATH-based mass spectrometric analysis detected 363 proteins involved in immune response, stress response, and cell signaling in various groups. Intergroup analysis showed that 52 proteins were significantly up- or downregulated in various groups. Of these 52 targets, 20 proteins were significantly altered in PDGBA cases only while 2 showed different levels in PDIdiopathic patients. Our results show that the levels of several pathologically relevant proteins, including Contactin-1, Selenium-binding protein 1, Adhesion G Protein-Coupled Receptor, and Apolipoprotein E are significantly different among the sporadic and genetic variants of PD and hint at aggravated synaptic damage, oxidative stress, neuronal loss, and aggregation of α-Syn in PDGBA cases.

Recombinant PrPSc shares structural features with brain-derived PrPSc: Insights from limited proteolysis.

Very solid evidence suggests that the core of full length PrPSc is a 4-rung ß-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the ß-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 ß-strands, helping us to predict the threading of the ß-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.

SFPQ and Tau: critical factors contributing to rapid progression of Alzheimer's disease.

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 DNA-related anomalies and aberrant dynamics of SGs in association with pathological tau represents a critical pathway which contributes to rapid progression of AD.

Nuclear face of Tau: an inside player in neurodegeneration.

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.

Differential interactome mapping of aggregation prone/prion-like proteins under stress: novel links to stress granule biology.

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.

Genetic Variants Associated with the Age of Onset Identified by Whole-Exome Sequencing in Fatal Familial Insomnia.

Fatal familial insomnia (FFI) is a rare autosomal-dominant inherited prion disease with a wide variability in age of onset. Its causes are not known. In the present study, we aimed to analyze genetic risk factors other than the prion protein gene (PRNP), in FFI patients with varying ages of onset. Whole-exome sequencing (WES) analysis was performed for twenty-five individuals with FFI (D178N-129M). Gene ontology enrichment analysis was carried out by Reactome to generate hypotheses regarding the biological processes of the identified genes. In the present study, we used a statistical approach tailored to the specifics of the data and identified nineteen potential gene variants with a potential effect on the age of onset. Evidence for potential disease modulatory risk loci was observed in two pseudogenes (NR1H5P, GNA13P1) and three protein coding genes (EXOC1L, SRSF11 and MSANTD3). These genetic variants are absent in FFI patients with early disease onset (19-40 years). The biological function of these genes and PRNP is associated with programmed cell death, caspase-mediated cleavage of cytoskeletal proteins and apoptotic cleavage of cellular proteins. In conclusions, our study provided first evidence for the involvement of genetic risk factors additional to PRNP, which may influence the onset of clinical symptoms in FFI.

α-Synuclein conformers reveal link to clinical heterogeneity of α-synucleinopathies.

α-Synucleinopathies, such as Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy, are a class of neurodegenerative diseases exhibiting intracellular inclusions of misfolded α-synuclein (αSyn), referred to as Lewy bodies or oligodendroglial cytoplasmic inclusions (Papp-Lantos bodies). Even though the specific cellular distribution of aggregated αSyn differs in PD and DLB patients, both groups show a significant pathological overlap, raising the discussion of whether PD and DLB are the same or different diseases. Besides clinical investigation, we will focus in addition on methodologies, such as protein seeding assays (real-time quaking-induced conversion), to discriminate between different types of α-synucleinopathies. This approach relies on the seeding conversion properties of misfolded αSyn, supporting the hypothesis that different conformers of misfolded αSyn may occur in different types of α-synucleinopathies. Understanding the pathological processes influencing the disease progression and phenotype, provoked by different αSyn conformers, will be important for a personalized medical treatment in future.

A new paradigm for diagnosis of neurodegenerative diseases: peripheral exosomes of brain origin.

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.

The prion protein and its ligands: Insights into structure-function relationships.

The prion protein is a multifunctional protein that exists in at least two different folding states. It is subject to diverse proteolytic processing steps that lead to prion protein fragments some of which are membrane-bound whereas others are soluble. A multitude of ligands bind to the prion protein and besides proteinaceous binding partners, interaction with metal ions and nucleic acids occurs. Although of great importance, information on structural and functional consequences of prion protein binding to its partners is limited. Here, we will reflect on the structure-function relationship of the prion protein and its binding partners considering the different folding states and prion protein fragments.

Molecular Profiles of Amyloid-ß Proteoforms in Typical and Rapidly Progressive Alzheimer's Disease.

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-ß (Aß) peptide is the most frequently studied candidate in this context. The Aß 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 Aß 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 Aß40, Aß42, Aß4-42, Aß11-42, and provide insight into their synthesis and quantities. Furthermore, we have validated the presence of highly hydrophobic Aß 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 Aß. These variations in the presence, synthesis, folding, and interactions of Aß 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.

Prion protein oligomers cause neuronal cytoskeletal damage in rapidly progressive Alzheimer's 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 ß-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.

Unveiling the Physical and Functional Niches of FAM26F by Analyzing Its Subcellular Localization and Novel Interacting Partners.

The knowledge of a protein's subcellular localization and interacting partners are crucial for elucidating its cellular function and associated regulatory networks. Although FAM26F (family with sequence similarity 26, member F) has been recognized as a vital player in various infections, stimulation studies, cancer, and immune pathogenesis, the precise location and function of FAM26F are not well understood. The current study is the first to focus on functional characterization of FAM26F by analyzing its subcellular localization and identifying its novel interacting partners using advanced proteome approaches. The immunofluorescence and confocal microscopy results revealed FAM26F to be largely localized within the Golgi apparatus of the cell. However, its minor presence in endoplasmic reticulum (ER) pointed toward the probable retrograde transfer of FAM26F from Golgi to ER during adverse conditions. Moreover, co-immunoprecipitation and MS/MS results demonstrated a total of 85 proteins, 44 of which significantly copurified with FAM26F. Interestingly, out of these 44 MS/MS identified proteins, almost 52% were involved in innate immunity, 38.6% in neutrophil degranulation, and remaining 10% were either involved in phosphorylation, degradation, or regulation of apoptosis. Further characterization through Ingenuity Pathway Analysis showed that majority of these proteins was involved in maintaining calcium homeostasis of cell. Consequently, the validation of selected proteins uncovered the key interaction of FAM26F with Thioredoxin, which essentially paved the way for depicting its mechanism of action under stress or disease conditions. It is proposed that activation and inhibition of the cellular immune response is essentially dependent on whether FAM26F or Thioredoxin considerably interact with CD30R.

Cytoskeleton-Associated Risk Modifiers Involved in Early and Rapid Progression of Sporadic Creutzfeldt-Jakob Disease.

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, PrPC interaction with cofilin-1 and phosphorylated form of cofilin (p-cofilin(Ser3)) was investigated in primary cultures of mouse cortex neurons (PCNs) of PrPC wild-type and knockout mice (PrP-/-). Short-interfering RNA downregulation of active form of cofilin-1 resulted in the redistribution/downregulation of PrPC, 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.

Prion Protein Interactome: Identifying Novel Targets in Slowly and Rapidly Progressive Forms of Alzheimer's Disease.

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.

Strain-Specific Altered Regulatory Response of Rab7a and Tau in Creutzfeldt-Jakob Disease and Alzheimer's Disease.

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 PrPC 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 PrPC wild type. This depleted expression of Rab7a led to the increased accumulation of PrPC in Rab9-positive endosomal compartments and consequently an increased co-localization between PrPC/Rab9; however, total tau level decreased. Interestingly, siRNA against tau gene in cortical primary neuronal cultures of PrPC 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.