Characterization of α-synuclein oligomers formed in the presence of lipid vesicles.

Aggregation of α-synuclein into oligomers and fibrils is associated with numerous neurodegenerative diseases such as Parkinson's disease (PD). Although the identity of the pathogenic species formed during the aggregation process is still under active debate, mounting evidence suggests that small oligomeric species rather than fibrillar aggregates are real toxic species. Isolation and characterization of small oligomers is essential to developing therapeutic strategies to prevent oligomer formation. Preparation of misfolded oligomeric species for biophysical characterization is, however, a great challenge due to their heterogenous, transient nature. Here we report the preparation of toxic and non-toxic α-synuclein oligomeric species formed at different pH values in the presence of lipid vesicles that mimic mitochondria membranes containing cardiolipin. Biophysical characterization of the lipid-induced α-synuclein oligomeric assemblies revealed that α-synuclein oligomers formed at pH 7.4 have higher surface hydrophobicity than the aggregates formed at pH 6.0. In addition, the high-pH oligomers were shown to exhibit higher toxicity than the low-pH aggregates. Structural, dynamic properties of the oligomers were also investigated by using circular dichroism (CD) and NMR spectroscopy. Our CD analyses revealed that the two oligomeric species have distinct molecular conformations, and 2D 1H/15N HSQC NMR experiments suggested that the high-pH oligomers have more extended dynamic regions than the low-pH aggregates. The distinct structural and dynamic properties of the oligomers might be associated with their different cytotoxic properties.

Tau Oligomers as Pathogenic Seeds: Preparation, Characterization, and Propagation In Vitro and In Vivo.

Tau oligomers have been shown to be the main toxic tau species in several neurodegenerative disorders. To study tau oligomers, we have developed reagents and established methods for the reliable preparation, isolation, and detection of tau oligomers as well as their seeding and propagation both in vitro and in vivo. Detailed below are methods for isolation of tau oligomers from brain tissues and detection of tau oligomers using tau oligomer-specific antibodies by biochemical, immunohistochemical, and biophysical methods. Further, methods for evaluating the biological activity of the tau oligomers including their effects on synaptic function, seeding, and propagation in cell models and in vivo are also described.

C9orf72-associated dipeptide protein repeats form A11-positive oligomers in amyotrophic lateral sclerosis and frontotemporal dementia.

Hexanucleotide repeat expansion in C9orf72 is one of the most common causes of amyotrophic lateral sclerosis and frontotemporal dementia. The hexanucleotide expansion, formed by GGGGCC (G4C2) repeats, leads to the production of five dipeptide protein repeats (DPRs) via repeat-associated non-AUG translation. Among the five dipeptide repeats, Gly-Arg, Pro-Arg, and Gly-Ala form neuronal inclusions that contain aggregates of the peptides. Several studies have attempted to model DPR-associated toxicity using various repeat lengths, which suggests a unique conformation that is cytotoxic and is independent of the repeat length. However, the structural characteristics of DPR aggregates have yet to be determined. Increasing evidence suggests that soluble species, such as oligomers, are the main cause of toxicity in proteinopathies, such as Alzheimer's and Parkinson's disease. To investigate the ability of DPRs to aggregate and form toxic oligomers, we adopted a reductionist approach using small dipeptide repeats of 3, 6, and 12. This study shows that DPRs, particularly glycine-arginine and proline-arginine, form oligomers that exhibit distinct dye-binding properties and morphologies. Importantly, we also identified toxic DPR oligomers in amyotrophic lateral sclerosis and frontotemporal dementia postmortem brains that are morphologically similar to those generated recombinantly. This study demonstrates that, similar to soluble oligomers formed by various amyloid proteins, DPR oligomers are toxic, independent of their repeat length.

Polymorphic Alpha-Synuclein Oligomers: Characterization and Differential Detection with Novel Corresponding Antibodies.

The pathological hallmark of many neurodegenerative diseases is the accumulation of characteristic proteinaceous aggregates. Parkinson's disease and dementia with Lewy bodies can be characterized as synucleinopathies due to the abnormal accumulation of the protein alpha-synuclein (α-Syn). Studies have shown amyloidogenic proteins such as α-Syn and tau can exist as polymorphic aggregates, a theory widely studied mostly in their fibrillar morphology. It is now well understood that an intermediate state of aggregates, oligomers, are the most toxic species. We have shown α-Syn, when modified by different physiological inducers, result in distinct oligomeric conformations of α-Syn. Polymorphic α-Syn oligomers exhibit distinct properties such as aggregate size, conformation, and differentially interact with tau. In this study, we confirm α-Syn oligomeric polymorphs furthermore using in-house novel α-Syn toxic conformation monoclonal antibodies (SynTCs). It is unclear the biological relevance of α-Syn oligomeric polymorphisms. Utilizing a combination of biochemical, biophysical, and cell-based assays, we characterize α-Syn oligomeric polymorphs. We found α-Syn oligomeric polymorphs exhibit distinct immunoreactivity and SynTCs exhibit differential selectivity and binding affinity for α-Syn species. Isothermal titration calorimetry experiments suggest distinct α-Syn:SynTC binding enthalpies in a species-specific manner. Additionally, we found SynTCs differentially reduce α-Syn oligomeric polymorph-mediated neurotoxicity and propagation in primary cortical neurons in a polymorph-specific manner. These studies demonstrate the biological significance of polymorphic α-Syn oligomers along with the importance of polymorph-specific antibodies that target toxic α-Syn aggregates. Monoclonal antibodies that can target the conformational heterogeneity of α-Syn oligomeric species and reduce their mediated toxicity have promising immunotherapeutic potential.

Pathological tau signatures and nuclear alterations in neurons, astrocytes and microglia in Alzheimer's disease, progressive supranuclear palsy, and dementia with Lewy bodies.

Accumulation of pathological tau aggregates is a prominent feature in tauopathies that leads during the course of the diseases to neuronal dysfunction before and cell death after. Microglia and astrocytes have been described as playing important roles in synaptic spreading of toxic tau in several neurodegenerative diseases (NDs). Here, we have investigated the immunological and biochemical properties of aggregated tau species in different brain cell types in tau-induced neurodegenerative diseases such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and dementia with Lewy bodies (DLB). Additionally, we examined nuclear size, nuclear density, and chromatin compaction in neuronal and glial cells from diseased brain tissues. Microscopic-histological examination was performed using in-house mouse monoclonal antibodies for toxic tau conformers (TTC-M1 and TTC-M2) and tau oligomers (TOMA1-4). By immunohistochemistry and co-immunofluorescence assays using TOMA/TTC-Ms and cell-type specific markers for neurons, astrocytes, and microglia, we observed that TOMA/TTC-Ms were immunoreactive to diverse tau species in different cell types. Analysis of colocalization coefficients indicated an increased pathological tau deposition mainly in the neurons. Western blot analysis of brain homogenates using TOMA/TTC-Ms revealed distinct patterns of tau aggregation in each disease, suggesting that TOMA/TTC-Ms can distinguish between different tau aggregates present in different tauopathies. Additionally, using DAPI staining, we observed that neuronal and astrocytic nuclei had significantly greater nuclear area and increased chromatin compaction in AD cortices compared to non-demented controls. In contrast, reduction in nuclear density/area and more relaxed chromatin was noticed in DLB neurons, astrocytes and microglia and PSP astrocytes and microglia. Cell-type specific tropism of toxic tau species in tauopathies will provide a greater understanding of the involvement of different brain cell types in tau pathology. In this study, we observed that each disease presented cell-type specific nuclear phenotype and tau deposition pattern.

Untwisted α-Synuclein Filaments Formed in the Presence of Lipid Vesicles.

Accumulation of filamentous aggregates of α-synuclein is a pathological hallmark of several neurodegenerative diseases, including Parkinson's disease (PD). The interaction between α-synuclein and phospholipids has been shown to play a critical role in the aggregation of α-synuclein. Most structural studies have, however, been focused on α-synuclein filaments formed in the absence of lipids. Here, we report the structural investigation of α-synuclein filaments assembled under the quiescent condition in the presence of anionic lipid vesicles using electron microscopy (EM), including cryogenic electron microscopy (cryo-EM). Our transmission electron microscopy (TEM) analyses reveal that α-synuclein forms curly protofilaments at an early stage of aggregation. The flexible protofilaments were then converted to long filaments after a longer incubation of 30 days. More detailed structural analyses using cryo-EM reveal that the long filaments adopt untwisted structures with different diameters, which have not been observed in previous α-synuclein fibrils formed in vitro. The untwisted filaments are rather similar to straight filaments with no observable twist that are extracted from patients with dementia with Lewy bodies. Our structural studies highlight the conformational diversity of α-synuclein filaments, requiring additional structural investigation of not only more ex vivo α-synuclein filaments but also in vitro α-synuclein filaments formed in the presence of diverse cofactors to better understand the molecular basis of diverse molecular conformations of α-synuclein filaments.

Amyloid ß, Tau, and α-Synuclein aggregates in the pathogenesis, prognosis, and therapeutics for neurodegenerative diseases.

Aggregation of specific proteins are histopathological hallmarks of several neurodegenerative diseases, such as, Amyloid ß (Aß) plaques and tau neurofibrillary tangles in Alzheimer's disease (AD); morphologically different inclusions of ratiometric 3 repeat (3 R) and 4 repeat (4 R) tau isoforms in progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick's disease (PiD); α-Synuclein (α-Syn) containing Lewy bodies (LBs) and dystrophic Lewy neurites (LNs) in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). However, mixed brain protein pathologies have been frequently observed in many of these diseases and in normal aging brains, among which Aß/tau and tau/α-Syn crosstalks have received increased attention. Interestingly, studies have also shown synergistic interplay among Aß, tau, and α-Syn in several neurodegenerative diseases, suggesting a protein triumvirate. In this review, we summarize the emerging evidence of Aß, tau, and α-Syn aggregation in pathophysiology, and their overlap in a spectrum of neurodegenerative diseases including AD, PSP, PiD, CBD, PD and DLB. We discuss the prognostic advancements made in biomarker and imaging techniques in the triumvirate proteinopathies. Finally, we discuss the combined therapeutic modality involving biomarkers and imaging techniques for future combinatorial immunotherapeutic targeting more than one protein aggregates. We hope that such a multitarget therapeutic approach will have synergistic or additive effects to manage neurodegenerative diseases with two or more protein pathologies that might uncover a promising strategy for personalized combination therapies. Managing neurodegenerative diseases by optimizing the diagnostic criteria and the correct combination of immunotherapies will be a key factor in the success of future treatment.

Lysine 63-linked ubiquitination of tau oligomers contributes to the pathogenesis of Alzheimer's disease.

Ubiquitin-modified tau aggregates are abundantly found in human brains diagnosed with Alzheimer's disease (AD) and other tauopathies. Soluble tau oligomers (TauO) are the most neurotoxic tau species that propagate pathology and elicit cognitive deficits, but whether ubiquitination contributes to tau formation and spreading is not fully understood. Here, we observed that K63-linked, but not K48-linked, ubiquitinated TauO accumulated at higher levels in AD brains compared with age-matched controls. Using mass spectrometry analyses, we identified 11 ubiquitinated sites on AD brain-derived TauO (AD TauO). We found that K63-linked TauO are associated with enhanced seeding activity and propagation in human tau-expressing primary neuronal and tau biosensor cells. Additionally, exposure of tau-inducible HEK cells to AD TauO with different ubiquitin linkages (wild type, K48, and K63) resulted in enhanced formation and secretion of K63-linked TauO, which was associated with impaired proteasome and lysosome functions. Multipathway analysis also revealed the involvement of K63-linked TauO in cell survival pathways, which are impaired in AD. Collectively, our study highlights the significance of selective TauO ubiquitination, which could influence tau aggregation, accumulation, and subsequent pathological propagation. The insights gained from this study hold great promise for targeted therapeutic intervention in AD and related tauopathies.

Dynamic interactions and Ca2+-binding modulate the holdase-type chaperone activity of S100B preventing tau aggregation and seeding.

The microtubule-associated protein tau is implicated in the formation of oligomers and fibrillar aggregates that evade proteostasis control and spread from cell-to-cell. Tau pathology is accompanied by sustained neuroinflammation and, while the release of alarmin mediators aggravates disease at late stages, early inflammatory responses encompass protective functions. This is the case of the Ca2+-binding S100B protein, an astrocytic alarmin which is augmented in AD and which has been recently implicated as a proteostasis regulator, acting over amyloid ß aggregation. Here we report the activity of S100B as a suppressor of tau aggregation and seeding, operating at sub-stoichiometric conditions. We show that S100B interacts with tau in living cells even in microtubule-destabilizing conditions. Structural analysis revealed that tau undergoes dynamic interactions with S100B, in a Ca2+-dependent manner, notably with the aggregation prone repeat segments at the microtubule binding regions. This interaction involves contacts of tau with a cleft formed at the interface of the S100B dimer. Kinetic and mechanistic analysis revealed that S100B inhibits the aggregation of both full-length tau and of the microtubule binding domain, and that this proceeds through effects over primary and secondary nucleation, as confirmed by seeding assays and direct observation of S100B binding to tau oligomers and fibrils. In agreement with a role as an extracellular chaperone and its accumulation near tau positive inclusions, we show that S100B blocks proteopathic tau seeding. Together, our findings establish tau as a client of the S100B chaperone, providing evidence for neuro-protective functions of this inflammatory mediator across different tauopathies.

Synaptic dysregulation and hyperexcitability induced by intracellular amyloid beta oligomers.

Intracellular amyloid beta oligomer (iAßo) accumulation and neuronal hyperexcitability are two crucial events at early stages of Alzheimer's disease (AD). However, to date, no mechanism linking iAßo with an increase in neuronal excitability has been reported. Here, the effects of human AD brain-derived (h-iAßo) and synthetic (iAßo) peptides on synaptic currents and action potential firing were investigated in hippocampal neurons. Starting from 500 pM, iAßo rapidly increased the frequency of synaptic currents and higher concentrations potentiated the AMPA receptor-mediated current. Both effects were PKC-dependent. Parallel recordings of synaptic currents and nitric oxide (NO)-associated fluorescence showed that the increased frequency, related to pre-synaptic release, was dependent on a NO-mediated retrograde signaling. Moreover, increased synchronization in NO production was also observed in neurons neighboring those dialyzed with iAßo, indicating that iAßo can increase network excitability at a distance. Current-clamp recordings suggested that iAßo increased neuronal excitability via AMPA-driven synaptic activity without altering membrane intrinsic properties. These results strongly indicate that iAßo causes functional spreading of hyperexcitability through a synaptic-driven mechanism and offers an important neuropathological significance to intracellular species in the initial stages of AD, which include brain hyperexcitability and seizures.

Tau oligomer induced HMGB1 release contributes to cellular senescence and neuropathology linked to Alzheimer's disease and frontotemporal dementia.

Aging, pathological tau oligomers (TauO), and chronic inflammation in the brain play a central role in tauopathies, including Alzheimer's disease (AD) and frontotemporal dementia (FTD). However, the underlying mechanism of TauO-induced aging-related neuroinflammation remains unclear. Here, we show that TauO-associated astrocytes display a senescence-like phenotype in the brains of patients with AD and FTD. TauO exposure triggers astrocyte senescence through high mobility group box 1 (HMGB1) release and inflammatory senescence-associated secretory phenotype (SASP), which mediates paracrine senescence in adjacent cells. HMGB1 release inhibition using ethyl pyruvate (EP) and glycyrrhizic acid (GA) prevents TauO-induced senescence through inhibition of p38-mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB)-the essential signaling pathways for SASP development. Despite the developed tauopathy in 12-month-old hTau mice, EP+GA treatment significantly decreases TauO and senescent cell loads in the brain, reduces neuroinflammation, and thus ameliorates cognitive functions. Collectively, TauO-induced HMGB1 release promotes cellular senescence and neuropathology, which could represent an important common pathomechanism in tauopathies including AD and FTD.

Tau induces formation of α-synuclein filaments with distinct molecular conformations.

Recent structural investigation of amyloid filaments extracted from human patients demonstrated that the ex vivo filaments associated with different disease phenotypes adopt diverse molecular conformations, which are different from those of in vitro amyloid filaments. A very recent cryo-EM structural study also revealed that ex vivo α-synuclein filaments extracted from multiple system atrophy patients adopt distinct molecular structures from those of in vitro α-synuclein filaments, suggesting the presence of co-factors for α-synuclein aggregation in vivo. Here, we report structural characterizations of α-synuclein filaments formed in the presence of a potential co-factor, tau, using cryo-EM and solid-state NMR. Our cryo-EM structure of the tau-promoted α-synuclein filaments reveals some similarities to one of the previously reported polymorphs of in vitro α-synuclein filaments in the core region, while illustrating distinct conformations in the N- and C-terminal regions. The structural study highlights the conformational plasticity of α-synuclein filaments and the importance of the co-factors, requiring additional structural investigation of not only more ex vivo α-synuclein filaments, but also in vitro α-synuclein filaments formed in the presence of diverse co-factors. The comparative structural analyses will help better understand molecular basis of diverse structures of α-synuclein filaments and possible relevance of each structure to the disease phenotype.

TDP-43 and Tau Oligomers in Alzheimer's Disease, Amyotrophic Lateral Sclerosis, and Frontotemporal Dementia.

Proteinaceous aggregates are major hallmarks of several neurodegenerative diseases. Aggregates of post-translationally modified transactive response (TAR)-DNA binding protein 43 (TDP-43) in cytoplasmic inclusion bodies are characteristic features in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Recent studies have also reported TDP-43 aggregation in Alzheimer's disease (AD). TDP-43 is an RNA/DNA binding protein (RBP) mainly present in the nucleus. In addition to several RBPs, TDP-43 has also been reported in stress granules in FTD and ALS pathologies. Despite knowledge of cytoplasmic mislocalization of TDP-43, the cellular effects of TDP-43 aggregates and their cytotoxic mechanism(s) remain to be clarified. We hypothesize that TDP-43 forms oligomeric assemblies that associate with tau, another key protein involved in ALS and FTD. However, no prior studies have investigated the interactions between TDP-43 oligomers and tau. It is therefore important to thoroughly investigate the cross-seeding properties and cellular localization of both TDP-43 and tau oligomers in neurodegenerative diseases. Here, we demonstrate the effect of tau on the cellular localization of TDP-43 in WT and P301L tau-inducible cell models (iHEK) and in WT HEK-293 cells treated exogenously with soluble human recombinant tau oligomers (Exo-rTauO). We observed cytoplasmic TDP-43 accumulation o in the presence of tau in these cell models. We also studied the occurrence of TDP-43 oligomers in AD, ALS, and FTD human brain tissue using novel antibodies generated against TDP-43 oligomers as well as generic TDP-43 antibodies. Finally, we examined the cross-seeding property of AD, ALS, and FTD brain-derived TDP-43 oligomers (BDT43Os) on tau aggregation using biochemical and biophysical assays. Our results allow us to speculate that TDP-43/tau interactions might play a role in AD, ALS, and FTD.

RNA-binding proteins Musashi and tau soluble aggregates initiate nuclear dysfunction.

Oligomeric assemblies of tau and the RNA-binding proteins (RBPs) Musashi (MSI) are reported in Alzheimer's disease (AD). However, the role of MSI and tau interaction in their aggregation process and its effects are nor clearly known in neurodegenerative diseases. Here, we investigated the expression and cellular localization of MSI1 and MSI2 in the brains tissues of Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) as well as in the wild-type mice and tau knock-out and P301L tau mouse models. We observed that formation of pathologically relevant protein inclusions was driven by the aberrant interactions between MSI and tau in the nuclei associated with age-dependent extracellular depositions of tau/MSI complexes. Furthermore, tau and MSI interactions induced impairment of nuclear/cytoplasm transport, chromatin remodeling and nuclear lamina formation. Our findings provide mechanistic insight for pathological accumulation of MSI/tau aggregates providing a potential basis for therapeutic interventions in neurodegenerative proteinopathies.

Internalization mechanisms of brain-derived tau oligomers from patients with Alzheimer's disease, progressive supranuclear palsy and dementia with Lewy bodies.

Tau aggregates propagate in brain cells and transmit to neighboring cells as well as anatomically connected brain regions by prion-like mechanisms. Soluble tau aggregates (tau oligomers) are the most toxic species that initiate neurodegeneration in tauopathies, such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and dementia with Lewy bodies (DLB). Exogenous tau aggregates have been shown to be internalized by brain cells; however, the precise cellular and molecular mechanisms that underlie the internalization of tau oligomers (TauO) remain elusive. Using brain-derived tau oligomers (BDTOs) from AD, PSP, and DLB patients, we investigated neuronal internalization mechanisms of BDTOs, including the heparan sulfate proteoglycan (HSPG)-mediated pathway, clathrin-mediated pathway, and caveolae-mediated pathway. Here, we demonstrated that the HSPG-mediated pathway regulates internalization of BDTOs from AD and DLB, while HSPG-mediated and other alternative pathways are involved in the internalization of PSP-derived tau oligomers. HSPG antagonism significantly reduced the internalization of TauO, prevented tau translocation to the endosomal-lysosomal system, and decreased levels of hyperphosphorylated tau in neurons, the well-known contributor for neurofibrillary tangles (NFT) accumulation, degeneration of neurons, and cognitive decline. Furthermore, siRNA-mediated silencing of heparan sulfate (HS)-synthesizing enzyme, exostosin-2, leads to decreased internalization of BDTOs, prevented tau-induced autophagy-lysosomal pathway impairment, and decreased hyperphosphorylated tau levels. Collectively, these findings suggest that HSPG-mediated endocytosis and exostsin-2 are involved in neuronal internalization of TauO and subsequent tau-dependent neuropathology in AD and DLB.

Polymorphic α-Synuclein Strains Modified by Dopamine and Docosahexaenoic Acid Interact Differentially with Tau Protein.

The pathological hallmark of synucleinopathies, including Parkinson's disease (PD), is the aggregation of α-synuclein (α-Syn) protein. Even so, tau protein pathology is abundantly found in these diseases. Both α-Syn and tau can exist as polymorphic aggregates, a phenomenon that has been widely studied, mostly in their fibrillar assemblies. We have previously discovered that in addition to α-Syn oligomers, oligomeric tau is also present in the brain tissues of patients with PD and dementia with Lewy bodies (DLB). However, the effect of interaction between polymorphic α-Syn oligomers and tau has not been scrupulously studied. Here, we have explored the structural and functional diversity of distinct α-Syn oligomers, prepared by modifying the protein with dopamine (DA) and docosahexaenoic acid (DHA). The two α-Syn oligomers differed in aggregate size, conformation, sensitivity to proteinase K digestion, tryptic digestion, and toxicity, suggesting them as distinct α-Syn oligomeric strains. We examined their internalization mechanisms in primary neurons and seeding propensity in inducing α-Syn aggregation. Using a combined approach of molecular and cellular techniques, we observed that the tau aggregates cross-seeded with the individual α-Syn oligomeric strains differed in their biochemical and biological properties, suggesting two distinct tau strains. The tau aggregate cross-seeded with the DA-modified α-Syn oligomeric strain possessed a potent intracellular tau seeding propensity. This study provides a comprehensive analysis of unique strain-specific interaction between oligomeric α-Syn and tau. Furthermore, this study allows us to speculate that distinct α-Syn-tau interactions inducing tau aggregation might be an underlying mechanism of neurodegeneration in PD.

Tau oligomers mediate aggregation of RNA-binding proteins Musashi1 and Musashi2 inducing Lamin alteration.

The exact mechanisms leading to neurodegeneration in Alzheimer's disease (AD) and other tauopathies are not yet entirely understood. However, it is known that several RNA-binding proteins (RBPs) form toxic aggregates and also interact with tau in such granules in tauopathies, including AD. The Musashi (MSI) family of RBPs, consisting of two homologues: Musashi1 and Musashi2, have not been extensively investigated in neurodegenerative diseases. Here, using a tau inducible HEK (iHEK) model we investigate whether MSI proteins contribute to the aggregation of toxic tau oligomers (TauO). Wild-type and mutant P301L tau iHEK cells are used to study the effect of different tau variants on the cellular localization of MSI proteins. Interestingly, we observe that tau co-localizes with MSI in the cytoplasm and nuclei, altering the nuclear transport of MSI. Furthermore, incremental changes in the size and density of nuclear MSI/tau foci are observed. We also report here that TauO interact with MSI to cause the formation of distinct nuclear aggregates. Moreover, tau/MSI aggregates induce structural changes to LaminB1, leading to nuclear instability. These results illustrate a possible mechanism of neurodegeneration mediated by the aggregation of MSI proteins and TauO, suggesting that MSI plays a critical role in cellular dysfunction.

Formation of Toxic Oligomeric Assemblies of RNA-binding Protein: Musashi in Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder associated with structural and functional alterations of brain cells causing progressive deterioration of memory and other cognitive functions. Recent studies demonstrate that several neurodegenerative diseases, including AD exhibit RNA-binding proteins (RBPs) pathologies, including TAR DNA -binding protein (TDP-43), fused in sarcoma (FUS), superoxide dismutase (SOD1) and T-interacting antigen-1 (TIA-1), highlighting the role of RBPs in neurodegeneration. One such group of RBPs, Musashi proteins comprised of MSI1 and MSI2, has been long studied in neurogenesis and cancer biology. Herein, we have investigated the aggregation properties of MSI1 and MSI2 by in vitro assays, their expression and accumulation as well as their possible interactions with other cellular proteins, such as tau in AD pathology. We have performed atomic force microscopy, Western blot, and immunoprecipitation to demonstrate the aggregation properties of recombinant Musashi proteins. Furthermore, we have studied cortical brain sections from AD (N = 4) and age-matched non-demented subjects (N = 4) by Western blot and immunofluorescence microscopy to investigate MSI1 and MSI2 levels and their localization in human brain tissues. Musashi proteins showed in vitro aggregation properties by forming oligomers. We have observed an increase in Musashi proteins levels in AD brain tissues as compared with age-matched non-demented subjects. Moreover, Musashi proteins are observed to form oligomers in the diseased brain tissues. Interestingly, the co-immunofluorescence study has revealed a change in fluorescence pattern of oligomeric Musashi proteins and tau with a high association in the perinuclear area of the cells suggesting changes in function of Musashi proteins. Our data have demonstrated for the first time that MSI1 and MSI2 are present in an oligomeric state in AD brains compared to the age-matched non-demented subjects and that these large assemblies co-localize with tau contributing to the neurodegenerative pathogenesis.

Study protocol: healthy urban living and ageing in place (HULAP): an international, mixed methods study examining the associations between physical activity, built and social environments for older adults the UK and Brazil.

BACKGROUND: The ability to 'age in place' is dependent on a range of inter-personal, social and built environment attributes, with the latter being a key area for potential intervention. There is an emerging body of evidence that indicates the type of built environment features that may best support age friendly communities, but there is a need to expand and consolidate this, while generating a better understanding of how on how research findings can be most effectively be translated in to policy and practice. METHODS: The study is based on two case study cities, Curtiba (Brazil) and Belfast (UK), which have highly contrasting physical, social and policy environments. The study deploys a mix methods approach, mirrored in each city. This includes the recruitment of 300 participants in each city to wear GPS and accelerometers, a survey capturing physical functioning and other personal attributes, as well as their perception of their local environment using NEWS-A. The study will also measure the built environments of the cities using GIS and develop a tool for auditing the routes used by participants around their neighbourhoods. The study seeks to comparatively map the policy actors and resources involved in healthy ageing in the two cities through interviews, focus groups and discourse analysis. Finally, the study has a significant knowledge exchange component, including the development of a tool to assess the capacities of both researchers and research users to maximise the impact of the research findings. DISCUSSION: The HULAP study has been designed and implemented by a multi-disciplinary team and integrates differing methodologies to purposefully impact on policy and practice on healthy ageing in high and low-middle income countries. It has particular strengths in its combination of objective and self-reported measures using validated tools and the integration of GPS, accelerometer and GIS data to provide a robust assessment of 'spatial energetics'. The strong knowledge exchange strand means that the study is expected to also contribute to our understanding of how to maximise research impact in this field and create effective evidence for linking older adult's physical activity with the social, built and policy environments.