AD Brain Research Articles

Properties of TCR gamma clonotypes in AD brain and peripheral blood

AbstractBackgroundThe immune system contributes to various neurodegenerative pathologies, including Alzheimer’s disease (AD). γδ T cells can mediate different pathological processes in the human brain, such as oncogenesis, autoimmunity, and stroke. However, the role of γδ T cells in AD have not yet been elucidated.MethodWe designed an approach based on multiplex PCR system with following library preparation and deep sequencing of the CDR3 region of TRGs. In total clonotype repertoires from 147 peripheral blood and postmortem brain samples of AD patients and control individuals without dementia were analyzed. Moreover 17 blood repertoires of patients with Parkinson’s disease (PD) were also tested.ResultOur results showed special features of peripheral blood (PB) and brain TRG repertoires: compared with the PB, the brain showed reduced TRGV9 clonotypes but was enriched in TRGV2/4/8 clonotypes. Age‐related changes were found. The reduced diversity and CDR3 length of human blood TRG repertoires and in the composition of the brain TRG repertoire, in particular, a decrease in the frequency of the TRGV9 segments and an increase in TRGV2/4/8 were identified. We developed a bioinformatic approach based on the physicochemical properties analysis of rare clones that are found in patients with AD but not in healthy individuals. Our findings revealed the enrichment of TRG clonotypes with specific properties in the brain or blood of patients with AD compared to those in controls. (Aliseychik et al. 2020) These AD‐associated clonotypes are not typical for patients with PD, for which accumulation of the AD specific clonotypes was not pronounced.ConclusionThe pilot study of TCR repertoires in AD revealed disease associated TRGs profile properties in brain and blood and implied putative AD‐associated immunogenic markers. This work was supported by the National Institute on Aging (NIA; R01 AG054712); the Russian Foundation for Basic Research (RFBR, grant no. 18‐29‐13051) and the Russian Science Foundation (RSF, grant no. 19‐75‐30039).

Development of [18F]SNFT‐1, a novel tau PET tracer with little off‐target binding

AbstractBackground[18F]THK‐5351, which was originally designed to detect tau aggregates, bound to monoamine oxidase B (MAO‐B) with high affinity. Lead optimization toward selective binding profiles to tau has resulted in the development of novel selective tau PET tracer named [18F]SNFT‐1 (THK‐5562). Here we present the preclinical characteristics of this tracer.MethodIn vitro competitive binding assays against MAO‐A, MAO‐B, amyloid, and tau were performed. In vitro autoradiography of the human brain tissues of various neurodegenerative diseases was performed. Receptor panel screen was performed to confirm the binding selectivity of this compound. Biodistribution study was performed in mice. In addition, an acute toxicity with intravenous administration of a single dose of this compound in mice was also investigated.ResultSNFT‐1 showed high affinity (K d = 0.47 nM) and selectivity for tau aggregates over other misfolding proteins as well as MAO enzymes. In vitro autoradiography demonstrated the intense laminar binding of [18F]SNFT‐1 to tau pathology in AD and less off‐target binding than currently available tau PET tracers. No remarkable binding inhibition on various receptors ion channels, and transporters was observed at 1 μM concentration. [18F]SNFT‐1 showed good initial brain uptake and rapid washout without defluorination and troublesome radiolabeled metabolites in mice. In acute toxicity study, no drug‐related changes were noted after intravenous administration of this compound in mice.Conclusion[18F]SNFT‐1 is highly selective tau PET tracer, which will enable accurate monitoring of abnormal tau pathology in AD brain.

Collagenous Alzheimer amyloid plaque component impacts on the compaction of amyloid-\u03b2 plaques

Massive deposition of amyloid β peptides (Aβ) as senile plaques (SP) characterizes the brain pathology of Alzheimer’s disease (AD). SPs exhibit a variety of morphologies, although little is known about the SP components that determine their morphology. Collagenous Alzheimer amyloid plaque component (CLAC) is one of the major non-Aβ proteinaceous components of SP amyloid in AD brains. Here we show that overexpression of CLAC precursor (CLAC-P) in the brains of APP transgenic mice results in a significant remodeling of amyloid pathology, i.e., reduction in diffuse-type amyloid plaques and an increase in compact plaques laden with thioflavin S-positive amyloid cores. In vivo microdialysis revealed a significant decrease in Aβ in the brain interstitial fluid of CLAC-P/APP double transgenic mice compared with APP transgenic mice. These findings implicate CLAC in the compaction of Aβ in amyloid plaques and the brain dynamics of Aβ.

Elucidating the relationship between TGF‐β signaling and Alzheimer’s disease

AbstractBackgroundThe TGF‐β signaling pathway represents a complex family and network of ligands and receptors. A body of evidence suggests that in Alzheimer’s Disease this signaling network may be perturbed and could contribute to disease development and progression. Other studies have shown a role for TGF‐β signaling in both Aβ clearance and deposition, as well as, in neuroprotection and neurotoxicity. It remains to be determined precisely the contributions of TGF‐β signaling to mechanisms underlying Alzheimer’s disease and other dementias and whether targeting TGF‐β may lead to suitable novel therapies.MethodsTo interrogate the contributions of TGF‐β signaling to Aβ and tau pathology in AD we developed several recombinant adeno‐associated virus (rAAV) constructs to express TGF‐β family ligands and decoy receptors. APP overexpressing (TgCRND8) mice were injected with rAAVs at postnatal day 0 or after 3 months of aging to examine contributions of the TGF‐β family prior to and during Aβ deposition. We also examined how TGF‐β family ligands and decoy receptors affect the development of tau pathology in tau brain slice culture (BSC) models.ResultsWe have successfully expressed TGF‐β family ligands and decoy receptors to determine their contributions to Aβ clearance and deposition in vivo in APP overexpressing mice, as well as, effects on tau pathology in ex vivo tau BSC models. Stimulating and blocking TGF‐β signaling cascades appears to have opposing effects on AD‐relevant pathology. Using this rAAV approach we can rapidly dissect how complex signaling cascades may contribute to the progression and development of human AD and how targeting these pathways may be therapeutically rewarding.ConclusionsThese findings suggest that interventions targeting TGF‐β signaling may be a novel therapeutic approach to AD. This data also enforces that pathways involved in immune function can have significant effects on the healthy and diseased brain and should be explored further in order to understand the development and progression of AD. Using a systems approach (NIH U01AG046139) we identified perturbed networks regulating innate immunity in the AD brain and have now validated how intervening with TGF‐β signaling may provide therapeutic benefit.

Modeling the effects of respirasome supercomplex formation on AD progression by incorporation of mitochondrial gene expression data

AbstractBackgroundAlzheimer’s disease is a specific form of dementia characterized by the aggregation of amyloid‐β plaques and tau tangles. New research has found that the formation of these aggregates occurs after dysregulation of respiratory chain activity. Proteomic and genomic data show that these changes are also related to unique gene expression patterns. Supercomplexes (SCs) are assemblages of multiple, functional respiratory complexes. After individual complexes have formed, respiratory complexes (RC) I, III, and IV begin to associate and form SCs. SCs are stable intermediates between respirasomes and individual complexes. We investigated the impact of our observed gene expression changes on new therapeutic options via metabolic flux analysis of SC assembly.MethodTotal RNA was isolated from 80‐180 mg of frozen brain tissue from 11 AD and 5 control samples. Total RNA was quantified and assessed for purity using Nanodrop and Bioanalyzer analysis. cDNA was prepared and applied to the Human Mitochondrial Energy Metabolism and the Mitochondria RT² Profiler™ PCR Arrays. The qPCR data was then analyzed using Qiagen RT2 Profiler PCR Array Data Analysis version 3.5. Gene expression changes were incorporated into the mathematical model created in CellDesigner 4.2 and these were converted into a series of mathematical equations by SBMLsqueezer. This file was imported into COPASI, a biological pathway simulation program. Treatment data were found using COPASI’s time course task. Each treatment represented a mathematical change in gene expression of a selected enzyme.ResultWe investigated 9 mitochondrial genes known to be important for efficient SC assembly. In all cases, the concentration of the gene products was decreased in the AD brain samples. After simulation of respirasome assembly using a reduced model only UQCRC1 treatment increased the percent of CI sequestration into respirasomes.ConclusionOur results indicate that protective changes in expression of UQCRCI, a component of electron transport chain respiratory complex III may serve as a potential avenue for AD therapeutics. This work also highlights the importance of metabolic enzyme activity in maintaining proper respiratory activity.We would like to acknowledge support for this work from the Alzheimer’s and Related Diseases Research Award Fund and the Commonwealth Health Research Board.

Exploring the role of RALYL in Alzheimer\u2019s disease reserve by network-based approaches

BackgroundAlzheimer’s disease (AD) reserve theory is based on specific individual characteristics that are associated with a higher resilience against neurodegeneration and its symptoms. A given degree of AD pathology may contribute to varying cognitive decline levels in different individuals. Although this phenomenon is attributed to reserve, the biological mechanisms that underpin it remain elusive, which restricts translational medicine research and treatment strategy development.MethodsNetwork-based approaches were integrated to identify AD reserve related genes. Then, AD brain transcriptomics data were clustered into co-expression modules, and a Bayesian network was developed using these modules plus AD reserve related phenotypes. The directed acyclic graph suggested that the module was strongly associated with AD reserve. The hub gene of the module of interest was filtered using the topological method. Validation was performed in the multi-AD brain transcriptomic dataset.ResultsWe revealed that the RALYL (RALY RNA Binding Protein-like) is the hub gene of the module which was highly associated with AD reserve related phenotypes. Pseudo-time projections of RALYL revealed the changes in relative expression drivers in the AD and control subjects over pseudo-time had distinct transcriptional states. Notably, the expression of RALYL decreased with the gradual progression of AD, and this corresponded to MMSE decline. Subjects with AD reserve exhibited significantly higher RALYL expression than those without AD reserve.ConclusionThe present study suggests that RALYL may be associated with AD reserve, and it provides novel insights into the mechanisms of AD reserve and highlights the potential role of RALYL in this process.

Alzheimer's genetic risk factor FERMT2 (kindlin‐2) controls axonal growth and synaptic plasticity in an APP‐dependent manner

AbstractBackgroundAlthough APP metabolism is being intensively investigated, a large fraction of its modulators are yet to be characterized. In this context, we combined two genome‐wide high‐content screenings to assess the functional impact of miRNAs and genes on APP metabolism and the signaling pathways involved.MethodWe combined genome‐wide high‐content screenings to identify miRNAs (n = 2,555) and target genes (n = 18,107) involved in the APP metabolism. To predict the function(s) of miRNAs that target these genes, pathway enrichment analysis was performed using DIANA Tools mirPath (v3.0). CRISPR/Cas9 technology has been used to generate HEK293 cell lines carrying rs7143400 variant located in the FERMT2 3’UTR and its impact on miRNA binding. Involvement of FERMT2 in axonal growth and synaptic connectivity was assessed using primary neurons cultured in microfluidic devices that fluidically isolate axons from their cell bodies. The functional impact of FERMT2 on CA1 basal synaptic transmission and long‐term potentiation (LTP) has been recorded in ex vivo mouse hippocampal slices, after stereotactic lentivirus injection allowing expression of shNT, shFERMT2 or shAPP.ResultOur systematic approaches led us to characterize 180 genes targeted by 41 miRNAs as modulators of APP metabolism. Among these genes, we focused on FERMT2, a known genetic risk factor of sporadic AD. We found that FERMT2 directly interacts with APP to modulate its metabolism and that FERMT2 under‐expression impacts axonal growth, synaptic connectivity and long‐term potentiation in an APP‐dependent manner.Lastly, the rs7143400‐T allele, which is associated with an increased AD risk and localized within the 3’UTR of FERMT2, induced a down‐regulation of FERMT2 expression through binding of miR‐4504. This miRNA is mainly expressed in neurons and significantly overexpressed in AD brains compared to controls.ConclusionAltogether, our data provide strong evidence for a detrimental effect of FERMT2 under‐expression in neurons and insight on how this may influence AD pathogenesis.

Presentation 3: Translational dysregulation promotes pathogenic events in tauopathies

AbstractBackgroundA major challenge in the field of tau‐associated neurodegeneration is the identification of the pathogenic mechanisms leading to disease. Tau is best known for associating and stabilizing microtubules; however, emerging results identifying alternate tau functions opens a new opportunity to understand the molecular basis of disease. Given the association of tau with ribosomes and RNA, and the enhancement of this interaction in Alzheimer’s disease, we hypothesized that the aberrant tau‐ribosome interaction impairs (increases and decreases) transcripts that are necessary for cellular homeostasis. For nearly thirty years, the consequences of tau‐ribosome complexes were unknown until now.MethodsWe used in vitro models such as GFP‐based cell‐free translation assays and surface sensing of translation (SUnSET) assays in immortalized and primary neuronal cultures. Moreover, we pioneered in vivo SUnSET in the brains of tau transgenic mice, and coupled these techniques with proteomics. We validated our results using human brain samples from AD and non‐demented controls. Finally, we performed ribosomal profiling assays using in vitro models of tauopathy.ResultsWe demonstrate that pathological tau species associate robustly with ribosomes profoundly shifting the translatome. Surprisingly, a significant number of proteins whose translation was impaired corresponded to ribosomal proteins. We also identified that the ribosomal protein S6, which regulates translation of the machinery necessary for translation (ribosomal proteins as well as elongation and initiation factors), was significantly reduced in vitro, in vivo, and in human brains. Finally, we also found major shifts in the integrity of AD brain polysomes as a consequence of tau aggregation.ConclusionPathological tau species negatively regulate ribosomal specificity thereby shifting the translatome.

Hyperphosphorylation, cytoskeleton and cell‐surface binding aptamers: Developing markers and understanding molecular changes in early stage Alzheimer’s disease

AbstractBackgroundHyperphosphorylated tau (pTau) is a characteristic of AD, leading to intracellular “tau tangles” followed by dementia. However, intracellular tau is difficult to measure in vivo, requiring a probe to enter the cell. Proteomic changes on the diseased cell‐surface however, provide a diagnostic target that eliminates the need for a probe to enter the cell. We therefore sought cell surface markers of pathological neurons in the process of hyperphosphorylating tau.MethodSH‐SY5Y cells differentiated to a neuronal phenotype were rendered hyperphosphorylative by okadaic acid, and aptamers specifically binding these cells were captured using a modified cell‐SELEX methodology. Mass spectrometric proteomics identified the binding targets. Target presence was validated on SH‐SY5Y cells, P301S mouse brain tissue and human AD brain tissue by immunofluorescence. Gene‐expression data were analysed to identify the utility of the target as a diagnostic marker. A Gd‐bearing nanoparticle targeted using the lead aptamers was injected i.v. in 2‐month P301S mice and T1‐weighted MRI imaging was used to assess binding as a predictor of future tau pathology.ResultAptamers identified by sequencing of the enriched aptamer pools were demonstrated to specifically bind hyperphosphorylative SH‐SY5Y cells . Proteomic analysis suggested Vimentin and keratin6A/6B as potential binding targets. Analysis of hyperphosphorylative SH‐SY5Ycells, human AD tissue and P301S mouse brain tissue confirmed increased levels of cell‐surface Vimentin. Meta‐analysis revealed that Vimentin is expressed at higher levels in the diseased state. Further pathway analysis identified alternative targets related to desmosomes. Two month old P301S mice had no tau pathology, but the targeted MRI particles were still ∼80% accurate in identifying the transgenic mice and predicting future tau pathology.ConclusionOur results suggest that Vimentin and other related desmosomal molecules could be prognostic markers of future tauopathy, and also suggest a need to investigate cell‐surface changes under conditions that cause tauopathies. Targeted MRI particles are promising as an indicator of future tau pathology.

Targeting porphyromonas gingivalis to treat Alzheimer’s disease and comorbid cardiovascular disease

AbstractBackgroundWe recently demonstrated the presence of the bacterial pathogen, Porphyromonas gingivalis (Pg), and its protease virulence factors, known as gingipains, in greater than 90% of AD brains. Brain gingipain levels significantly correlated with AD diagnosis and tau and ubiquitin pathology. Mechanistic studies in wild type mice demonstrated that Pg invades the brain after infection of the oral cavity, resulting in the development of neuropathology consistent with that of AD. These effects were blocked in mice after oral administration of the gingipain inhibitor COR388. In addition to neuropathology, AD is often accompanied by the comorbidity of cardiovascular disease. Pg has been linked to the development of atherosclerosis and the bacteria are found abundant within atheromatous plaque. We investigated if COR388 was efficacious in treating Pg‐accelerated atherosclerosis in a rabbit model.MethodEighteen New Zealand White rabbits on a 13‐week regimen of 0.5% cholesterol diet to induce atherosclerosis were randomized in the study. Twelve rabbits were orally infected with Pg 3x/week for 6 weeks for oral disease induction and 6 rabbits were sham‐infected. Six Pg –infected rabbits were orally administered COR388 10mg/kg once daily for the entire 13 weeks of the study (prevention protocol), and 6 Pg‐infected rabbits received vehicle for 13 weeks. Six sham‐infected rabbits received vehicle for 13 weeks. At 13 weeks, atherosclerotic plaques were quantified by assessing Sudan IV staining, histology, and ex vivo MRI. Serum levels of C‐reactive protein (CRP) were evaluated as a measure of systemic inflammation.ResultPg‐infected rabbits exhibited accelerated atherosclerotic disease severity than sham‐infected animals. COR388 diminished this atherogenesis compared to vehicle controls as evidenced by significantly less arterial plaque and a lower intima/media ratio. COR388 treatment also significantly reduced systemic levels of CRP.ConclusionCOR388, a novel lysine‐gingipain inhibitor, is currently being tested in a Phase 2/3 clinical trial to target Pg for the treatment of AD. Based on the current preclinical data reported here, COR388 has the potential to attenuate atheroma formation and systemic inflammation in Pg‐ induced atherosclerosis and therefore may have beneficial effects on comorbid cardiovascular disease in Pg‐associated AD.

The Dysregulation of OGT/OGA Cycle Mediates Tau and APP Neuropathology in Down Syndrome

Protein O-GlcNAcylation is a nutrient-related post-translational modification that, since its discovery some 30 years ago, has been associated with the development of neurodegenerative diseases. As reported in Alzheimer’s disease (AD), flaws in the cerebral glucose uptake translate into reduced hexosamine biosynthetic pathway flux and subsequently lead to aberrant protein O-GlcNAcylation. Notably, the reduction of O-GlcNAcylated proteins involves also tau and APP, thus promoting their aberrant phosphorylation in AD brain and the onset of AD pathological markers. Down syndrome (DS) individuals are characterized by the early development of AD by the age of 60 and, although the two conditions present the same pathological hallmarks and share the alteration of many molecular mechanisms driving brain degeneration, no evidence has been sought on the implication of O-GlcNAcylation in DS pathology. Our study aimed to unravel for the first time the role of protein O-GlcNacylation in DS brain alterations positing the attention of potential trisomy-related mechanisms triggering the aberrant regulation of OGT/OGA cycle. We demonstrate the disruption of O-GlcNAcylation homeostasis, as an effect of altered OGT and OGA regulatory mechanism, and confirm the relevance of O-GlcNAcylation in the appearance of AD hallmarks in the brain of a murine model of DS. Furthermore, we provide evidence for the neuroprotective effects of brain-targeted OGA inhibition. Indeed, the rescue of OGA activity was able to restore protein O-GlcNAcylation, and reduce AD-related hallmarks and decreased protein nitration, possibly as effect of induced autophagy.

Electroacupuncture Protects Cognition by Regulating Tau Phosphorylation and Glucose Metabolism via the AKT/GSK3β Signaling Pathway in Alzheimer's Disease Model Mice.

BackgroundAlzheimer’s disease (AD) is mainly manifested as a continuous and progressive decline in cognitive ability. Neurofibrillary tangles (NFTs) are pathological hallmarks of AD and due to accumulated phosphorylated Tau. Glycogen synthase kinase-3β (GSK3β), as a major Tau kinase and a downstream target of the serine protein kinase B (AKT) signaling pathway, can regulate Tau phosphorylation in AD. Importantly, the AKT/GSK3β signaling pathway is involved in glucose metabolism, and abnormal glucose metabolism is found in the AD brain. Numerous studies have shown that electroacupuncture (EA), which is thought to be a potential complementary therapeutic approach for AD, can protect cognitive ability to a certain extent.ObjectiveThe purpose of this experiment was to investigate whether the protective and beneficial mechanism of EA on cognition was mediated by the AKT/GSK3β signaling pathway, thereby improving glucose metabolism and Tau phosphorylation in the brain.MethodsEA was applied to the Baihui (GV20) and Yintang (GV29) acupoints of 6-month-old amyloid precursor protein (APP)/presenilin-1 (PS1) mice for 20 min, and then quickly prick Shuigou (GV26) acupoint. The intervention was performed once every other day for 28 days. The Morris water maze (MWM) test was performed on C57BL/6N (Non-Tg) mice, APP/PS1 (Tg) mice and EA-treated Tg (Tg + EA) mice to evaluate the effect of EA therapy on cognitive function. 18F-FDG positron emission tomography (PET), immunohistochemistry, and western blotting (WB) were used to investigate the possible mechanism underlying the effect of EA on AD.ResultsEA treatment significantly improved the cognition of APP/PS1 mice and the glucose uptake rate in the hippocampus. Furthermore, EA inhibited the phosphorylation of Tau (Ser199 and Ser202) proteins by inducing AKT (Ser473) and GSK3β (Ser9) phosphorylation.ConclusionThese results demonstrate that EA intervention protects cognition by enhancing glucose metabolism and inhibiting abnormal phosphorylation of Tau protein in the AD model mice, and the AKT/GSK3β pathway might play an irreplaceable role in the regulation process.

Comparison of size distribution and (Pro249-Ser258) epitope exposure in in vitro and in vivo derived Tau fibrils

BackgroundAlthough several studies demonstrate prion-like properties of Tau fibrils, the effect of size in the seeding capacity of these aggregates is not fully understood. The aim of this study is to characterize Tau seeds by their size and seeding capacity.MethodsTau aggregates were isolated from postmortem AD brain tissue and separated from low molecular weight species by sucrose gradient ultracentrifugation. Biochemical characterization of the different fractions was done by non-reducing Western blotting and aggregate-specific immuno-assays using in house developed anti-Tau monoclonal antibodies, including PT76 which binds to an epitope close to the microtubule-binding domain and, hence, also to K18. Seeding efficiency was then assessed in HEK293 cells expressing K18 FRET sensors.ResultsWe observed that upon sonication of Tau aggregates different size-distributed tau aggregates are obtained. In biochemical assays, these forms show higher signals than the non-sonicated material in some aggregation-specific Tau assays. This could be explained by an increased epitope exposure of the smaller aggregates created by the sonication. By analyzing human brain derived and recombinant (K18) Tau aggregates in a cellular FRET assay, it was observed that, in the absence of transfection reagent, sonicated aggregates showed higher aggregation induction. Preparations also showed altered profiles on native PAGE upon sonication and we could further separate different aggregate species based on their molecular weight via sucrose gradients.ConclusionsThis study further elucidates the molecular properties regarding relative aggregate size and seeding efficiency of sonicated vs. non-sonicated high molecular weight Tau species. This information will provide a better knowledge on how sonication, a commonly used technique in the field of study of Tau aggregation, impacts the aggregates. In addition, the description of PT76-based aggregation specific assay is a valuable tool to quantify K18 and human AD Tau fibrils.

Alzheimer\u2019s genetic risk factor FERMT2 (Kindlin-2) controls axonal growth and synaptic plasticity in an APP-dependent manner

Although APP metabolism is being intensively investigated, a large fraction of its modulators is yet to be characterized. In this context, we combined two genome-wide high-content screenings to assess the functional impact of miRNAs and genes on APP metabolism and the signaling pathways involved. This approach highlighted the involvement of FERMT2 (or Kindlin-2), a genetic risk factor of Alzheimer’s disease (AD), as a potential key modulator of axon guidance, a neuronal process that depends on the regulation of APP metabolism. We found that FERMT2 directly interacts with APP to modulate its metabolism, and that FERMT2 underexpression impacts axonal growth, synaptic connectivity, and long-term potentiation in an APP-dependent manner. Last, the rs7143400-T allele, which is associated with an increased AD risk and localized within the 3′UTR of FERMT2, induced a downregulation of FERMT2 expression through binding of miR-4504 among others. This miRNA is mainly expressed in neurons and significantly overexpressed in AD brains compared to controls. Altogether, our data provide strong evidence for a detrimental effect of FERMT2 underexpression in neurons and insight into how this may influence AD pathogenesis.

High\xa0Glucose and Hypoxia-Mediated Damage to Human Brain Microvessel Endothelial Cells Induces an Altered, Pro-Inflammatory Phenotype in BV-2 Microglia In Vitro

Diabetes is strongly linked to the development of Alzheimer’s disease (AD), though the mechanisms for this enhanced risk are unclear. Because vascular inflammation is a consistent feature of both diabetes and AD, the cerebral microcirculation could be a key target for the effects of diabetes in the brain. The goal of this study is to explore whether brain endothelial cells, injured by diabetes-related insults, glucose and hypoxia, can affect inflammatory and activation processes in microglia in vitro. Human brain microvascular endothelial cells (HBMVECs) were either treated with 5 mM glucose (control), 30 mM glucose (high glucose), exposed to hypoxia, or exposed to hypoxia plus high glucose. HBMVEC-conditioned medium was then used to treat BV-2 microglia. Alterations in microglia phenotype were assessed through measurement of nitric oxide (NO), cytokine production, microglial activation state markers, and microglial phagocytosis. HBMVECs were injured by exposure to glucose and/or hypoxia, as assessed by release of LDH, interleukin (IL)-1β, and reactive oxygen species (ROS). HBMVECs injured by glucose and hypoxia induced increases in microglial production of NO, tumor necrosis factor-α (TNFα) and matrix metalloproteinase (MMP)-9. Injured HBMVECs significantly increased microglial expression of CD11c and CLEC7A, and decreased expression of the homeostatic marker P2RY12. Finally, bead uptake by BV-2 cells, an index of phagocytic ability, was elevated by conditioned media from injured HBMVECs. The demonstration that injury to brain endothelial cells by diabetic-associated insults, glucose and hypoxia, promotes microglial inflammation supports the idea that the cerebral microcirculation is a critical locus for the deleterious effects of diabetes in the AD brain.

Spatial cell type composition in normal and Alzheimers human brains is revealed using integrated mouse and human single cell RNA sequencing

Single-cell RNA sequencing (scRNA-seq) resolves heterogenous cell populations in tissues and helps to reveal single-cell level function and dynamics. In neuroscience, the rarity of brain tissue is the bottleneck for such study. Evidence shows that, mouse and human share similar cell type gene markers. We hypothesized that the scRNA-seq data of mouse brain tissue can be used to complete human data to infer cell type composition in human samples. Here, we supplement cell type information of human scRNA-seq data, with mouse. The resulted data were used to infer the spatial cellular composition of 3702 human brain samples from Allen Human Brain Atlas. We then mapped the cell types back to corresponding brain regions. Most cell types were localized to the correct regions. We also compare the mapping results to those derived from neuronal nuclei locations. They were consistent after accounting for changes in neural connectivity between regions. Furthermore, we applied this approach on Alzheimer’s brain data and successfully captured cell pattern changes in AD brains. We believe this integrative approach can solve the sample rarity issue in the neuroscience.

Discovery and Functional Characterization of hPT3, a Humanized Anti-Phospho Tau Selective Monoclonal Antibody.

As a consequence of the discovery of an extracellular component responsible for the progression of tau pathology, tau immunotherapy is being extensively explored in both preclinical and clinical studies as a disease modifying strategy for the treatment of Alzheimer's disease. Describe the characteristics of the anti-phospho (T212/T217) tau selective antibody PT3 and its humanized variant hPT3. By performing different immunization campaigns, a large collection of antibodies has been generated and prioritized. In depth, in vitro characterization using surface plasmon resonance, phospho-epitope mapping, and X-ray crystallography experiments were performed. Further characterization involved immunohistochemical staining on mouse- and human postmortem tissue and neutralization of tau seeding by immunodepletion assays. Various in vitro experiments demonstrated a high intrinsic affinity for PT3 and hPT3 for AD brain-derived paired helical filaments but also to non-aggregated phospho (T212/T217) tau. Further functional analyses in cellular and in vivo models of tau seeding demonstrated almost complete depletion of tau seeds in an AD brain homogenate. Ongoing trials will provide the clinical evaluation of the tau spreading hypothesis in Alzheimer's disease.

GSK3‐ARC/Arg3.1 and GSK3‐Wnt signaling axes trigger amyloid‐β accumulation and neuroinflammation in middle‐aged Shugoshin 1 mice

The cerebral amyloid‐β accumulation that begins in middle age is considered the critical triggering event in the pathogenesis of late‐onset Alzheimer's disease (LOAD). However, the molecular mechanism remains elusive. The Shugoshin 1 (Sgo1−/+) mouse model, a model for mitotic cohesinopathy‐genomic instability that is observed in human AD at a higher rate, showed spontaneous accumulation of amyloid‐β in the brain at old age. With the model, novel insights into the molecular mechanism of LOAD development are anticipated. In this study, the initial appearance of cerebral amyloid‐β accumulation was determined as 15‐18 months of age (late middle age) in the Sgo1−/+ model. The amyloid‐β accumulation was associated with unexpected GSK3α/β inactivation, Wnt signaling activation, and ARC/Arg3.1 accumulation, suggesting involvement of both the GSK3‐Arc/Arg3.1 axis and the GSK3‐Wnt axis. As observed in human AD brains, neuroinflammation with IFN‐γ expression occurred with amyloid‐β accumulation and was pronounced in the aged (24‐month‐old) Sgo1−/+ model mice. AD‐relevant protein panels (oxidative stress defense, mitochondrial energy metabolism, and β‐oxidation and peroxisome) analysis indicated (a) early increases in Pdk1 and Phb in middle‐aged Sgo1−/+ brains, and (b) misregulations in 32 proteins among 130 proteins tested in old age. Thus, initial amyloid‐β accumulation in the Sgo1−/+ model is suggested to be triggered by GSK3 inactivation and the resulting Wnt activation and ARC/Arg3.1 accumulation. The model displayed characteristics and affected pathways similar to those of human LOAD including neuroinflammation, demonstrating its potential as a study tool for the LOAD development mechanism and for preclinical AD drug research and development.

Regional correlation of biochemical measures of amyloid and tau phosphorylation in the brain

Alzheimer’s disease (AD) neuropathologic change is characterized by amyloid plaques and neurofibrillary tangles (NFTs) that consist of aggregated amyloid beta (Abeta) and hyperphosphorylated tau proteins (p-tau), respectively. Although the global relationship between Abeta and p-tau has been studied for decades, it is still unclear whether a regional correlation exists between Abeta and p-tau in the human brain. Recent studies in cerebrospinal fluid (CSF) have suggested that tau phosphorylation at specific sites such as T217 is modified at an early stage of AD when amyloid plaques become detectable. We applied biochemical and mass spectrometry methods in human brain samples with and without Abeta plaque pathology to measure site-specific phosphorylation occupancies in soluble and insoluble tau. Our quantitative results identified multiple residues specifically hyper-phosphorylated in AD, including at sites T111, T153, S184 (or S185), T205, S208, T217, S262, and S285 in brain soluble tau. In contrast, the most enriched phosphorylated residues in brain insoluble tau were T111, S113, T153, T181, S199, S202, T205, T217, T231, S262, and S396. Tau phosphorylation occupancies in the insoluble fraction were relatively constant across brain regions, suggesting that tau has a consistent phosphorylation pattern once it has aggregated into NFTs. We did not find regional association between Abeta42 and insoluble tau. However, the phosphorylation profile of soluble tau in AD brain was highly correlated to that in AD CSF, which was analyzed in a previous study. We also found a higher regional association between total Abeta42 and soluble tau phosphorylation occupancy at residues T111, T153 and T217 in the brain. This study provides insights into regional interactions between amyloidosis and specific tau phosphorylated residues in the human brain and may explain the specific increases of tau species phosphorylation observed in AD CSF.

Phosphorylated A\u03b2 peptides in human Down syndrome brain and different Alzheimer\u2019s-like mouse models

The deposition of neurotoxic amyloid-β (Aβ) peptides in extracellular plaques in the brain parenchyma is one of the most prominent neuropathological features of Alzheimer’s disease (AD), and considered to be closely related to the pathogenesis of this disease. A number of recent studies demonstrate the heterogeneity in the composition of Aβ deposits in AD brains, due to the occurrence of elongated, truncated and post-translationally modified Aβ peptides that have peculiar characteristics in aggregation behavior and biostability. Importantly, the detection of modified Aβ species has been explored to characterize distinct stages of AD, with phosphorylated Aβ being present in the clinical phase of AD. People with Down syndrome (DS) develop AD pathology by 40 years of age likely due to the overproduction of Aβ caused by the additional copy of the gene encoding the amyloid precursor protein on chromosome 21. In the current study, we analysed the deposition of phosphorylated and non-phosphorylated Aβ species in human DS, AD, and control brains. In addition, deposition of these Aβ species was analysed in brains of a series of established transgenic AD mouse models using phosphorylation-state specific Aβ antibodies. Significant amounts of Aβ phosphorylated at serine residue 8 (pSer8Aβ) and unmodified Aβ were detected in the brains of DS and AD cases. The brains of different transgenic mouse models with either only human mutant amyloid precursor protein (APP), or combinations of human mutant APP, Presenilin (PS), and tau transgenes showed distinct age-dependent and spatiotemporal deposition of pSer8Aβ in extracellular plaques and within the vasculature. Together, these results demonstrate the deposition of phosphorylated Aβ species in DS brains, further supporting the similarity of Aβ deposition in AD and DS. Thus, the detection of phosphorylated and other modified Aβ species could contribute to the understanding and dissection of the complexity in the age-related and spatiotemporal deposition of Aβ variants in AD and DS as well as in distinct mouse models.