AD Pathogenesis Research Articles

Insights into Novel Therapies and the Pathogenesis of Alzheimer's disease, and the Possible Role of COVID-19

The Wisniewski laboratory has helped develop novel biomarkers and therapeutic approaches to AD, particular immunotherapeutic approaches that affect both the adaptive and innate immune systems. We have developed a means to stimulate innate immunity to ameliorate AD pathology, which we have tested in aged squirrel monkeys, and now are conducting a phase I clinical trial. We have also developed both active and passive immunization that specifically targets the dominant β-sheet secondary structure of multiple toxic oligomers concurrently (including both Aβ and tau oligomers). Recently we have also documented the neurological manifestations of SARS-CoV-2 infection (in particular cognitive dysfunction) and associated biomarkers of neurodegeneration and neuroinflammation. We have also developed an unbiased proteomic methodology that produces robust data utilizing archival formalin, fixed paraffin embedded human tissue, using this method to perform the most extensive proteomic analysis of amyloid plaques and the phosphorylated tau interaction, as well as to characterize the amyloid proteome of distinct subtypes of AD including rapidly progressive AD and AD in Down syndrome. These studies have helped enhance the understanding of the pathogenesis of AD, and also with the development of novel therapies.

The association between BCG treatment in patients with bladder cancer and subsequent risk of developing Alzheimer and other dementia.—A Swedish nationwide cohort study from 1997 to 2019

Background Alzheimer’s disease (AD) affects 50 million people worldwide. The immune system plays a major role in the pathogenesis of AD. Several retrospective analyses have reported a decreased risk of AD and other dementias in bladder cancer patients treated with immunotherapy in the form of Bacillus Calmette-Guerin (BCG) bladder instillations. We tested this hypothesis in a Swedish population-based prospective cohort of patients with non-muscle invasive bladder cancer (NMIBC). Methods and findings We utilized the BladderBaSe 2.0 database, which contains tumor-specific, health-related, and socio-demographic information for patients diagnosed with NMIBC between 1997 and 2019. The database also includes a matched comparison cohort sampled from the general population, consisting of individuals free from urinary tract cancer at the time of the index case’s diagnosis. Five controls were randomly selected for each index case without replacement on the date of the index case’s diagnosis. Our inclusion criteria identified participants diagnosed with NMIBC who had received BCG as primary treatment, along with their corresponding comparison cohort. We excluded those diagnosed with dementia before or within 6 months of NMIBC diagnosis. To compare the NMIBC cohort with their matched comparison cohort, we used a stratified Cox model, treating each case with its controls as a stratum. We identified 38,934 patients in the NMIBC cohort, with 6,496 receiving BCG after primary diagnosis (cases). AD/dementia was diagnosed during follow-up in 6.1% of cases and 7.4% of controls. Cases had a slightly lower risk of dementia than controls, with a hazard ratio (HR) of 0.88 (95% confidence interval [CI] 0.780–0.991), and a HR of 0.89 (CI 0.703–1.119) for AD. Subgroup analysis for dementia showed that age over 75 years had an HR of 0.73 (CI 0.616–0.863), and female gender had an HR of 0.73 (CI 0.552–0.971). The associations were similar for AD specifically, but not statistically significant. Similar to previous studies, we analyzed bladder cancer patients treated with and without BCG therapy. Multivariate Cox analysis indicated that those treated with BCG had a lower risk of dementia (HR 0.81, 95% CI 0.71–0.92), and an HR of 0.98 (95% CI 0.75–1.27) for AD specifically. High age was a significant risk modifier; the HR was 3.8 (CI 3.44–4.11) for dementia and 3.1 (CI 2.59–3.73) for AD. Even patients not receiving BCG had a significantly lower risk for AD than controls (HR 0.86, CI 0.77–0.96). Conclusions This study observed a marginally decreased risk of developing AD/dementia associated with earlier intravesical BCG treatment in NMIBC patients. This small benefit was most pronounced in those with high age and female gender. The disparity from previous highly positive studies underscores the importance of using an appropriate control cohort.

Ethanol extract of Andrographis paniculata alleviates aluminum-induced neurotoxicity and cognitive impairment through regulating the p62-keap1-Nrf2 pathway

BackgroundAlzheimer’s disease (AD) is the most prevalent neurodegenerative and remains incurable. Aluminum is a potent neurotoxin associated with AD. The main pathological features of AD are extracellular amyloid-β protein deposition and intracellular hyperphosphorylated Tau protein. A body of evidence suggest that oxidative stress and autophagy are involved in the pathogenesis of AD. Andrographis paniculata (AP) is a native plant with anti-inflammatory, anti-oxidative stress, and regulation of autophagy properties. AP significantly alleviated cognitive impairments, reduced Aβ deposition and has neuroprotective effect. However, its effects on aluminum-induced AD model have not been studied much. In this study, we investigated whether AP protect against aluminum-induced neurotoxicity through regulation of p62-Kelch-like ECH-associated protein 1(Keap1)-Nuclear factor E2 related factor 2 (Nrf2) pathway and activation autophagy in vivo and in vitro.MethodsUPLC-ESI-qTOF-MS/MS was used to identify the chemical constituents of AP ethanol extract. The mice with cognitive deficit were established by injecting aluminum chloride and D-galactose, and treated with either AP extract (200, 400, or 600 mg/kg/d) or andrographolide (2 mg/kg/2d).The spatial memory ability was detected by Morris water maze, HE staining were used to detect in brain tissue,Oxidative stress indexs and SOD activity in both serum and brain tissue were detected by kit.The expression of p62-Nrf2 pathway proteins were measured via western blotting. Furthermore, the neurotoxicity model was induced by aluminum maltolate (700 µM) in PC12 cells. Following AP and andrographolide treatment, the cell viability was detected. The relevant mRNA and protein expressions were detected in cells transfected with the p62 siRNA.ResultsThe main active components of AP included andrographolide, neoandrographolide and deoxyandrographolide as identified. AP and andrographolide significantly improved the spatial memory ability of mice, attenuated pathological changes of hippocampal cells, reduced the level of malondialdehyde, and increased superoxide dismutase activity in serum or brain tissue as compared to model control. In addition, the Nrf2, p62 and LC3B-II proteins expression were increased, and p-Tau and Keap1 proteins were decreased in the hippocampus after AP and andrographolide treatment.Furthermore, AP increased aluminum maltolate-induced cell viability in PC12 cells. Silencing p62 could reverse the upregulation expression of Nrf2 and downregulation of Keap1 and Tau proteins induced by AP in aluminum maltolate-treated cells.ConclusionsAP had neuroprotective effects against aluminum -induced cognitive dysfunction or cytotoxicity, which was involved in the activation of the p62-keap1-Nrf2 pathway and may develop as therapeutic drugs for the treatment of AD. However, this study has certain limitations, further optimize the protocol or model and study the molecular mechanism of AP improving AD.

Differences in the proteomic profile of the brain in Alzheimer’s disease in Down syndrome and early‐onset Alzheimer’s disease

AbstractBackgroundDown syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and is a leading genetic cause of Alzheimer’s disease (AD‐DS). People with DS have a third copy of amyloid precursor protein (APP) which causes the increased risk to develop AD neuropathology and dementia, decades earlier than in the general population. However, other genes on Hsa21 have been shown to modify AD‐DS development compared to the development of AD in the general population. Here, we aim to understand how AD‐DS differs to EOAD at end‐stage disease.MethodLabel‐free mass spectrometry was performed using a SYNAPT G2‐Si High‐Definition machine with 2D fractionation on prefrontal cortex tissue (BA10) from individuals with AD‐DS, early‐onset AD (EOAD) (both Braak‐stage VI), and healthy age‐matched controls. Proteins identified were ranked by significance (p < 0.05) and a fold‐change of difference between groups was calculated. qPCR was used to measure transcript levels of altered proteins in the same samples and to assess if transcript levels of raised proteins were altered in the Dp1Tyb (MGI:5703798) mouse model of DS which has three‐copies of ∼148 Hsa21 orthologous genes.ResultRaised abundance of Hsa21 encoded proteins, including PDXK, APP and S100B, were identified in individuals with AD‐DS compared to EOAD. Importantly, non‐chromosome 21 encoded proteins were also shown to be raised in individuals with AD‐DS compared to EOAD, including APOE, NLRP8 and PTN. Raised APOE protein in AD‐DS compared to EOAD brain tissue is Apolipoprotein E genotype independent. Apoe is not raised in the Dp1Tyb hippocampus or cortex at 6‐months of age.ConclusionPeople with AD‐DS exhibit an altered proteomic profile compared to people with EOAD, likely driven by having three‐copies of Hsa21 genes. Raised APOE may be driven by an interaction of trisomy 21 and AD pathogenesis, as this phenotype is not seen in the Dp1Tyb mouse model. Understanding the differences in AD pathogenesis is key for including people with DS in future AD clinical trials. Future work will validate protein expression of candidates of interest in an independent cohort of human post‐mortem brain samples, as well as determine the cell types in which proteins of interest are expressed.

Ferroptosis in the Pathogenesis of Alzheimer’s Disease: The New Evidence for Validation of FAB Model

Alzheimer’s disease is an age-associated progressive disorder, characterized by neurodegeneration and following cognitive decline. Several pathological alterations are implicated in its pathogenesis, hence etiology is still poorly understood. Ferroptosis is an alternative form of cell death, driven by intracellular accumulation of iron with subsequent reactive oxygen species formation, which damages membranes, proteins, and DNA, causing cell death. The imbalance in iron homeostasis is rapidly gaining weight as a neurodegeneration cause, increasing the need to develop in vivo and in vitro models to understand the role of ferroptosis in Alzheimer’s disease pathogenesis. This review focuses on the mechanisms of ferroptosis in the pathogenesis of AD, giving a detailed overview of the available in vivo and in vitro methods and their applications, as well as describing in detail the ferrous amyloid buthionine (FAB) model.

Roles of HDAC6 in the pathogenesis of Alzheimer’s disease

AbstractBackgroundIt has recently been demonstrated that HDAC6 depletion ameliorates the impairment of memory function and mitochondrial axonal transport induced by the accumulation of Aβ. However, very little is known about molecular mechanisms through which HDAC6 regulates APP processing. Also, it remains controversial whether HDAC6 induces inflammation or reduces it.MethodWe analyzed the expression of HDAC6 in AD patients and 5xFAD mice. We confirmed that HDAC6 regulates BACE1 in primary neurons and NLRP3 inflammasome in primary microglia. And we crossed HDAC6 knock‐out mice with 5xFAD mice and got 5xFAD/HDAC6 KO. We compared 5xFAD (n = 11) and 5xFAD/HDAC6 KO (n = 13). We did a nest building test, open field test, Morris water maze, Y‐maze, passive avoidance, and elevated plus maze. After finishing all these behaviour tests, we sacrificed the mouse and checked protein and mRNA. We did Immunohistochemistry using Aβ plaque, NLRP3 inflammasome and synapse‐related markers. We also did RNA‐seq analysis in these mice’s brains.ResultWe confirmed that the expression of HDAC6 was increased in the AD cortex compared to neuropathologically normal brains. The stability of BACE1 is regulated by its C‐terminal lysine, and HDAC6 deacetylates the C‐terminal lysine of BACE1 by direct binding. Also, HDAC6 deficiency reduced NLRP3 inflammasome activation‐, and subsequent inflammatory responses in microglia. By crossing HDAC6 knock‐out mice with 5xFAD mice, the role of HDAC6 was confirmed in an animal model of AD. HDAC6 deletion improved cognitive function and reduced the protein expression of BACE1 and inflammatory factors in 5xFAD mice. Also, HDAC6‐deficient 5xFAD mice developed fewer dense‐core plaques, IL‐1β, and ASC specks than 5xFAD mice. Furthermore, RNA‐sequencing was analyzed from the hippocampus of 5xFAD and 5xFAD/HDAC6 KO mice. Synapse and neuron development‐related genes were significantly up‐regulated, and type I interferon, immune response, and prion disease‐related genes were down‐regulated in HDAC6‐depleted mice. Finally, we confirmed that the ratio of PSD95 colocalized with C1q was significantly decreased in 5xFAD/HDAC6 KO compared to 5xFAD mice, which suggested that synapse loss was inhibited in AD mice.ConclusionThis study reveals distinct functions of HDAC6 and suggests HDAC6 as a significant regulator in the pathogenesis of AD that could be exploited therapeutically.

Differential Microglial activation and Astrocyte reactivity in Alzheimer’s trajectory

AbstractBackgroundMicroglial activation and astrocyte activation plays a significant role in the pathogenesis of AD. Here, we evaluate microglial activation, the interrelationship between microglial activation, amyloid and tau pathologies, and the impact of microglial activation in Alzheimer’s disease trajectory. We specifically focus on microglia as a major player in neuroinflammation and evaluate the spatial and temporal variation of microglial activation in the disease trajectory. Additionally, we evaluate astroglial activation using novel astrocyte marker and evaluate the relationship between amyloid, MRI volume changes and glucose metabolism in Alzheimer’s trajectory.MethodWe evaluated AD, MCI and cognitively normal subjects using amyloid PET, tau PET and neuroinflammation using TSPO PET scans. A separate group of AD, MCI and control subjects were evaluated using novel astrocyte marker [11C]BU99008 PET along with [18F]FDG PET and amyloid PET. [11C]PBR28 and [11C]BU99008 PET scans were analysed using two tissue compartmental model, while [18F]Flutemetamol was analysed using target to cerebellar ratio. [18F]FDG PET was analysed using pons as a reference region. We performed regional and voxel wise analysis for all the tracers.ResultMicroglial activation was elevated in different cortical regions in both AD and MCI subjects. Microglial activation was associated with lower grey matter density in later stages of the disease, while higher grey matter volume was associated with increased microglial activation in early mild cognitive impairment subjects. There were areas of increased astrocyte activation in both AD and MCI. Higher microglial activation was associated with increased tau formation and neuronal damage in later stages of the disease. Interestingly, there was increased astrocyte reactivity in the early stages of the disease.ConclusionWhile microglial activation in the late phase of the disease is associated with neuronal damage, however, in the early stages microglial activation was associated with higher grey matter volume. Microglial activation demonstrated a biphasic pattern of activation. In contrast to microglial activation, astrocyte activation was prominent in the early stage of the disease and may be associated with neuroprotection. This suggests that in targeting glial activation as a therapeutic strategy, we must target astrocyte and microglia separately and in different stages of the disease.

The AI‐based AlphaFold 2 algorithm predicts an open structure for tauC3

AbstractBackgroundTauC3 is an especially noxious C‐terminal tau fragment produced by caspase‐3 cleavage at Asp421. Two decades of accumulating evidence support its importance in the pathogenesis of AD and other tauopathies, notably FTD and PSP. TauC3 promotes aggregation and filament formation, recruits full length tau (FLT) to incipient fibrils, and despite low concentration in AD brain tissue relative to total tau, has been reported to account for substantially all the seeding activity in AD brain extracts. TauC3 has also been shown to slow fast axonal transport (FAT), with deleterious consequences. Although tau’s largely disordered structure prevents analysis by X‐ray crystallography, FRET analysis suggested the existence of a hairpin conformation in which the N‐terminus folds over the C‐terminus close to the MTBR. In this model, Ser432 is the point of closest contact with Pro301. Opening of the hairpin has been hypothesized to 1) make FLT prone to aggregation by exposing the MTBR, and 2) impair FAT by overexposing the phosphatase activating domain near the N‐terminus. We postulated that tauC3 cannot form the hairpin because it lacks Ser432 and thus has an “open” conformation. This would explain tauC3’s substantially increased pathogenicity compared to FLT. The AI‐based tool AlphaFold 2 (AF2) is being used to predict the conformation of many proteins with great accuracy. Applying it to FLT and tauC3 could provide valuable data about the hairpin and whether caspase cleavage alters it.MethodWe tested AF2’s ability to predict the conformation of 4‐repeat FLT and tauC3 from FASTA sequences, aligning the models produced with the MTBR.ResultAF2 modeling revealed a dramatically different conformation for tauC3 compared to FLT, specifically, an open conformation with the MTBR and N‐terminus far apart (Figure). Interestingly, it also revealed that the entire C‐terminus region of FLT starting at Gln423 is composed of a single α‐helix.ConclusionAF2 predictions support the existence of the hairpin conformation for tau and underscore the critical importance of residue 432 in maintaining it. The extended C‐terminus α‐helix may have functional implications. TauC3’s open conformation is consistent with its slowing of FAT and its ability to seed aggregation even at low concentrations.

Spatial Extent as a more sensitive amyloid biomarker for early stages of Alzheimer’s disease

AbstractBackgroundDespite considerable advances in beta amyloid (Aβ)‐PET imaging over the last decade, the standard approach of estimating the average neocortical Aβ burden remains largely unchanged. However, as research and clinical trials increasingly shift earlier in the disease process, measures of how far Aβ has spread throughout the cortex (spatial extent) may prove more sensitive than average neocortical magnitude for detecting and quantifying early Aβ deposits and their association with future tau proliferation and cognitive decline.MethodClinically‐normal individuals (n = 214) were included from the Harvard Aging Brain Study with longitudinal PIB‐PET (2‐4 scans, median = 4.7±2.7 years) and annual cognitive data (median = 5.2±2 years), as well as a subset (n = 181) with flortaucipir (FTP)‐PET. Spatial extent (EXT) was computed as the number of cortical ROIs (n = 62, Desikan atlas) above their ROI‐specific threshold for Aβ positivity. EXT was compared to a traditional mean neocortical DVR using logistic growth modeling. Receiver Operator Characteristic (ROC) curve analyses evaluated EXT’s ability to identify baseline PIB‐ individuals (<1.19DVR/24CL) who progressed to PIB+ at 3‐year follow‐up. Linear Mixed Effects (LME) modeling assessed baseline EXT (or DVR) as a predictor of increasing inferior temporal tau (IT FTP SUVR) and cognitive decline on the Preclinical Alzheimer’s Cognitive Composite (PACC).ResultEXT begins rising below the neocortical DVR threshold (Figure1A), reaching a maximal growth rate of 14 ROIs per 0.1 DVR increase (∼10CL) and plateauing as full cortical EXT is achieved starting at ∼1.5DVR/68CL. A 3‐ROI EXT threshold predicts progression from PIB‐ to PIB+ in 3 years (AUC = .97, SE = .82,SP = .97), outperforming neocortical DVR (AUC = .92, SE = .65,SP = .94, Figure1B). EXT provides a stronger biomarker of Aβ change than DVR (lower coefficient of variation, Figure2) across the Aβ continuum due to its low variance, even after EXT has plateaued. Baseline EXT is also a stronger predictor of increases in IT FTP SUVR (Figure 3A, η2 EXT = .25, η2 DVR = .20) and PACC decline (Figure3B, η2 EXT = .28, η2 DVR = .22).ConclusionBy describing the spread of Aβ throughout the cortex rather than average neocortical Aβ burden, spatial extent provides a more sensitive measure of Aβ at early, preclinical stages of AD that may improve design of AD prevention trials and open new avenues for research into AD pathogenesis.

Multiomics of iPSC‐derived ApoE3‐ and ApoE4 astrocytes and microglia uncovers cell type specific dysregulation of immunometabolism

AbstractBackgroundApoE4 is the major genetic risk factor for Alzheimer’s disease and the main lipid transporter in brain. How ApoE4 drives AD pathogenesis, and how this is related to changes in lipid metabolism, is not well understood. This lack of mechanistic insight has hampered the development of interventions that can reverse the pathological effects of ApoE4.MethodsHere we perform integrated multiomic analysis (lipidomics, metabolomics, transcriptomics and proteomics) on isogenic iPSC‐derived glia carrying an ApoE4/4, ApoE3/3 or ApoE‐knockout genotype. We employ pathway enrichment to identify candidate biological processes that contribute to AD pathogenesis and perform a number of biochemical‐ and intervention experiments to gain insight into the biological mechanism. We feed iPSC‐derived glia with different lipid species to better understand the interaction between lipids and immune dysfunction. Lastly, we generate a new open‐access resource (the Neurolipid atlas), to allow others in the field to interact with our lipid data.ResultsOur lipidomic analysis shows that ApoE4 has a major effect on lipid metabolism in human iPSC‐derived astrocytes and microglia. In both astrocytes and microglia, ApoE4 causes accumulation of cholesteryl esters (CE), triglycerides (TAG) as well changes in ceramide metabolism. Interestingly, transcriptomic and proteomic analysis in both cell types shows that ApoE4 glia have strongly upregulated cholesterol synthesis despite having already high levels of stored cholesterol (CE). Astrocytes and microglia show very distinct adaptations to ApoE4 in terms of energy production. Whereas ApoE4 astrocytes have higher levels of mitochondrial proteins, ApoE4 microglia downregulate mitochondrial proteins but have increased levels of key glycolytic proteins. Immune profiles, including DAM signatures in microglia, are changed in a similar direction in both ApoE4 astrocytes and microglia. Lipid feeding also directly affect these immune pathways.ConclusionOur data indicate that astrocytes and microglia display both common and distinct immunometabolic adaptations to the ApoE4 genotype. Interestingly, both ApoE4 microglia and astrocytes have increased stored cholesterol yet keep upregulating cholesterol synthesis, indicating a possibly pathogenic broken feedback loop. We also show that this altered lipid metabolism is tightly coupled to immune dysfunction and propose novel mechanisms and interventions that may normalize ApoE4 dysfunction in human glia in AD.

Neural responses to peripheral infection in Alzheimer’s disease model mice

AbstractBackgroundClinical and animal studies have suggested that peripheral inflammation affects the central nervous system (CNS) and may be a major factor in the pathophysiology of neurodegenerative diseases like AD. Furthermore, a variety of signaling factors originating in the periphery, such as inflammatory mediators, have been associated with the regulation of brain barrier functions. Here, we hypothesized that the presence of peripheral inflammation dysregulates brain barriers’ integrity and induces neuroinflammation with consequences on amyloid pathology.MethodIn this study, we employed an AD mouse model nasally infected with Staphylococcus aureus to assess the impact of chronic or acute peripheral inflammation on brain transcriptome and amyloid pathology. We utilized both single cell and Spatial Transcriptomics to better determine transcriptional response to peripheral inflammation.ResultThe chronic exposure increased the diffuse and compact amyloid plaques and blood cytokine levels. Following a short‐term exposure, single‐cell and spatial transcriptomics uncovered cell type‐ and spatial‐specific transcriptional changes indicating a dysregulation of the brain barriers, including the blood‐brain and the blood‐cerebrospinal fluid barriers. Brain macrophages exhibited increased Apoe expression and macrophage‐specific genes were upregulated at ventricular areas of infected mice. In addition, we report an increase of disease associated microglia genes around the Ab plaques, together with disbalances in neuronal expression in response to peripheral inflammation.ConclusionWe observed that low‐grade peripheral infection triggers transcriptional responses linked to brain barriers and brain infiltrated/resident macrophages indicate a fundamental importance of them in the mechanisms linking peripheral inflammation and AD pathogenesis. Finally, we showed that bacterial infection caused molecular changes restricted to specific cell types and/or brain spatial microenvironments, which highlights the relevance of studying brain molecular responses with high resolution in order to have new insights on AD etiology.

Capturing polygenic risk for Alzheimer’s disease using induced pluripotent stem cells

AbstractBackgroundOver 70 loci have been implicated in AD pathogenesis through genome‐wide association studies. Understanding of how these genetic variants interact with one another, as well as with unknown genetic variants of weak effect size to impinge upon biological processes and molecular pathway to affect risk and resilience to AD is key to unlocking appropriate therapeutic strategies for AD.MethodWe have generated iPSC lines from over 50 deceased individuals in the Religious Order Study (ROS) or Rush Memory and Aging Project (MAP) that span the clinical and neuropathological spectrum of aging. About one third of these individuals had clinical and neuropathological diagnoses of AD and Alzheimer’s dementia. We differentiated these lines into cortical neuronal fates, astrocyte fates and microglial fates and measured RNA and protein profiles as well as quantitative measures of APP cleavage products and tau species. In addition, we have measured autophagic and proteasome capacity, as well as synatic vesicle release and cytokine production.ResultWe identify specific proteins and pathways in neuronal and glial cultures that are associated with AD polygenic risk score (PRS), neuropathological burden, and/or cognitive trajectory in the donors and validate these findings through analyses of brain tissue. We identify an association of cognitive decline and measures of specific amyloid beta peptides and phospho‐tau species in iPSC‐derived neurons. We find that proteasome capacity and autophagic flux are influenced by genetic background, which in turn affects tau proteostasis. Intriguingly, different genetic backgrounds respond differentially to modulation of these protein degradation networks.ConclusionThe data presented establish this iPSC collection as a resource for investigating person‐specific processes in the brain that are encoded in part by polygenic risk for AD that can aid in identifying and validating molecular pathways underlying AD.

Assessing amyloid plaque burden in aging marmosets and genetically engineered PSEN1 mutation carriers using 11C‐PiB PET

AbstractBackgroundThere is an urgent need to develop novel animal models to investigate the underlying cellular and molecular root causes of the pathogenesis and progression of AD. Our group has worked to establish the common marmoset as the first primate‐specific model of AD to reveal the earliest cellular and molecular events of AD processes and allow charting of AD progression from its inception. For this, we established a colony of outbred marmosets and used genetic engineering techniques to generate marmosets carrying the PSEN1‐C410Y and PSEN1‐A426P mutations that cause early‐onset, familial AD. The present study aims to use PET imaging to access and track the progression of Ab plaque burden non‐invasively and longitudinally in our marmoset models of AD.MethodMarmosets anesthetized with isoflurane delivered via a facemask were placed in a preclinical PET/CT scanner customized with marmoset‐specific beds. Respiratory rate, heart rate, arterial O2 saturation, and rectal temperature were monitored and maintained at normal physiological values. Our PET imaging protocol follows the Alzheimer’s Disease Neuroimaging Initiative (ADNI). The amyloid‐binding 11C‐Pittsburgh Compound B (11C‐PiB) was delivered intravenously in doses varying from 18.5 – 185 MBq (0.5 – 5 mCi). A 90‐min dynamic acquisition was performed immediately following the radiotracer injection, followed by a 3D CT scan acquired at 200 µm isotropic resolution. After the PET/CT neuroimaging scan, the marmosets were recovered from anesthesia and returned to their home cages.ResultWe observed positive 11C‐PiB binding and retention in the brains of aging outbred marmosets and in marmosets harboring PSEN1 mutations. Significant 11C‐PiB retention was observed in the cortex and white matter tracts. The areas with the highest 11C‐PiB retention were the lateral frontal cortex, anterior cingulate cortex, and temporal lobe regions.ConclusionThis study demonstrates using 11C‐PiB to track Ab plaque burden in the brains of aging marmosets and marmosets harboring PSEN1 mutations. As observed in human patients of AD, the amount of 11C‐PiB bound (measured in SUV and SUVR units) varied from animal to animal, and we are currently performing group analysis to investigate the dependence of 11C‐PiB retention on age and sex. This research was funded by NIH/NIA grants R24AG073190 and U19AG074866.

Assessing amyloid plaque burden in aging marmosets and genetically engineered PSEN1 mutation carriers using 11C‐PiB PET

AbstractBackgroundThere is an urgent need to develop novel animal models to investigate the underlying cellular and molecular root causes of the pathogenesis and progression of AD. Our group has worked to establish the common marmoset as the first primate‐specific model of AD to reveal the earliest cellular and molecular events of AD processes and allow charting of AD progression from its inception. For this, we established a colony of outbred marmosets and used genetic engineering techniques to generate marmosets carrying the PSEN1‐C410Y and PSEN1‐A426P mutations that cause early‐onset, familial AD. The present study aims to use PET imaging to access and track the progression of Ab plaque burden non‐invasively and longitudinally in our marmoset models of AD.MethodMarmosets anesthetized with isoflurane delivered via a facemask were placed in a preclinical PET/CT scanner customized with marmoset‐specific beds. Respiratory rate, heart rate, arterial O2 saturation, and rectal temperature were monitored and maintained at normal physiological values. Our PET imaging protocol follows the Alzheimer’s Disease Neuroimaging Initiative (ADNI). The amyloid‐binding 11C‐Pittsburgh Compound B (11C‐PiB) was delivered intravenously in doses varying from 18.5 – 185 MBq (0.5 – 5 mCi). A 90‐min dynamic acquisition was performed immediately following the radiotracer injection, followed by a 3D CT scan acquired at 200 µm isotropic resolution. After the PET/CT neuroimaging scan, the marmosets were recovered from anesthesia and returned to their home cages.ResultWe observed positive 11C‐PiB binding and retention in the brains of aging outbred marmosets and in marmosets harboring PSEN1 mutations. Significant 11C‐PiB retention was observed in the cortex and white matter tracts. The areas with the highest 11C‐PiB retention were the lateral frontal cortex, anterior cingulate cortex, and temporal lobe regions.ConclusionThis study demonstrates using 11C‐PiB to track Ab plaque burden in the brains of aging marmosets and marmosets harboring PSEN1 mutations. As observed in human patients of AD, the amount of 11C‐PiB bound (measured in SUV and SUVR units) varied from animal to animal, and we are currently performing group analysis to investigate the dependence of 11C‐PiB retention on age and sex.This research was funded by NIH/NIA grants R24AG073190 and U19AG074866.

NEUROPROTECTIVE EFFECT OF PALIPERIDONE IN LPS INDUCED ANIMAL MODEL OF AD

AbstractBackgroundAlzheimer’s disease is a progressive neurological condition, that impairs cognition and memory. Extracellular beta‐amyloid plaques and intracellular neurofibrillary tangle formation are the two hallmarks while cognitive decline, neurotransmitter deficit, neuroinflammation, mitochondrial dysfunction, decreased cerebral glucose uptake, and oxidative stress has been known to contribute to the pathogenesis of AD. The present study was designed to investigate the neuroprotective potential of Paliperidone on intracerebroventricular (i.c.v.) Lipopolysaccharide‐induced experimental dementia of Alzheimer’s type.MethodLPS was administered by the i.c.v route on 1st day of the study in rats. Paliperidone was administered (1mg/Kg and 2mg/kg/day i.p) from the 1st to 21st day in LPS‐infused rats. Morris water maze, open‐field test, and object recognition parameters were used to assess learning, memory, behavioral changes and locomotor activity in rats. LPS infusion rapidly causes neuroinflammation and oxidative‐nitrosative stress.ResultThese markers were determined in the cortical and hippocampal brain regions of rats. While paliperidone treatment attenuates the LPS‐induced behavioral and biochemical abnormalities in rats by reducing levels of Malondialdehyde (MDA), reduced glutathione (GSH), and increasing levels of catalase. The neurodegeneration in Hippocampus and cortex regions was observed through histopathological examination. The observed cognitive improvement following paliperidone in LPStreated rats may be due to its antioxidant and anti‐inflammatory activity and restoration of cholinergic functions.ConclusionOur results suggest the therapeutic potential of paliperidone in cognitive disorders such as AD. However, more investigations are required to evaluate the potential of paliperidone in boosting memory and cognition and preventing neuronal damage.

Tracking the intracellular itinerary of APP and de novo Aβ generation using click chemistry

AbstractBackgroundAmyloid‐beta (Aβ), is generated by sequential cleavage of the amyloid precursor protein (APP) by beta‐ and gamma‐secretase. The intracellular sites of cleavage are unknown due to the lack of tools that can detect de novo Aβ production.MethodUsing unnatural amino acid (UAA) mutagenesis and click chemistry, a single fluorophore (magenta) site‐specifically binds to Aβ within the APP construct. The C‐terminus of APP is fused with the green fluorescent protein mClover, allowing for a distinction between full‐length APP and Aβ, as the uncleaved APP will appear white (magenta+green) from the bifluorescence, contrasting with the magenta of Aβ alone when separated from the green mClover C‐terminus. Live cells expressing the APP‐mClover construct (green) were incubated with cell‐impermeable tetrazine‐AlexaFluor 594 (magenta) at 4°C, which click‐labeled the UAA on Aβ on cell surface APP. The cells were then incubated at 37°C, resuming endocytosis of bifluorescent APP.ResultAfter click‐labeling, the APP construct resides exclusively at the cell surface. Once warmed to 37°C, the intracellular bifluorescence increases with time, suggesting APP endocytosis (Fig 1). Using object‐based analyses, the magenta click‐label:green mClover ratio was determined from super‐resolution z‐stack images (Fig 2). The magenta:green ratio was lower in cells treated with gamma‐secretase inhibitor (GSI).ConclusionA qualitative and semi‐quantitative picture can be created of Aβ’s localization by following the separation of the bifluorescent signal into intraluminal magenta click‐label and membrane‐bound green mClover. This technology will be coupled with immunocytochemistry of subcellular markers, which will allow for further specificity on the location of Aβ travel within a cell, with regards to the endolysosomal pathway. Furthermore, this technology can be applied to examine Aβ within the unique cytoarchitecture of primary neurons which may help inform the design of new therapeutics in this cell type central to AD pathogenesis.

C/EBPβ/δ‐secretase Signaling Drives Neurodegenerative Diseases

AbstractBackgroundC/EBPb/AEP pathway are escalated in the brain in an age‐dependent manner and proteolytically cleaves APP and Tau and a‐Synuclein, facilitating AD and PD pathologies.MethodC/EBPb/AEP pathway couples different risk factors that elicit inflammation to Alzheimer’s disease. To explore why women are more vulnerable for AD, we investigated different hormones altered during menopause and found FSH (follicle‐stimulating hormone) strongly activated this signalling, leading to AD pathogenesis in different mouse models. Employing immuno‐EM and knockout tissues, we demonstrated that FSHR is expressed in mouse and human brain neurons. Notably, FSH administration drives AD onset in young male and female mice, whereas anti‐FSH robustly diminishes AD progression in overiactomized female 3xTg mice. Moreover, deletion of FSHR or C/EBPb from the neurons of 3xTg mice attenuates AD pathologies.ResultElevated FSH during menopause activates C/EBPb/AEP signalling, which subsequently cleaves both APP and Tau, triggering AD onset. Blockade of FSH abrogates overiactomy‐induced AD pathologies and restores the cognitive dysfunctions in AD mouse models.ConclusionFSH elevation after menopause elicits women susceptibility to AD via activating C/EBPb/AEP pathway. Inhibition of FSH/FSHR may provide an innovative therapeutic strategy for treating AD.

The gingipain inhibitor atuzaginstat and results of the GAIN trial

AbstractThe gingipain inhibitor atuzaginstat is a novel protease inhibitor that blocks a neurotoxic virulence factor known to trigger AD pathogenesis. Because the link between periodontal disease and AD is mechanistic and not incidental, it seems logical to develop drugs that target the virulence factors of P. gingivalis infection, such as gingipains. Atuzaginstat is appealing because it is orally bioavailable and blood–brain barrier penetrant. Clinical trials of atuzaginstat have been completed, with no evidence of amyloid‐related imaging abnormalities. However, a hepatotoxic signal has emerged that requires monitoring and could potentially influence further development of this potential treatment. Since the patients most likely to benefit from the results of the clinical trials underway are those with mild‐to‐moderate AD, more studies are being planned. Atuzaginstat can address the ongoing need for treatment for most patients for whom anti‐amyloids are not suitable.

Cortical gray to white matter signal intensity ratio as a sign of neurodegeneration and cognition independent of β-amyloid in dementia.

Cortical gray to white matter signal intensity ratio (GWR) measured from T1-weighted magnetic resonance (MR) images was associated with neurodegeneration and dementia. We characterized topological patterns of GWR during AD pathogenesis and investigated its association with cognitive decline. The study included a cross-sectional dataset and a longitudinal dataset. The cross-sectional dataset included 60 cognitively healthy controls, 61 mild cognitive impairment (MCI), and 63 patients with dementia. The longitudinal dataset included 26 participants who progressed from cognitively normal to dementia and 26 controls that remained cognitively normal. GWR was compared across the cross-sectional groups, adjusted for amyloid PET. The correlation between GWR and cognition performance was also evaluated. The longitudinal dataset was used to investigate GWR alteration during the AD pathogenesis. Dementia with β-amyloid deposition group exhibited the largest area of increased GWR, followed by MCI with β-amyloid deposition, MCI without β-amyloid deposition, and controls. The spatial pattern of GWR-increased regions was not influenced by β-amyloid deposits. Correlation between regional GWR alteration and cognitive decline was only detected among individuals with β-amyloid deposition. GWR showed positive correlation with tau PET in the left supramarginal, lateral occipital gyrus, and right middle frontal cortex. The longitudinal study showed that GWR increased around the fusiform, inferior/superior temporal lobe, and entorhinal cortex in MCI and progressed to larger cortical regions after progression to AD. The spatial pattern of GWR-increased regions was independent of β-amyloid deposits but overlapped with tauopathy. The GWR can serve as a promising biomarker of neurodegeneration in AD.

Bone Marrow-Derived GCA+ Immune Cells Drive Alzheimer's Disease Progression.

Alzheimer's disease (AD) is an age-related degenerative disease of the central nervous system (CNS), whereas the role of bone marrow immune cells in the pathogenesis of AD remains unclear. Here, the study reveals that compared to matched healthy individuals, AD patients have higher circulating grancalcin (GCA) levels, which negatively correlate with cognitive function. Bone marrow-derived GCA+ immune cells, which secret abundant GCA and increase during aging, preferentially invaded the hippocampus and cortex of AD mouse model in a C-C Motif Chemokine Receptor 10 (CCR10)-dependent manner. Transplanting GCA+ immune cells or direct stereotaxic injection of recombinant GCA protein intensified amyloid plaque load and aggravated cognitive and memory impairments. In contrast, genetic ablation of GCA in the hematopoietic compartment improves cognitive and memory function. Mechanistically, GCA competitively binds to the low-density lipoprotein receptor-related protein 1 (LRP1) in microglia, thus inhibiting phagocytosis and clearance of Aβ and potentiating neuropathological changes. Importantly, GCA-neutralizing antibody treatment rejuvenated cognitive and memory function and constrained AD progression. Together, the study demonstrates a pathological role of GCA+ immune cells instigating cognitive and memory decline, suggesting that GCA+ immune cells can be a potential target for innovative therapeutic strategies in AD.