Citations

AbstractBackgroundImaging markers are central to diagnose and monitor disease progression in dementia with Lewy bodies (DLB). This presentation provides an up‐to‐date overview of the contributions of positron emission tomography (PET) and magnetic resonance imaging (MRI) to the field of DLB.MethodsExpert opinion and road‐map for the future based on the current state‐of‐the‐art.ResultsPET for Lewy pathology is an active area of research but a reliable tracer is still lacking. Tracers for glucose metabolism and dopaminergic deficits have merited inclusion in diagnostic criteria for DLB. Alzheimer’s disease (AD) co‐pathology is common in DLB. Amyloid PET has been used in DLB for a long time and most studies show an elevated frequency of a positive amyloid scan in DLB. Tau PET has recently attracted attention, with studies suggesting an atypical pattern of tau PET uptake in DLB, different from the pattern seen in AD. Newest advances include PET tracers for synaptic activity, microglial activation, and neuromelanin. MRI findings in DLB traditionally include preservation of hippocampal volume, as compared with AD. More recent findings encourage the inclusion of a pattern of cortical neurodegeneration, mostly affecting posterior cortical areas, with sparing of the hippocampus. This finding is consistent across MRI markers (volume, thickness, functional MRI, diffusion, perfusion, and connectivity). Advanced techniques for cholinergic system have shown vulnerability of cholinergic basal forebrain and its white matter projections.ConclusionsNeuroimaging is central for DLB diagnosis and for advancing mechanistic knowledge about the disease. Biomarkers of Lewy pathology are emerging and will revolutionize the field, but in the absence of topographical markers, the reviewed techniques will have an important role in precision medicine, for example, helping to identify subgroups with different disease trajectories or treatment response.

AbstractBackgroundThe meningeal layers form a unique interface between the central nervous and peripheral immune systems. The lymphatic vasculature that is present in the meningeal dura and drains cerebrospinal fluid (CSF) becomes dysregulated during aging and in models of neurodegenerative disorders such as Alzheimer’s disease (AD). Apolipoprotein E (APOE), and the isoform APOE4 in particular, increases the risk for late‐onset sporadic AD. However, it is still poorly understood whether changes in meningeal lymphatic morphology and drainage are mediating the deleterious effects of APOE4 expression. In the present study, we investigate the effects of human APOE3 or APOE4 on meningeal lymphatic function and immunity.MethodWe evaluated the morphology of lymphatic vessels in dural whole mounts of age‐matched wild type, Apoe‐KO, APOE3, and APOE4 mice at 2‐3, and 11‐12 months. This assessment was performed in both male and female mice. The drainage function of meningeal lymphatics is being assessed in the same mice by in vivo imaging of CSF‐derived fluorescent tracers into the deep cervical lymph nodes. We have also performed RNAscope and protein measurements using murine meningeal tissue to investigate the main cellular source(s) of Apoe, and single‐cell RNA sequencing to explore the cell‐specific transcriptomic signatures linked to the changes in meningeal lymphatic function.ResultLymphatic vessel length is increased in 11‐12‐month‐old APOE4 male mice, when compared to their APOE3 counterparts. Interestingly, the same was not detected in female mice. Dural myeloid cells, expressing Itgam (encoding for CD11b), are the main source of Apoe mRNA transcripts, and one of the main sources of apoE protein in the brain dura mater. APOE4 expression and age markedly alter the transcriptional profile of meningeal myeloid cells, which show overexpression of lysosomal, antigen presentation, and complement genes.ConclusionAPOE4 expression leads to a sex‐dependent meningeal lymphatic vascular remodeling, which is associated with an exacerbated activation of meningeal macrophages. In future studies, we will test whether the changes in meningeal lymphatics observed in mice expressing APOE4 are mediated by myeloid cell derived APOE.

AbstractBackgroundIndividuals with neuropathological criteria for Alzheimer’s disease (AD) may manifest with atypical clinical syndromes. Past work showed that the neurobiological basis for these differences is related to specific neuronal vulnerabilities for tau pathology. For instance, amnestic cases have a higher burden of neurofibrillary changes in CA1. In contrast, logopenic variant primary progressive aphasia (lvPPA) cases have a higher tau burden in the superior temporal gyrus (STG). Single‐cell technology enables investigations on the molecular basis of differential neuronal vulnerability in AD. Therefore, we investigated factors underlying selective vulnerability using brain samples from individuals with pure AD with different clinical manifestations.MethodsnRNA Sequencing using the Chromium Single Cell 3′ (10X Genomics, USA) on nuclei cells extracted from the CA1 sector and posterior STG of postmortem brain tissue of 24 individuals either meeting pathological criteria for AD (A3B3C3; 12 amnestic and eight lvPPA) and healthy controls (A≤1B≤1C≤1; n = 4) (Table 1, Fig. 1A/B). Bioinformatics analyses were conducted using Cell Ranger and R software. Comparisons between cell subpopulations were conducted with Wald statistical test, and p‐values < .05 were considered significant.ResultAfter quality control, we recovered 98,180 nuclei with a mean of 2,130 genes per nuclei. Upon cross‐sample alignment and t‐stochastic neighborhood embedding clustering (Fig. 1C), we found 21 excitatory neuronal subpopulations (Exc‐sub) in CA1 and 26 in STG, and 22 and 27 inhibitory neuronal subpopulations (Inh‐sub) in CA1 and STG, respectively; 16 astrocytes subpopulations in both areas and 20 microglia subpopulations in CA1 and 17 in STG (Fig. 2). One STG Exc‐sub, expressing CUX2 and LAMP5 genes showed vulnerability in lvPPA patients. Also, one STG Inh‐sub, expressing the ADARB2 gene, showed vulnerability for all AD patients. Furthermore, two astrocytes subpopulations (one in both areas and one in STG) and three STG microglia subpopulations showed proportional composition changes comparing AD patients and HC.ConclusionOur preliminary study identified a vulnerable population of excitatory neurons related to lvPPA. We are conducting validation studies using quantitative pathology to confirm these results. Furthermore, analysis of a higher number of cases is ongoing and will continue to inform on factors associated with neuronal vulnerability.

AbstractBackgroundIn Alzheimer’s disease (AD), tau accumulates with a stereotyped progressive involvement of brain regions. The cellular mechanism for this is the neuron‐to‐neuron propagation of tau, but the factors that influence tau propagation are not fully understood. We hypothesize that AD‐associated post‐translational modifications (PTMs) of tau reduce its propagation since lysines are critical for LRP1‐mediated tau uptake.MethodA construct was designed to model tau propagation in Chinese hamster ovary (CHO) cells and allow identification of donor and recipient cells by immunocytochemistry and flow cytometry. In addition to the wildtype (WT) tau sequence, constructs were made with pseudophosphorylation and pseudoacetylation mutations corresponding to the phosphorylations and acetylations found on low molecular weight (LMW) and high molecular weight (HMW) tau species in AD brains. Additionally, individual PTM mimic mutations corresponding to PTMs of the LMW tau species were made. The constructs were transfected into CHO cells and tau propagation was assessed by flow cytometry after 48 hours. Statistical analysis was performed by ANOVA with adjustment for multiple comparisons.ResultTau propagation using this cell culture model was demonstrated by immunocytochemistry and flow cytometry. Comparison of the WT, LMW‐mimic (8D2Q mutant), and HMW‐mimic (20D3Q mutant) tau propagation constructs revealed that the 8D2Q and 20D3Q mutants displayed reduced tau propagation compared to WT by 23.7% (p = 0.0186) and 26.5% (p = 0.0087), respectively. Comparing the ten single mutant constructs to WT, T181D and T231D showed increased tau propagation by 20.3% (p = 0.0002) and 15.9% (p = 0.0094), respectively. Compared to WT, T217D, K281Q, K353Q, and S404D showed reduced tau propagation by 17.3% (p = 0.0030), 15.3% (p = 0.0123), 23.7% (p <0.0001), and 16.6% (p = 0.0046), respectively.ConclusionThe mimics of AD‐associated tau PTMs found on LMW and HMW tau reduce tau propagation, consistent with the initial hypothesis. The impact of individual PTM mimics on tau propagation is variable, but notably both pseudoacetylation mimics reduce propagation, possibly due to lower affinity with LRP1. Understanding how AD‐associated tau PTMs impact tau propagation may suggest novel targets to slow the progression of AD pathology.

AbstractBackgroundPSEN1 mutations are the most common genetic cause for autosomal dominant forms of Alzheimer’s disease patients with an age at onset below 65 years (ADAD). Even though all PSEN1 mutations lead to an increased Aβ processing and production, there is a large variability in phenotypic presentation even in individuals with the same mutation. Splicing isoforms in AD genes are reported to be associated with the disease, however little is known about correlation between splicing pattern and phenotypic presentation (e.g. variable age onset). We hypothesized that cellular and transcriptomic alterations differ amongst patients with different phenotypic presentation. In a group of 8 ADAD with the same PSEN1 p.G206A mutation, we investigate the association between brain cell type proportion, gene expression, and splicing isoforms with age at onset, Aβ plaque type and severity of AD neuropathologic change.MethodsEight Hispanic ADAD cases with PSEN1 p.G206A mutation were identified in the Mayo Clinic brain bank housed in Jacksonville, FL. Standard (immuno)histochemical stains, including H&E and thioflavin S, were performed to evaluate AD neuropathologic change. We focused on distinct Aβ plaque types in an association cortex (inferior parietal gyrus) and primary cortex (motor). We carried‐out single‐nuclei short and long read RNA sequencing on both brain regions. We will stratify our transcriptomic analyses based on neuropathology and phenotypic presentation.ResultsWe observed large variability in age at onset (median = 53 years; range: 42‐59 years). Furthermore, disease duration was reportedly variable (median = 10 years; range: 8‐17). All cases had a Braak tangle stage VI and Thal amyloid phase 5. Only 3 patients carried at least one APOE*ε4 risk allele, suggesting that its effect was minimal on ages at onset and death. Different plaque types were observed for short versus long survivors. In short survivors, plaques were predominantly fibrillar with an eosinophilic radial component, whereas in longer survivors predominantly cored plaques were observed.ConclusionLarge phenotypic variability within patients with the same mutation is observed suggesting the involvement of yet to be discovered genetic modifiers. Understanding cellular transcriptomic events in ADAD is essential to future development of therapeutic approaches and development of biomarkers.

AbstractBackgroundAlzheimer’s disease (AD) is characterized by the accumulation of neurofibrillary tangles (NFTs), comprised primarily of microtubule‐associated protein tau. NFTs are defining hallmarks of AD and its accumulation into pathological oligomers and fibrils has been associated with cognitive decline in AD and other tauopathies. Despite its pivotal role in the disease process, we still have a poor understanding of how their formation, toxicity, and spread across brain regions is regulated via tau‐associated proteins that regulate the oligomerization, pathological accumulation, and seeding of tau in affected cell types and vulnerable brain regions.MethodWe have established two proximity‐dependent biotin‐identification (BioID) models as novel approaches to identify the composition and proximal molecular environment of tau aggregates. Using an in vitro and ex vivo model approach coupled with mass spectrometry, we have identified proteins that were primarily associated with mutant tau aggregates vs. soluble wildtype tau and stratified them based on significance, molecular pathway and druggability. Immunoblotting and immunofluorescence were performed to assess the interaction of endogenous and overexpressed top candidates with tau, along with visualization with NFTs in the context of human AD pathology to assess their impact on tau‐aggregate formation and toxicity.ResultTo determine the physiological and aggregate specific interacting partners of tau, we are comparing wild type tau (TauWT) with mutant tau (Tau3xMUT) carrying three tauopathy‐associated mutations (A152T/P301L/S320F). The proteomic analysis from our combined proteomic datasets revealed that significantly changed proteins included candidates involved in RNA processing, as well as protein ubiquitination and proteasome degradation. We have begun validating proteins that are enriched in mutant tau in human tissue of AD, as well as primary tauopathies, that may be druggable targets for tauopathies.ConclusionThis study provides novel insights into cellular pathways and molecular mechanisms of neurodegeneration, by identifying in the context of living neurons and brain tissue different functional classes of tau‐associated proteins with relevance for AD pathophysiology. These are expected to include proteins that may mediate the toxic effect of NFTs, facilitate the formation or degradation of pathological tau aggregates, and catalyze posttranslational modifications of tau oligomers and associated proteins.

AbstractBackgroundTau aggregation is the underlying cause of several neurodegenerative tauopathies. Formation of tau aggregates with seeding activity involves interactions with other proteins, but with unknown effects on tau‐seed properties. Our previous study found that Bassoon (BSN), a presynaptic protein, interacts with tau‐seed, exacerbating its toxicity in vivo. Bsn downregulation reduced tau spreading and overall pathology. A previous study also linked missense mutations in BSN to the aggregation of 3‐ and 4‐repeat tau in patients. However, it is unknown how genetic mutations in BSN influence tau pathology in vivo.MethodWe developed a knock‐in mouse model harboring a mutation in endogenous BSN (p.Pro3882Ala), equivalent to p.Pro3866Ala BSN mutation identified in patients. We evaluated the cognitive and motor abilities of aged heterozygous and homozygous BSNKI mice and also we analyzed BSN and tau patterns in the brains of these mice. Additionally, we examined BSN and tau patterns in the BSN mutation carrier.ResultAt 10 months, BSNKI mice did not exhibit anxiety‐related behavior and had similar activity as WT littermates. They also displayed similar cognitive abilities as the WT mice. Interestingly, the BSNKI mice displayed lower latency to fall off the rotarod and reduced grip strength compared to the WT mice. We also observed an increase in the accumulation of pathological tau and BSN deposits in the brains of the BSNKI mice. The BSN mutation carrier displayed inclusions of BSN and tau pathology.ConclusionWe developed a mouse model with a knock‐in mutation in the Bassoon gene, allowing us to evaluate the effects of this disease‐associated mutation in vivo. Our results have shown that the mice with Bsn mutation have impairments in motor coordination, and postural instability, similar to BSN mutation carriers and show pathological tau and BSN deposits in the brain. We found inclusions of BSN and tau pathology in BSN mutation the carrier that resembled certain pathological features of AD and PSP. These findings suggest that BSN plays a role in tau pathology and targeting it may represent a promising therapeutic strategy for tauopathies. Further research is needed to fully understand our findings' underlying mechanisms and potential therapeutic implications.

AbstractBackgroundRecent studies increased the number of genes that are associated with the co‐existence of vascular pathologies and Alzheimer’s disease (AD). However, the biological functions and the mechanism of action by which they contribute to the disease pathology are still to be further elucidated. Therefore, animal models that allow streamlined large‐throughput functional screens in a biologically relevant manner are essential. Towards this goal, we generated adult zebrafish models of amyloid toxicity to functionally analyze candidate genes and how they relate to disease perturbations.MethodThrough cerebroventricular injection of human amyloid‐beta42 monomers into the cerebrospinal fluid of the adult zebrafish brain, we inflict acute amyloid toxicity. By performing immunohistological analyses, single cell transcriptomics, gene editing for generating gene variants or full knockouts, transient functional knockdown of candidate genes, pharmacological intervention of selected signalling pathways and comparing the findings to human AD brain transcriptomics datasets, we determined the similarities and differences of the effects of amyloid pathology on the vasculature in zebrafish in relation to the human conditions.ResultsWe found that amyloid pathology in zebrafish brain shows histological and molecular similarities to human brains. Integration of single cell transcriptomics showed remarkable similarities in neurons to amyloid in humans and zebrafish. We identified several molecular mechanisms that underlie this pathology‐induced cellular response and translated to mammalian models including rodent brains. By performing genome‐wide association in AD patients, we identified that the FMNL2 links cerebrovascular disease and AD, by regulating the astroglial and blood vessel interactions and controlling the efficient clearance of toxic proteins from the brain. Our new single cell transcriptomics analyses of the gliovascular niche cells of the zebrafish brain revealed potential cellular interactions and the pathways perturbed in vascular disease and AD.ConclusionWe propose zebrafish as a useful model for functional investigation of vascular pathology and AD‐related genes identified in clinical studies by providing in vivo biological knowledge on disease mechanisms, on which novel drug development strategies can be based.

AbstractBackgroundInter‐cellular communication within the gliovascular unit (GVU) is critical for cerebral blood flow regulation, and maintenance of the blood‐brain‐barrier (BBB) properties. The breakdown of BBB in Alzheimer’s disease (AD) is well‐established, but precise underlying molecular changes remain unclear. Additionally, whether GVU molecular alterations observed in AD brains are also detected in blood from living patients is unknown. Further, these GVU molecular perturbations require further investigation in different model systems to identify both human brain‐specific and cross‐species conserved alterations. In this study, we investigated prioritized GVU molecules altered in AD brains for their conservation in blood and cross‐species model systems.MethodsWe performed single nucleus RNA sequencing (snRNAseq) of temporal cortex tissue in AD and control brains. We analyzed this data to detect cell‐specific GVU molecular perturbations and their interactions. We investigated molecular interactions between vascular and astrocyte clusters, the major cell types of the GVU of the BBB. To determine whether GVU transcriptional alterations detected in the brain are preserved in the blood, existing blood expression, genetic, and neuroimaging data from two longitudinal antemortem cohorts were analyzed. Using model systems, including mouse, drosophila, and zebrafish, we evaluated the cross‐species conservation of the top GVU alterations detected in AD brains.ResultsBrain snRNAseq revealed transcriptional profiles of 6,541 astrocytes and 2,210 vascular cells. The latter formed three distinct vascular clusters characterized as pericytes, endothelia and perivascular fibroblasts. We identified differentially expressed genes and their enriched pathways within these clusters and observed the highest levels of transcriptional changes within pericytes. Vascular targets that interact with astrocytic ligands have biological functions in cell signaling, angiogenesis, amyloid ß metabolism, and cytoskeletal architecture. We discovered that genetic variants influencing blood expression levels of some of the prioritized GVU genes were associated with neuroimaging burden of cerebrovascular disease in living human cohorts. Our ongoing studies in model systems revealed conservation of some of the top prioritized molecular perturbations across species.ConclusionOur findings prioritized by multiscale, cross‐tissue human data revealed GVU perturbations within interacting pericyte and astrocyte molecules, which are conserved across multiple cross‐species models. These results nominate new molecular targets and mechanistic insights for BBB disruptions in AD.

AbstractBackgroundAlzheimer Disease (AD) currently has no effective treatments. A consistent failure of recent clinical trials focused on Aβ or pTau necessitates the identification of novel therapeutic targets and strategies. Since abnormal brain energy homeostasis and mitochondria dysfunction underlie early stages of AD, the development of treatments to restore these defects could be beneficial. We synthesized small molecules that selectively and mildly inhibit mitochondrial complex I (MCI) which restored synaptic and cognitive function in APP/PS1 and 3xTgAD mice. One of the most important mechanisms improved by the treatment included restoration of glucose uptake/utilization that resulted in augmented cellular energetics in mouse brains. However, mechanistic relationship between MCI inhibition, glucose uptake/utilization and mitochondrial function remains to be determined.MethodNeuroblastoma SH‐SY5Y cells that express mutant human APP protein (APPswe) and WT cells were treated with MCI inhibitors in a time‐ and dose‐dependent manner. Flow cytometry was used to measure the translocation of glucose transporters to the cell surface. Changes in protein expression in response to treatment were assessed using Western blot analysis. Changes in mitochondrial morphology were monitored using electron microscopy. Cellular energetics was assessed using a Seahorse Extracellular Flux Analyzer. The non‐radioactive Glucose Uptake‐Glo™ assay was utilized for measuring glucose uptake in cells.ResultsTreatment of with SH‐SY5Y‐APPswe cells with MCI inhibitors within an hour increased surface translocation of GLUTs enhancing glucose uptake and utilization. The effect persisted for 48 hrs. The mechanism of enhanced glucose uptake was associated with AMPK‐dependent activation of NRF2 pathway but not AKT. Furthermore, the improvement in the bioenergetic parameters including Spare Respiratory Capacity, an indicator of the mitochondrial ability to produce energy under stress conditions, and ATP levels were found 24 hrs after the compound treatment. Treatment activated mitochondrial morphofunctional pathway (e.g., fission, fusion, biogenesis and turnover) consistent with the improved cellular bioenergetics. Similar mechanisms were confirmed in the brain tissue of 3xTgAD mice treated with one of the compounds.ConclusionsPartial MCI inhibition activates multiple mechanisms essential for improved energy homeostasis and mitochondrial function. These mechanisms could be consistently induced by our compounds in vivo and in human brain cells support translational potential of this therapeutic strategy.