Citations

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.

AbstractBackgroundGlial activation plays a critical role in Alzheimer’s disease (AD) pathogenesis and is associated with amyloid‐β plaques and neurofibrillary tangles composed of phosphorylated tau (phospho‐tau). With approximately 40%‐60% of AD cases having limbic‐predominant age‐related TDP‐43 encephalopathy neuropathologic change (LATE‐NC), TDP‐43 pathology has become more the rule than the exception in AD brains. However, the effect of LATE‐NC in AD‐related glial activation is unknown.MethodWe analyzed the effect of TDP‐43 pathology on glial activation in three AD subtypes: hippocampal sparing (HpSp) AD, typical AD, and limbic predominant AD. Twenty cases of each subtype (n = 60 total) were selected from the FLorida Autopsied Multi‐Ethnic (FLAME) cohort. Cases were immunohistochemically evaluated on serial sections of the hippocampus in the CA1 and subiculum subsectors, and superior temporal, inferior parietal, and mid‐frontal cortices with antibodies to CD68 (phagocytosis), GFAP (astrogliosis), GT‐38 (conformation‐specific AD‐type tau), 6F/3D (amyloid‐β), and phospho‐Serine 409/410 of TDP‐43 (pTDP‐43). Digital pathology was employed to quantify immunohistochemical staining. Multivariable regression modeling was utilized to understand the relationship between glial activation and AD pathologies.ResultThe level of glial activation, defined as % of immunoreactive surface area for CD68 and GFAP, was lowest in HpSp AD (0.54%, 13%), whereas typical AD (1.3%, 33%) and limbic predominant AD (1.3%, 30%) had similar levels suggesting a ceiling effect, or plateau, of gliosis levels (both p<0.001). HpSp AD was more often male (p<0.001) and younger at disease onset (p<0.001) compared to both typical AD and limbic predominant AD. Multivariable regression modeling of CD68 (subiculum R2 = 0.60) identified advanced tau to be a significant predictor of CD68 in all brain regions. LATE‐NC stage predicted increased CD68 levels in limbic brain regions. Multivariable regression modeling of GFAP (CA1 R2 = 0.60) showed that Aβ was the strongest predictor of astrogliosis in CA1 and subiculum (p<0.001). Advanced tau in CA1 and parietal cortex, LATE‐NC in subiculum and temporal cortex, and APOE4 genotype in CA1 predicted a significant increase in GFAP.ConclusionThese findings highlight the significant role TDP‐43 pathology plays in affecting the microenvironment and interplay between neuroinflammation and neurodegeneration and have important implications for therapeutic strategies in AD with co‐morbid LATE‐NC.

AbstractBackgroundTau proteins aggregate into spatially and morphologically distinct lesions in primary and secondary tauopathies. These signature lesions result from deposition of tau proteins that are distinctly modified via missense mutation and/or post‐translational modification for each disease strain. Identifying the unique tau protein abnormalities that lead to specific neurodegenerative diseases is beneficial in their early diagnosis and treatment. This is especially true for Progressive Supranuclear Palsy a primary tauopathy with clinical symptoms mirroring Parkinsonian syndromes and frontotemporal dementias.MethodBiochemical analysis of tau aggregates from patients with a pathological diagnosis of Progressive Supranuclear Palsy (n = 60) demonstrate that the tau protein modifications observed in this disease result from tau that is specifically modified and thereby forms the basis of the distinct disease strains observed. Here data collected using the sarkosyl‐insoluble tau fractions from tissue samples of pathologically diagnosed patients with Progressive Supranuclear Palsy without comorbidities allows for the direct observation of the modifications. Most interesting is the variability of the tau proteins that actually accumulate for this disease opening up the possibility that tau strains may exist within any given tauopathy even as the clinical manifestations appear similar. This indicates that early and specific identification and detection of these strains is critical for the development of therapies for these diseases.ResultExtraction and examination of sarkosyl‐insoluble tau proteins from deceased patients pathologically diagnosed with Progressive Supranuclear Palsy indicate that multiple tau strains exist within this primary tauopathy. Western blotting of these extracts from patient tissues demonstrates a unique banding pattern of insoluble tau in a subset of the cases that exhibit extreme hyperphosphorylation as indicated by additional high molecular weight tau banding. These patients also had an earlier age of onset and age of death indicating a more aggressive disease strain. Additional data is presented comparing the structure and seeding capability of this insoluble tau and the distribution and burden of insoluble tau in these Progressive Supranuclear Palsy patients compared to those with typical onset and insoluble tau signature.ConclusionMultiple pathological tau strains exist in Progressive Supranuclear Palsy.

AbstractBackgroundThe coarse‐grained plaque is a biochemically and morphologically distinct amyloid‐β (Aβ) deposit that was discovered in clinically‐defined atypical Alzheimer’s disease (AD). Unlike other Aβ deposits, this plaque is not observed in cognitively normal people. This study investigates the hypothesis that the coarse‐grained plaque is distinctly associated with AD neuropathologic change and glial activation within the context of AD subtypes.MethodSixty AD cases from the FLorida Autopsied Multi‐Ethnic (FLAME) cohort were assessed. We included 20 hippocampal sparing (HpSp) AD, 20 typical AD, and 20 limbic predominant AD. Immunohistochemistry was performed on the middle frontal, inferior temporal, and inferior parietal cortex for AD neuropathology (Aβ [6F/3D], phospho‐tau [AT8], tau [GT38]) and glial activation (CD68 [KP1] and GFAP). Digital pathology was used to quantify burden. Thioflavin‐S tangle counts were performed at time of neuropathologic diagnosis. Distinct deposit types, including classic cored plaques, coarse‐grained plaques, cerebral amyloid angiopathy (CAA)‐Type 1, and CAA‐Type 2, were semi‐quantitatively scored on Aβ (Fig. 1). Correlations between demographic, clinical, and neuropathologic characteristics were assessed using Spearman rho analysis and reported if p<0.05. An independent cohort of ∼100 HpSp AD cases will be assessed to validate initial findings.ResultIn the frontal cortex of HpSp AD, higher scores of classic cored plaques correlated with older age at onset and death, non‐APOE ε4 carriership, and less CAA‐Type 1 and Type 2 (Fig. 2). On the contrary, the severity of coarse‐grained plaques correlated with a younger age at onset and death, APOE ε4 carriership, higher scores for CAA‐Type 1, CAA‐Type 2, and increased burden of Aβ, GFAP, and CD68. Classic cored plaques correlated negatively with phospho‐tau and tau, whereas coarse‐grained plaques correlated positively with phospho‐tau, tau, and tangles. The two deposit types were negatively correlated with each other. Correlations were weaker but showed similar trends in other AD subtypes (Fig. 2) and regions.ConclusionWithin HpSp AD, classic cored and coarse‐grained plaques show distinct correlations with glial activation, (phospho‐)tau, and tangles. These results suggest opposite directions of association for these deposits on AD neuropathologic change that may impact clinical symptoms. These findings are relevant to therapies targeting overall Aβ pathology.

AbstractBackgroundAlzheimer’s disease (AD) is neuropathologically characterized by amyloid‐β (Aβ) plaques and tau neurofibrillary tangles often quantified by Thal phase and Braak stage, respectively. Aβ also frequently deposits in the cerebrovasculature with severity categorized by a cerebral amyloid angiopathy (CAA) score. These and related measures often show high variability within AD suggesting distinct underlying mechanisms, the identification of which may reveal important biological insights into AD pathophysiology. We hypothesize that, within the AD brain, neuropathology and levels of core AD‐related proteins are influenced by variations in DNA methylation (DNAm). To test this, we performed epigenome‐wide association studies (EWAS) using DNAm clusters from the temporal cortex (TCX) and cerebellum (CER) with AD‐related neuropathologic measures (Braak, Thal, and CAA) and brain biochemical levels of five proteins (apoE, Aβ40, Aβ42, tau, and p‐tau).MethodsDNAm from neuropathologically‐confirmed AD cases available through the Mayo Clinic brain bank was measured by reduced representation bisulfite sequencing (RRBS) from 471 brain samples, 200 of which had both TCX and CER measures. Neuropathology was scored by an experienced neuropathologist. TCX levels of five AD‐related proteins from three tissue fractions (buffer‐, detergent‐, and in‐soluble) were measured by ELISA (Liu 2020). CpG methylation (CpGm) was binned by a 15‐state chromatin model (Kundaje 2015) and averaged. Averaged CpGm clusters were tested for their association with each endophenotype through multi‐variable linear regression. The influence of CpGm clusters on expression levels of nearby genes was also investigated through multi‐variable regression.ResultsOur innovative binning method demonstrated expected and biologically relevant CpGm patterns. We identified multiple epigenome‐wide significant CpGm clusters unique to each AD‐related endophenotype and brain region. Comparisons between the TCX and CER further revealed brain region‐specific enrichment of distinct CpGm clusters. Significant CpGm clusters were further characterized by their influence on gene expression.ConclusionsAlthough all neuropathologic and biochemical endophenotypes tested are core to AD pathophysiology, our results suggest each has a distinct epigenetic architecture underlying their variability in the AD brain. By discovering brain region‐specific DNAm changes that contribute to each neuropathologic or biochemical outcome, we can identify core components of complex mechanisms that individually and jointly contribute to AD.

AbstractBackgroundNeuropathologically, Alzheimer’s disease (AD) is characterized by the presence of senile plaques composed of beta‐amyloid (Aβ) and neurofibrillary tangles made of hyperphosphorylated tau. Aβ and p‐tau have been shown to impact endoplasmic reticulum homeostasis, DNA damage response and autophagic functions. Intriguingly, alterations in these systems have also been connected to dysfunctions in UFMylation, a ubiquitin‐like posttranslational modification. Analogous to ubiquitin, UFM1, is conjugated to substrates via a catalytic cascade involving UFM1‐specific set of E1 (UBA5), E2 (UFC1), and a complex that consists of E3 ligase (UFL1), DDRGK1 and CDK5RAP3. UFMylation is reversible, and this is mediated by UFSP2. The UFMylation pathway is essential for brain development as complete loss of function of any of its components result in severe neurodevelopmental disorders. However, alterations in UFMylation and their role in and significance for age‐related AD remains unknown.MethodWe performed Western blot and Meso Scale Discovery based ELISA to measure the protein level of UFM1, UFSP2, tau, p‐tau and other UFMylation pathway components in human post‐mortem brain. Analyses were performed with RIPA‐ and 2% SDS‐soluble fraction from frontal cortex. To validate our finding in human tissues, we knocked out UFM1 and UFSP2 in ReN cells, a neural progenitor cell model that has already been used to study AD and that can be terminally differentiated in neurons.ResultWe found a significant reduction of RIPA UFSP2, but an increase of RIPA and SDS UFM1 in AD compared to controls. Strikingly, RIPA UFSP2 had a strong negative correlation with both RIPA and SDS UFM1. Furthermore. UFM1 was significantly increased in UFSP2‐KO neurons compared to WT, mirroring the negative correlation between UFM1 and UFSP2 observed in human brain. In addition, RIPA and SDS UFM1 had a strong positive correlation with p‐tau, indicating that dysfunctional UFMylation might play an important role for disease pathogenesis.ConclusionReduction of RIPA soluble UFSP2 in human AD frontal cortex might result in the abnormal accumulation of UFM1 and this might further facilitate the AD pathogenesis. Because of its role for several central AD‐relevant pathways, the UFMylation pathway might be a potential therapeutic target for AD.