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  Glial and vascular contributions to neurodegenerative diseases
Long-read isoform sequencing implicates PSEN2 cryptic splicing in sporadic Alzheimer’s disease
Meredith Course1,2, Jenna Somberg1, Kathryn Gudsnuk1, C.
Dirk Keene1, Thomas Bird1, Suman Jayadev1, Paul Valdmanis1
1University Of Washington, Seattle, USA, 2Colorado College, Colorado Springs, USA
 Background: Pathogenic variants in presenilin-1 (PSEN1) and presenilin-2 (PSEN2) cause early-onset Alzheimer’s disease (AD). Sporadic AD shares the same hallmarks of pathology including amyloid beta aggregates and tau neurofibrillary tangles. We hypothesized that improper PSEN splicing in sporadic AD could generate aberrant transcripts that act mechanistically in a manner similar to pathogenic variants in these genes.
Methods: We used a probe-based pull-down strategy and leveraged PacBio long-read isoform sequencing (Iso-Seq) to characterize tens of thousands of complete PSEN1 and PSEN2 transcripts in the prefrontal cortex of individuals with sporadic AD, early-onset AD (carrying PSEN1 and PSEN2 variants), and controls.
Results: We found that sporadic AD samples had significant enrichment in a human-specific cryptic exon in intron 9 of PSEN2: 11.8% of full- length reads in sporadic AD samples relative to 1.9% of controls (p=0.002) and 3.4% of PSEN variant carriers (p=0.005) as well as a 77bp intron retention product prior to PSEN2 exon 6 (6.6% of sporadic AD reads versus 0.8% of controls; p=0.005). Conversely, PSEN1 splicing patterns were no different between sporadic AD, familial AD and controls. An independent cerebellum RNA-seq dataset of ~80 AD cases and ~80 controls revealed a significant increase in cryptic exon 9B inclusion in sporadic AD (17.4%) versus controls (9.8%; p<0.0001) as well as increased alternative splice products at exon 6 in AD (4.4%) versus control (2.5%; p=0.045). Exon 9B was also significantly elevated in AD relative to controls in a set of parietal lobe tissue samples (p=0.02). We found evidence of allele-specific expression in PSEN2 variant carriers and common RNA editing events within PSEN2.
Discussion: Cryptic splicing in PSEN2 could adversely impact its function, especially given its role in microglia biology.
Conclusions: Our findings demonstrate that transcript differences in PSEN2 may play a role in sporadic AD, and reveal novel therapeutic targets for AD.
Background: The gut microbiome (GMB) has been implicated in numerous disease etiologies, including Alzheimer’s disease (AD). Previous studies show that antibiotic-mediated (abx) alteration of the gut microbiome (GMB) results in a reduction of amyloid beta plaques (Aβ) and a change in microglial phenotype in male APPPS1-21 mice [1, 2]. However, the effect of GMB perturbation on astrocyte phenotypes in the context of amyloidosis has not been examined. Herein, we have investigated the effect of GMB manipulation on astrocyte phenotypes in APPPS1-21 mice, a well-studied mouse model of amyloidosis. Materials and methods: To study the GMB’s role in regulating astrocyte phenotypes, we treated APPPS1-21 male and female mice with a previously established abx cocktail or vehicle control and quantified GFAP+ astrocytes, plaque-associated-astrocytes (PAA), and PAA morphological parameters using a combination of immunohistochemistry, widefield imaging, and confocal imaging.
Additionally, we have also studied the same astrocyte phenotypes in separate cohorts from the University of Chicago, which include APPPS1-21 abx-treated mice that were given fecal matter transplants (FMT) from untreated donor APPPS1-21 mice [2]. Lastly, we studied astrocyte phenotypes in a cohort of APPPS1-21 male mice raised in germ-free raised or specific pathogen free (SPF) environments.
Results: We observed that treatment of male but not female APPPS1-21 mice with broad-spectrum abx leading to GMB perturbation reduces Aβ plaques, GFAP+ astrocytes, and PAAs. Additionally, we found that PAAs in abx-treated male APPPS1-21 mice exhibit an altered morphology. These phenotypes were restored in abx-treated mice that were subjected to FMT from APPPS1-21 male donor mice. Finally, we found that APPPS1-21 male mice raised in germ-free (GF) conditions have similar astrocyte phenotypes compared to abx-treated mice. Conclusion: Our results indicate that the GMB plays an important role in controlling reactive astrocyte induction, morphology, and astrocyte recruitment to Aβ plaques.
Background: Dysfunction of neurovascular unit represents a major pathological hallmark of Alzheimer’s disease (AD) and age-related cognitive impairment. Diminished blood flow and blood-brain barrier breakdown have been linked to the accumulation of amyloid-β (Aβ) peptide in brain parenchyma and cerebral blood vessels1. Additionally, cerebrovascular dysfunction has been observed in primary tauopathies, including progressive supranuclear palsy (PSP) where amyloid deposition is not present2 3. Despite the significance of brain vasculature in maintaining the proper neuronal function, the proteomic profiles of cerebrovasculature remain poorly characterized.
Methods: We isolated cerebrovasculature from frontal cortex of individuals with AD, PSP, and controls (N=20-24 cases/diagnosis) using density-mediated separation to purify blood vessels from parenchymal components, followed by the multiplex tandem-mass-tag (TMT) labeled mass spectrometry technique.
Results: The deep proteome of brain vasculature consisted of 9,853 proteins of which ~3,500 were differentially altered in AD brains compared to controls. Co-expression network analysis of cerebrovasculature resolved biologically diverse modules linked to vascular cell types, including endothelial cells, pericytes, and smooth muscle cells distinct from the bulk tissue proteome from the same cases. Specific modules were highly correlated to Braak and/or CERAD score, and clinical phenotypes. We replicated previously identified dysregulation of extracellular matrix in AD4 with SMOC1 and MDK showing significant increase in AD vasculature compared to PSP. Discussion: Our comprehensive proteomic study of brain vasculature identified disease-specific protein changes strongly associated with AD or PSP suggesting distinct key protein of disease pathology in these disorders. Importantly, many AD-specific modules were uniquely found in brain vasculature and not present in bulk analysis highlighting the utility of this approach to resolve the proteomes of less abundant brain cells.
Conclusions: Our findings provide insights into disease-related pathophysiology and are a promising source of vascular protein targets for future AD biomarker and therapeutic studies.
Deep quantitative proteomics analysis of the brain vasculature in Alzheimer’s disease and related tauopathies.
Aleksandra Wojtas1, Eric Dammer1, Duc Duong1, Luming
Yin1, Edward Lee2, Nicholas Seyfried1
1Emory University, Atlanta, USA, 2University of Pennsylvania, Philadelphia, USA
Gut microbiome perturbations alter astrocyte phenotypes in the APPPS1-21 mouse model of amyloidosis
Sidhanth Chandra1, Antonio Di Meco1, Hemraj Dodiya2, Jelena Popovic1, Xiaoqiong Zhang2, Katherine Sadleir1,
Sangram Sisodia2, Robert Vassar1
1Northwestern University, Chicago, USA, 2University of Chicago, Chicago, USA
 78 • ISMND 2022

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