Gliovascular transcriptional perturbations in Alzheimer's disease reveal molecular mechanisms of blood brain barrier dysfunction.

To uncover molecular changes underlying blood-brain-barrier dysfunction in Alzheimer's disease, we performed single nucleus RNA sequencing in 24 Alzheimer's disease and control brains and focused on vascular and astrocyte clusters as main cell types of blood-brain-barrier gliovascular-unit. The majority of the vascular transcriptional changes were in pericytes. Of the vascular molecular targets predicted to interact with astrocytic ligands, SMAD3, upregulated in Alzheimer's disease pericytes, has the highest number of ligands including VEGFA, downregulated in Alzheimer's disease astrocytes. We validated these findings with external datasets comprising 4,730 pericyte and 150,664 astrocyte nuclei. Blood SMAD3 levels are associated with Alzheimer's disease-related neuroimaging outcomes. We determined inverse relationships between pericytic SMAD3 and astrocytic VEGFA in human iPSC and zebrafish models. Here, we detect vast transcriptome changes in Alzheimer's disease at the gliovascular-unit, prioritize perturbed pericytic SMAD3-astrocytic VEGFA interactions, and validate these in cross-species models to provide a molecular mechanism of blood-brain-barrier disintegrity in Alzheimer's disease.

ABCA7-dependent Neuropeptide-Y signalling is a resilience mechanism required for synaptic integrity in Alzheimer's disease.

Alzheimer's disease (AD) remains a complex challenge characterized by cognitive decline and memory loss. Genetic variations have emerged as crucial players in the etiology of AD, enabling hope for a better understanding of the disease mechanisms; yet the specific mechanism of action for those genetic variants remain uncertain. Animal models with reminiscent disease pathology could uncover previously uncharacterized roles of these genes. Using CRISPR/Cas9 gene editing, we generated a knockout model for abca7, orthologous to human ABCA7 - an established AD-risk gene. The abca7 +/- zebrafish showed reduced astroglial proliferation, synaptic density, and microglial abundance in response to amyloid beta 42 (Aß42). Single-cell transcriptomics revealed abca7 -dependent neuronal and glial cellular crosstalk through neuropeptide Y (NPY) signaling. The abca7 knockout reduced the expression of npy, bdnf and ngfra , which are required for synaptic integrity and astroglial proliferation. With clinical data in humans, we showed reduced NPY in AD correlates with elevated Braak stage, predicted regulatory interaction between NPY and BDNF , identified genetic variants in NPY associated with AD, found segregation of variants in ABCA7, BDNF and NGFR in AD families, and discovered epigenetic changes in the promoter regions of NPY, NGFR and BDNF in humans with specific single nucleotide polymorphisms in ABCA7 . These results suggest that ABCA7-dependent NPY signaling is required for synaptic integrity, the impairment of which generates a risk factor for AD through compromised brain resilience.

Cross species systems biology discovers glial DDR2, STOM, and KANK2 as therapeutic targets in progressive supranuclear palsy.

Progressive supranuclear palsy (PSP) is a neurodegenerative parkinsonian disorder characterized by cell-type-specific tau lesions in neurons and glia. Prior work uncovered transcriptome changes in human PSP brains, although their cell-specificity is unknown. Further, systematic data integration and experimental validation platforms to prioritize brain transcriptional perturbations as therapeutic targets in PSP are currently lacking. In this study, we combine bulk tissue (n = 408) and single nucleus RNAseq (n = 34) data from PSP and control brains with transcriptome data from a mouse tauopathy and experimental validations in Drosophila tau models for systematic discovery of high-confidence expression changes in PSP with therapeutic potential. We discover, replicate, and annotate thousands of differentially expressed genes in PSP, many of which reside in glia-enriched co-expression modules and cells. We prioritize DDR2, STOM, and KANK2 as promising therapeutic targets in PSP with striking cross-species validations. We share our findings and data via our interactive application tool PSP RNAseq Atlas ( https://rtools.mayo.edu/PSP_RNAseq_Atlas/ ). Our findings reveal robust glial transcriptome changes in PSP, provide a cross-species systems biology approach, and a tool for therapeutic target discoveries in PSP with potential application in other neurodegenerative diseases.

Nerve growth factor receptor (Ngfr) induces neurogenic plasticity by suppressing reactive astroglial Lcn2/Slc22a17 signaling in Alzheimer's disease.

Neurogenesis, crucial for brain resilience, is reduced in Alzheimer's disease (AD) that induces astroglial reactivity at the expense of the pro-neurogenic potential, and restoring neurogenesis could counteract neurodegenerative pathology. However, the molecular mechanisms promoting pro-neurogenic astroglial fate despite AD pathology are unknown. In this study, we used APP/PS1dE9 mouse model and induced Nerve growth factor receptor (Ngfr) expression in the hippocampus. Ngfr, which promotes neurogenic fate of astroglia during the amyloid pathology-induced neuroregeneration in zebrafish brain, stimulated proliferative and neurogenic outcomes. Histological analyses of the changes in proliferation and neurogenesis, single-cell transcriptomics, spatial proteomics, and functional knockdown studies showed that the induced expression of Ngfr reduced the reactive astrocyte marker Lipocalin-2 (Lcn2), which we found was sufficient to reduce neurogenesis in astroglia. Anti-neurogenic effects of Lcn2 was mediated by Slc22a17, blockage of which recapitulated the pro-neurogenicity by Ngfr. Long-term Ngfr expression reduced amyloid plaques and Tau phosphorylation. Postmortem human AD hippocampi and 3D human astroglial cultures showed elevated LCN2 levels correlate with reactive gliosis and reduced neurogenesis. Comparing transcriptional changes in mouse, zebrafish, and human AD brains for cell intrinsic differential gene expression and weighted gene co-expression networks revealed common altered downstream effectors of NGFR signaling, such as PFKP, which can enhance proliferation and neurogenesis in vitro when blocked. Our study suggests that the reactive non-neurogenic astroglia in AD can be coaxed to a pro-neurogenic fate and AD pathology can be alleviated with Ngfr. We suggest that enhancing pro-neurogenic astroglial fate may have therapeutic ramifications in AD.

Profiling of d-alanine production by the microbial isolates of rat gut microbiota.

d-alanine (d-Ala) and several other d-amino acids (d-AAs) act as hormones and neuromodulators in nervous and endocrine systems. Unlike the endogenously synthesized d-serine in animals, d-Ala may be from exogenous sources, e.g., diet and intestinal microorganisms. However, it is unclear if the capability to produce d-Ala and other d-AAs varies among different microbial strains in the gut. We isolated individual microorganisms of rat gut microbiota and profiled their d-AA production in vitro, focusing on d-Ala. Serial dilutions of intestinal contents from adult male rats were plated on agar to obtain clonal cultures. Using MALDI-TOF MS for rapid strain typing, we identified 38 unique isolates, grouped into 11 species based on 16S rRNA gene sequences. We then used two-tier screening to profile bacterial d-AA production, combining a d-amino acid oxidase-based enzymatic assay for rapid assessment of non-acidic d-AA amount and chiral LC-MS/MS to quantify individual d-AAs, revealing 19 out of the 38 isolated strains as d-AA producers. LC-MS/MS analysis of the eight top d-AA producers showed high levels of d-Ala in all strains tested, with substantial inter- and intra-species variations. Though results from the enzymatic assay and LC-MS/MS analysis aligned well, LC-MS/MS further revealed the existence of d-glutamate and d-aspartate, which are poor substrates for this enzymatic assay. We observed large inter- and intra-species variation of d-AA production profiles from rat gut microbiome species, demonstrating the importance of chemical profiling of gut microbiota in addition to sequencing, furthering the idea that microbial metabolites modulate host physiology.

Transcriptional landscape of human microglia implicates age, sex, and APOE-related immunometabolic pathway perturbations.

Microglia have fundamental roles in health and disease; however, effects of age, sex, and genetic factors on human microglia have not been fully explored. We applied bulk and single-cell approaches to comprehensively characterize human microglia transcriptomes and their associations with age, sex, and APOE. We identified a novel microglial signature, characterized its expression in bulk tissue and single-cell microglia transcriptomes. We discovered microglial co-expression network modules associated with age, sex, and APOE-ε4 that are enriched for lipid and carbohydrate metabolism genes. Integrated analyses of modules with single-cell transcriptomes revealed significant overlap between age-associated module genes and both pro-inflammatory and disease-associated microglial clusters. These modules and clusters harbor known neurodegenerative disease genes including APOE, PLCG2, and BIN1. Meta-analyses with published bulk and single-cell microglial datasets further supported our findings. Thus, these data represent a well-characterized human microglial transcriptome resource and highlight age, sex, and APOE-related microglial immunometabolism perturbations with potential relevance in neurodegeneration.

Alzheimer's disease and progressive supranuclear palsy share similar transcriptomic changes in distinct brain regions.

Vast numbers of differentially expressed genes and perturbed networks have been identified in Alzheimer's disease (AD), however, neither disease nor brain region specificity of these transcriptome alterations has been explored. Using RNA-Seq data from 231 temporal cortex and 224 cerebellum samples from patients with AD and progressive supranuclear palsy (PSP), a tauopathy, we identified a striking correlation in the directionality and magnitude of gene expression changes between these 2 neurodegenerative proteinopathies. Further, the transcriptomic changes in AD and PSP brains ware highly conserved between the temporal and cerebellar cortices, indicating that highly similar transcriptional changes occur in pathologically affected and grossly less affected, albeit functionally connected, areas of the brain. Shared up- or downregulated genes in AD and PSP are enriched in biological pathways. Many of these genes also have concordant protein changes and evidence of epigenetic control. These conserved transcriptomic alterations of 2 distinct proteinopathies in brain regions with and without significant gross neuropathology have broad implications. AD and other neurodegenerative diseases are likely characterized by common disease or compensatory pathways with widespread perturbations in the whole brain. These findings can be leveraged to develop multifaceted therapies and biomarkers that address these common, complex, and ubiquitous molecular alterations in neurodegenerative diseases.

Rapid Screening of Lanthipeptide Analogs via In-Colony Removal of Leader Peptides in Escherichia coli.

Most native producers of ribosomally synthesized and post-translationally modified peptides (RiPPs) utilize N-terminal leader peptides to avoid potential cytotoxicity of mature products to the hosts. Unfortunately, the native machinery of leader peptide removal is often difficult to reconstitute in heterologous hosts. Here we devised a general method to produce bioactive lanthipeptides, a major class of RiPP molecules, in Escherichia coli colonies using synthetic biology principles, where leader peptide removal is programmed temporally by protease compartmentalization and inducible cell autolysis. We demonstrated the method for producing two lantibiotics, haloduracin and lacticin 481, and performed analog screening for haloduracin. This method enables facile, high throughput discovery, characterization, and engineering of RiPPs.

Profiling of Microbial Colonies for High-Throughput Engineering of Multistep Enzymatic Reactions via Optically Guided Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS) imaging has been used for rapid phenotyping of enzymatic activities, but is mainly limited to single-step conversions. Herein we report a label-free method for high-throughput engineering of multistep biochemical reactions based on optically guided MALDI-ToF MS analysis of bacterial colonies. The bacterial cells provide containment of multiple enzymes and access to substrates and cofactors via metabolism. Automated MALDI-ToF MS acquisition from randomly distributed colonies simplifies procedures to prepare strain libraries without liquid handling. MALDI-ToF MS profiling was utilized to screen both substrate and enzyme libraries for natural product biosynthesis. Computational algorithms were developed to process and visualize the resulting mass spectral data sets. For analogues of the peptidic antibiotic plantazolicin, multivariate analyses by t-distributed stochastic neighbor embedding were used to group similar spectra for rapid identification of nonisobaric variants. After MALDI-ToF MS screening, follow-up analyses using high-resolution MS and tandem MS were readily performed on the same sample target. Separately, relative ion intensities of rhamnolipid congeners with various lipid moieties were evaluated to engineer enzymatic specificity. The glycolipid profiles of each colony were overlaid with optical images to facilitate the recovery of desirable mutants. For both the antibiotic and rhamnolipid cases, large populations of colonies were rapidly surveyed at the molecular level, providing information-rich insights not easily obtained with traditional screening assays. Utilizing standard microbiological techniques with routine microscopy and MALDI-ToF MS instruments, this simple yet effective workflow is applicable for a wide range of screening campaigns targeting multistep enzymatic reactions.

Automated multiplex genome-scale engineering in yeast.

Genome-scale engineering is indispensable in understanding and engineering microorganisms, but the current tools are mainly limited to bacterial systems. Here we report an automated platform for multiplex genome-scale engineering in Saccharomyces cerevisiae, an important eukaryotic model and widely used microbial cell factory. Standardized genetic parts encoding overexpression and knockdown mutations of >90% yeast genes are created in a single step from a full-length cDNA library. With the aid of CRISPR-Cas, these genetic parts are iteratively integrated into the repetitive genomic sequences in a modular manner using robotic automation. This system allows functional mapping and multiplex optimization on a genome scale for diverse phenotypes including cellulase expression, isobutanol production, glycerol utilization and acetic acid tolerance, and may greatly accelerate future genome-scale engineering endeavours in yeast.