Burgess, Jeremy D.

Abstract Background Mesenchymal stromal cells (MSCs) have a dynamic secretome that plays a critical role in tissue repair and regeneration. However, studying the MSC secretome in mixed-culture disease models remains challenging. This study aimed to develop a mutant methionyl-tRNA synthetase-based toolkit (MetRSL274G) to selectively profile secreted proteins from MSCs in mixed-culture systems and demonstrate its potential for investigating MSC responses to pathological stimulation.Methods We used CRISPR/Cas9 homology-directed repair to stably integrate MetRSL274G into cells, enabling the incorporation of the non-canonical amino acid, azidonorleucine (ANL), and facilitating selective protein isolation using click chemistry. MetRSL274G was integrated into both in H4 cells and induced pluripotent stem cells (iPSCs) for a series of proof-of-concept studies. Following iPSC differentiation into induced-MSCs, we validated their identity and co-cultured MetRSL274G-expressing iMSCs with naïve or lipopolysaccharide- (LPS) treated THP-1 cells. We then profiled the iMSC secretome using antibody arrays.Results Our results showed successful integration of MetRSL274G into targeted cells, allowing specific isolation of proteins from mixed-culture environments. We also demonstrated that the secretome of MetRSL274G-expressing iMSCs can be differentiated from that of THP-1 cells in co-culture, and is altered when co-cultured with LPS-treated THP-1 cells compared to naïve THP-1 cells.Conclusions The MetRSL274G-based toolkit we have generated enables selective profiling of the MSC secretome in mixed-culture disease models. This approach has broad applications for examining not only MSC responses to models of pathological conditions, but any other cell type that can be differentiated from iPSCs. This can potentially reveal novel MSC-mediated repair mechanisms and advancing our understanding of tissue regeneration processes.

AbstractIntron retention (IR) has been implicated in the pathogenesis of complex diseases such as cancers; its association with Alzheimer's disease (AD) remains unexplored. We performed genome‐wide analysis of IR through integrating genetic, transcriptomic, and proteomic data of AD subjects and mouse models from the Accelerating Medicines Partnership‐Alzheimer's Disease project. We identified 4535 and 4086 IR events in 2173 human and 1736 mouse genes, respectively. Quantitation of IR enabled the identification of differentially expressed genes that conventional exon‐level approaches did not reveal. There were significant correlations of intron expression within innate immune genes, like HMBOX1, with AD in humans. Peptides with a high probability of translation from intron‐retained mRNAs were identified using mass spectrometry. Further, we established AD‐specific intron expression Quantitative Trait Loci, and identified splicing‐related genes that may regulate IR. Our analysis provides a novel resource for the search for new AD biomarkers and pathological mechanisms.

Traumatic brain injury (TBI) is traditionally characterized by primary and secondary injury phases, both contributing to pathological and morphological changes. The mechanisms of damage and chronic consequences of TBI remain to be fully elucidated, but synaptic homeostasis disturbances and impaired energy metabolism are proposed to be a major contributor. It has been proposed that an increase of extracellular (eATP) adenosine triphosphate (ATP) in the area immediately surrounding impact may play a pivotal role in this sequence of events. After tissue injury, rupture of cell membranes allows release of intracellular ATP into the extracellular space, triggering a cascade of toxic events and inflammation. ATP is a ubiquitous messenger; however, simple and reliable techniques to measure its concentration have proven elusive. Here, we integrate a sensitive bioluminescent eATP sensor known as pmeLUC, with a controlled cortical impact mouse model to monitor eATP changes in a living animal after injury. Using the pmeLUC probe, a rapid increase of eATP is observed proximal to the point of impact within minutes of the injury. This event is significantly attenuated when animals are pretreated with an ATP hydrolyzing agent (apyrase) before surgery, confirming the contribution of eATP. This new eATP reporter could be useful for understanding the role of eATP in the pathogenesis in TBI and may identify a window of opportunity for therapeutic intervention.

AbstractIntroductionAbnormal gene expression patterns may contribute to the onset and progression of late‐onset Alzheimer's disease (LOAD).MethodsWe performed transcriptome‐wide meta‐analysis (N = 1440) of blood‐based microarray gene expression profiles as well as neuroimaging and cerebrospinal fluid (CSF) endophenotype analysis.ResultsWe identified and replicated five genes (CREB5, CD46, TMBIM6, IRAK3, and RPAIN) as significantly dysregulated in LOAD. The most significantly altered gene, CREB5, was also associated with brain atrophy and increased amyloid beta (Aβ) accumulation, especially in the entorhinal cortex region. cis‐expression quantitative trait loci mapping analysis of CREB5 detected five significant associations (P < 5 × 10−8), where rs56388170 (most significant) was also significantly associated with global cortical Aβ deposition measured by [18F]Florbetapir positron emission tomography and CSF Aβ1‐42.DiscussionRNA from peripheral blood indicated a differential gene expression pattern in LOAD. Genes identified have been implicated in biological processes relevant to Alzheimer's disease. CREB, in particular, plays a key role in nervous system development, cell survival, plasticity, and learning and memory.

AbstractIntroductionMAPT H1 haplotype is implicated as a risk factor for neurodegenerative diseases including Alzheimer's disease (AD).MethodsUsing Alzheimer's Disease Genetics Consortium (ADGC) genome‐wide association study (GWAS) data (n = 18,841), we conducted a MAPT H1/H2 haplotype–stratified association to discover MAPT haplotype–specific AD risk loci.ResultsWe identified 11 loci—5 in H2‐non‐carriers and 6 in H2‐carriers—although none of the MAPT haplotype–specific associations achieved genome‐wide significance. The most significant H2 non‐carrier–specific association was with a NECTIN2 intronic (P = 1.33E‐07) variant, and that for H2 carriers was near NKX6‐1 (P = 1.99E‐06). The GABRG2 locus had the strongest epistasis with MAPT H1/H2 variant rs8070723 (P = 3.91E‐06). Eight of the 12 genes at these loci had transcriptome‐wide significant differential expression in AD versus control temporal cortex (q < 0.05). Six genes were members of the brain transcriptional co‐expression network implicated in “synaptic transmission” (P = 9.85E‐59), which is also enriched for neuronal genes (P = 1.0E‐164), including MAPT.DiscussionThis stratified GWAS identified loci that may confer AD risk in a MAPT haplotype–specific manner. This approach may preferentially enrich for neuronal genes implicated in synaptic transmission.

AbstractMisfolding and aggregation of alpha‐synuclein (α‐synuclein) with concomitant cytotoxicity is a hallmark of Lewy body related disorders such as Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy. Although it plays a pivotal role in pathogenesis and disease progression, the function of α‐synuclein and the molecular mechanisms underlying α‐synuclein‐induced neurotoxicity in these diseases are still elusive. Many in vitro and in vivo experimental models mimicking α‐synuclein pathology such as oligomerization, toxicity and more recently neuronal propagation have been generated over the years. In particular, cellular models have been crucial for our comprehension of the pathogenic process of the disease and are beneficial for screening of molecules capable of modulating α‐synuclein toxicity. Here, we review α‐synuclein based cell culture models that reproduce some features of the neuronal populations affected in patients, from basic unicellular organisms to mammalian cell lines and primary neurons, to the cutting edge models of patient‐specific cell lines. These reprogrammed cells known as induced pluripotent stem cells (iPSCs) have garnered attention because they closely reproduce the characteristics of neurons found in patients and provide a valuable tool for mechanistic studies. We also discuss how different cell models may constitute powerful tools for high‐throughput screening of molecules capable of modulating α‐synuclein toxicity and prevention of its propagation. image This article is part of the Special Issue “Synuclein”.