Pitx2 Expression Defines a Left Cardiac Lineage of Cells: Evidence for Atrial and Ventricular Molecular Isomerism in the iv/iv Mice

AbstractThe homeobox gene Pitx2 has been characterized as a mediator of left-right signaling in heart, gut, and lung morphogenesis. However, the relationship between the developmental role of Pitx2 and its expression pattern at the organ level has not been explored. In this study we focus on the role of Pitx2 in heart morphogenesis. Chicken Pitx2 transcripts are present in the left portion of the cardiac crescent and in the left side of the heart tube. Through looping Pitx2 is present in the left atrium, in the ventral portion of the ventricles and in the left-ventral part of the outflow tract. Mouse Pitx2 shows a similar developmental profile of expression. To test whether Pitx2 represents a lineage marker we have tagged the left portion of the chicken cardiac tube with fluorescent DiD. Labeled cells were found at HH16 in the left atrium and in the ventral region of the ventricles and the outflow tract. In the iv/iv mouse model of cardiac heterotaxia Pitx2 was abnormally expressed in the atrial and in the ventricular chambers. Furthermore, altered Pitx2 expression correlated with the occurrence of DORV. Our data reveal the existence of molecular isomerism not only in the atrial, but also in the ventricular compartment of the heart

Localization of Acetylcholine, Alpha 7-NAChR and the Antimicrobial Peptide Piscidin 1 in the Macrophages of Fish Gut: Evidence for a Cholinergic System, Diverse Macrophage Populations and Polarization of Immune Responses

20 pages, 9 figures, 2 tables.-- Data Availability Statement: Not applicableThe recognition and elimination of invading pathogens are vital for host survival. Macrophages play a central role in host protection and cells functionally reminiscent of vertebrate macrophages are present in all multicellular organisms. A pattern responsible for bacterial recognition found on the surface of macrophages is CD14. These cells possess a repertoire of antimicrobial molecules stored in their granules and lysosomes. Polarization states observed in mammalian macrophages termed M1 and M2 also likely exist in fish macrophages. Markers for macrophage subtypes are slowly but definitively emerging in fish species. In the present study cell markers such as CD14, acetylcholine, alpha 7 acetylcholine nicotinic receptor (nAChR) subtype, the inducible nitric oxidase synthase (iNOS), and the antimicrobial peptide piscidin 1 are reported for the first time in the intestinal macrophages of both catfish Heteropneustes fossilis (Bloch, 1794) and the African bonytongue Heterotis niloticus (Cuvier, 1829) along the anterior and the posterior axis and the concentric muscle layers. Many antimicrobial effector responses of vertebrate macrophages including respiratory burst and NO induction are similar across the diverse animal taxa. Antibodies against calbindin coupled with ones to VAChT and tubulin revealed the localization of myenteric and submucosal plexuses, which are made up of enteric neurons, glial cells, and nerves near macrophages. Current studies allow for the elucidation of multiple roles of macrophages in disease models providing an insight into their in vivo function in fishPeer reviewe

Identification and distribution of neuronal nitric oxide synthase and neurochemical markers in the neuroepithelial cells of the gill and the skin in the giant mudskipper, Periophthalmodon schlosseri

Mudskippers are amphibious fishes living in mudflats and mangroves. These fishes hold air in their large buccopharyngeal-opercular cavities where respiratory gas exchange takes place via the gills and higher vascularized epithelium lining the cavities and also the skin epidermis. Although aerial ventilation response to changes in ambient gas concentration has been studied in mudskippers, the localization and distribution of respiratory chemoreceptors, their neurochemical coding and function as well as physiological evidence for the gill or skin as site for O2 and CO2 sensing are currently not known. In the present study we assessed the distribution of serotonin, acetylcholine, catecholamines and nitric oxide in the neuroepithelial cells (NECs) of the mudskipper gill and skin epithelium using immunohistochemistry and confocal microscopy. Colocalization studies showed that 5-HT is coexpressed with nNOS, Na+/K+-ATPase, TH and VAChT; nNOS is coexpressed with Na+/K+-ATPase and TH in the skin. In the gill 5-HT is coexpressed with nNOS and VAhHT and nNOS is coexpressed with Na+/K+-ATPase and TH. Acetylcholine is also expressed in chain and proximal neurons projecting to the efferent filament artery and branchial smooth muscle. The serotonergic cells c labeled with VAChT, nNOS and TH, thus indicating the presence of NEC populations and the possibility that these neurotransmitters (other than serotonin) may act as primary transmitters in the hypoxic reflex in fish gills. Immunolabeling with TH antibodies revealed that NECs in the gill and the skin are innervated by catecholaminergic nerves, thus suggesting that these cells are involved in a central control of branchial functions through their relationships with the sympathetic branchial nervous system. The Na+/K+-ATPase in mitochondria-rich cells (MRCs), which are most concentrated in the gill lamellar epithelium, is colabeled with nNOS and associated with TH nerve terminals. TH-immunopositive fine varicosities were also associated with the numerous capillaries in the skin surface and the layers of the swollen cells. Based on the often hypercapnic and hypoxic habitat of the mudskippers, these fishes may represent an attractive model for pursuing studies on O2 and CO2 sensing due to the air-breathing that increases the importance of acid/base regulation and the O2-related drive including the function of gasotransmitters such as nitric oxide that has an inhibitory (regulatory) function in ionoregulation.This research was supported by project PAN LAB PONA3_00166. The authors would like to thank Michał Ignaszewski (TDT) for his kind help in statistical analysis

Mitochondria function associated genes contribute to Parkinson's Disease risk and later age at onset

Mitochondrial dysfunction has been implicated in the etiology of monogenic Parkinson’s disease (PD). Yet the role that mitochondrial processes play in the most common form of the disease; sporadic PD, is yet to be fully established. Here, we comprehensively assessed the role of mitochondrial function-associated genes in sporadic PD by leveraging improvements in the scale and analysis of PD GWAS data with recent advances in our understanding of the genetics of mitochondrial disease. We calculated a mitochondrial-specific polygenic risk score (PRS) and showed that cumulative small effect variants within both our primary and secondary gene lists are significantly associated with increased PD risk. We further reported that the PRS of the secondary mitochondrial gene list was significantly associated with later age at onset. Finally, to identify possible functional genomic associations we implemented Mendelian randomization, which showed that 14 of these mitochondrial functionassociated genes showed functional consequence associated with PD risk. Further analysis suggested that the 14 identified genes are not only involved in mitophagy, but implicate new mitochondrial processes. Our data suggests that therapeutics targeting mitochondrial bioenergetics and proteostasis pathways distinct from mitophagy could be beneficial to treating the early stage of PD

Moving beyond neurons: the role of cell type-specific gene regulation in Parkinson's disease heritability

Parkinson’s disease (PD), with its characteristic loss of nigrostriatal dopaminergic neurons and deposition of α-synuclein in neurons, is often considered a neuronal disorder. However, in recent years substantial evidence has emerged to implicate glial cell types, such as astrocytes and microglia. In this study, we used stratified LD score regression and expression-weighted cell-type enrichment together with several brain-related and cell-type-specific genomic annotations to connect human genomic PD findings to specific brain cell types. We found that PD heritability attributable to common variation does not enrich in global and regional brain annotations or brain-related cell-type-specific annotations. Likewise, we found no enrichment of PD susceptibility genes in brain-related cell types. In contrast, we demonstrated a significant enrichment of PD heritability in a curated lysosomal gene set highly expressed in astrocytic, microglial, and oligodendrocyte subtypes, and in LoF-intolerant genes, which were found highly expressed in almost all tested cellular subtypes. Our results suggest that PD risk loci do not lie in specific cell types or individual brain regions, but rather in global cellular processes detectable across several cell types

Identification of novel risk loci, causal insights, and heritable risk for Parkinson's disease: a meta-analysis of genome-wide association studies

Background Genome-wide association studies (GWAS) in Parkinson's disease have increased the scope of biological knowledge about the disease over the past decade. We aimed to use the largest aggregate of GWAS data to identify novel risk loci and gain further insight into the causes of Parkinson's disease. Methods We did a meta-analysis of 17 datasets from Parkinson's disease GWAS available from European ancestry samples to nominate novel loci for disease risk. These datasets incorporated all available data. We then used these data to estimate heritable risk and develop predictive models of this heritability. We also used large gene expression and methylation resources to examine possible functional consequences as well as tissue, cell type, and biological pathway enrichments for the identified risk factors. Additionally, we examined shared genetic risk between Parkinson's disease and other phenotypes of interest via genetic correlations followed by Mendelian randomisation. Findings Between Oct 1, 2017, and Aug 9, 2018, we analysed 7·8 million single nucleotide polymorphisms in 37 688 cases, 18 618 UK Biobank proxy-cases (ie, individuals who do not have Parkinson's disease but have a first degree relative that does), and 1·4 million controls. We identified 90 independent genome-wide significant risk signals across 78 genomic regions, including 38 novel independent risk signals in 37 loci. These 90 variants explained 16–36% of the heritable risk of Parkinson's disease depending on prevalence. Integrating methylation and expression data within a Mendelian randomisation framework identified putatively associated genes at 70 risk signals underlying GWAS loci for follow-up functional studies. Tissue-specific expression enrichment analyses suggested Parkinson's disease loci were heavily brain-enriched, with specific neuronal cell types being implicated from single cell data. We found significant genetic correlations with brain volumes (false discovery rate-adjusted p=0·0035 for intracranial volume, p=0·024 for putamen volume), smoking status (p=0·024), and educational attainment (p=0·038). Mendelian randomisation between cognitive performance and Parkinson's disease risk showed a robust association (p=8·00 × 10−7). Interpretation These data provide the most comprehensive survey of genetic risk within Parkinson's disease to date, to the best of our knowledge, by revealing many additional Parkinson's disease risk loci, providing a biological context for these risk factors, and showing that a considerable genetic component of this disease remains unidentified. These associations derived from European ancestry datasets will need to be followed-up with more diverse data. Funding The National Institute on Aging at the National Institutes of Health (USA), The Michael J Fox Foundation, and The Parkinson's Foundation (see appendix for full list of funding sources)

Regulatory sites for splicing in human basal ganglia are enriched for disease-relevant information

Genome-wide association studies have generated an increasing number of common genetic variants associated with neurological and psychiatric disease risk. An improved understanding of the genetic control of gene expression in human brain is vital considering this is the likely modus operandum for many causal variants. However, human brain sampling complexities limit the explanatory power of brain-related expression quantitative trait loci (eQTL) and allele-specific expression (ASE) signals. We address this, using paired genomic and transcriptomic data from putamen and substantia nigra from 117 human brains, interrogating regulation at different RNA processing stages and uncovering novel transcripts. We identify disease-relevant regulatory loci, find that splicing eQTLs are enriched for regulatory information of neuron-specific genes, that ASEs provide cell-specific regulatory information with evidence for cellular specificity, and that incomplete annotation of the brain transcriptome limits interpretation of risk loci for neuropsychiatric disease. This resource of regulatory data is accessible through our web server, http://braineacv2.inf.um.es/

Exome sequencing identifies rare damaging variants in ATP8B4 and ABCA1 as risk factors for Alzheimer’s disease

Alzheimer’s disease (AD), the leading cause of dementia, has an estimated heritability of approximately 70%1. The genetic component of AD has been mainly assessed using genome-wide association studies, which do not capture the risk contributed by rare variants2. Here, we compared the gene-based burden of rare damaging variants in exome sequencing data from 32,558 individuals—16,036 AD cases and 16,522 controls. Next to variants in TREM2, SORL1 and ABCA7, we observed a significant association of rare, predicted damaging variants in ATP8B4 and ABCA1 with AD risk, and a suggestive signal in ADAM10. Additionally, the rare-variant burden in RIN3, CLU, ZCWPW1 and ACE highlighted these genes as potential drivers of respective AD-genome-wide association study loci. Variants associated with the strongest effect on AD risk, in particular loss-of-function variants, are enriched in early-onset AD cases. Our results provide additional evidence for a major role for amyloid-β precursor protein processing, amyloid-β aggregation, lipid metabolism and microglial function in AD

Exome sequencing identifies rare damaging variants in ATP8B4 and ABCA1 as novel risk factors for Alzheimers Disease

The genetic component of Alzheimer’s disease (AD) has been mainly assessed using Genome Wide Association Studies (GWAS), which do not capture the risk contributed by rare variants. Here, we compared the gene-based burden of rare damaging variants in exome sequencing data from 32,558 individuals —16,036 AD cases and 16,522 controls— in a two-stage analysis. Next to known genes TREM2, SORL1 and ABCA7, we observed a significant association of rare, predicted damaging variants in ATP8B4 and ABCA1 with AD risk, and a suggestive signal in ADAM10. Next to these genes, the rare variant burden in RIN3, CLU, ZCWPW1 and ACE highlighted these genes as potential driver genes in AD-GWAS loci. Rare damaging variants in these genes, and in particular loss-of-function variants, have a large effect on AD-risk, and they are enriched in early onset AD cases. The newly identified AD-associated genes provide additional evidence for a major role for APP-processing, Aβ-aggregation, lipid metabolism and microglial function in AD

Identification of candidate Parkinson disease genes by integrating genome-wide association study, expression, and epigenetic data sets

Importance Substantial genome-wide association study (GWAS) work in Parkinson disease (PD) has led to the discovery of an increasing number of loci shown reliably to be associated with increased risk of disease. Improved understanding of the underlying genes and mechanisms at these loci will be key to understanding the pathogenesis of PD. Objective To investigate what genes and genomic processes underlie the risk of sporadic PD. Design and Setting This genetic association study used the bioinformatic tools Coloc and transcriptome-wide association study (TWAS) to integrate PD case-control GWAS data published in 2017 with expression data (from Braineac, the Genotype-Tissue Expression [GTEx], and CommonMind) and methylation data (derived from UK Parkinson brain samples) to uncover putative gene expression and splicing mechanisms associated with PD GWAS signals. Candidate genes were further characterized using cell-type specificity, weighted gene coexpression networks, and weighted protein-protein interaction networks. Main Outcomes and Measures It was hypothesized a priori that some genes underlying PD loci would alter PD risk through changes to expression, splicing, or methylation. Candidate genes are presented whose change in expression, splicing, or methylation are associated with risk of PD as well as the functional pathways and cell types in which these genes have an important role. Results Gene-level analysis of expression revealed 5 genes (WDR6 [OMIM 606031], CD38 [OMIM 107270], GPNMB [OMIM 604368], RAB29 [OMIM 603949], and TMEM163 [OMIM 618978]) that replicated using both Coloc and TWAS analyses in both the GTEx and Braineac expression data sets. A further 6 genes (ZRANB3 [OMIM 615655], PCGF3 [OMIM 617543], NEK1 [OMIM 604588], NUPL2 [NCBI 11097], GALC [OMIM 606890], and CTSB [OMIM 116810]) showed evidence of disease-associated splicing effects. Cell-type specificity analysis revealed that gene expression was overall more prevalent in glial cell types compared with neurons. The weighted gene coexpression performed on the GTEx data set showed that NUPL2 is a key gene in 3 modules implicated in catabolic processes associated with protein ubiquitination and in the ubiquitin-dependent protein catabolic process in the nucleus accumbens, caudate, and putamen. TMEM163 and ZRANB3 were both important in modules in the frontal cortex and caudate, respectively, indicating regulation of signaling and cell communication. Protein interactor analysis and simulations using random networks demonstrated that the candidate genes interact significantly more with known mendelian PD and parkinsonism proteins than would be expected by chance. Conclusions and Relevance Together, these results suggest that several candidate genes and pathways are associated with the findings observed in PD GWAS studies