Search Results (96)

Down syndrome (DS), stemming from the triplication of human chromosome 21, results in intellectual disability, with early mid-life onset of Alzheimer’s disease (AD) pathology. Early interventions to reduce cognitive impairments and neuropathology are lacking. One modality, maternal choline supplementation (MCS), has shown beneficial effects on behavior and gene expression in neurodevelopmental and neurodegenerative disorders, including trisomic mice. Loss of basal forebrain cholinergic neurons (BFCNs) and other DS/AD relevant hallmarks were observed in a well-established trisomic model (Ts65Dn, Ts). MCS attenuates these endophenotypes with beneficial behavioral effects in trisomic offspring. We postulate MCS ameliorates dysregulated cellular mechanisms within vulnerable BFCNs, with attenuation driven by novel gene expression. Here, choline acetyltransferase immunohistochemical labeling identified BFCNs in the medial septal/ventral diagonal band nuclei of the basal forebrain in Ts and normal disomic (2N) offspring at ~11 months of age from dams exposed to MCS or normal choline during the perinatal period. BFCNs (~500 per mouse) were microisolated and processed for RNA-sequencing. Bioinformatic assessment elucidated differentially expressed genes (DEGs) and pathway alterations in the context of genotype (Ts, 2N) and maternal diet (MCS, normal choline). MCS attenuated select dysregulated DEGs and relevant pathways in aged BFCNs. Trisomic MCS-responsive improvements included pathways such as cognitive impairment and nicotinamide adenine dinucleotide signaling, among others, indicative of increased behavioral and bioenergetic fitness. Although MCS does not eliminate the DS/AD phenotype, early choline delivery provides long-lasting benefits to aged trisomic BFCNs, indicating that MCS prolongs neuronal health in the context of DS/AD. Full article

Wood is an important raw material for industrial applications. Its fiber-specific genetic modification provides an effective strategy to alter wood characteristics in tree breeding. Here, we performed a cross-analysis of previously reported single-cell RNA sequencing and the AspWood database during wood formation to identify potential xylem fiber-dominant expressing genes in poplar. As a result, 32 candidate genes were obtained, and subsequently, we further examined the expression of these genes in fibers and/or vessels of stem secondary xylem using the laser capture microdissection technique and RT-qPCR. Analysis identified nine candidate genes, including PtrFLA12-2, PtrIRX12, PtrFLA12-6, PtrMYB52, PtrMYB103, PtrMAP70, PtrLRR-1, PtrKIFC2-3, and PtrNAC12. Next, we cloned the promoter regions of the nine candidate genes and created promoter::GUS transgenic poplars. Histochemical GUS staining was used to investigate the tissue expression activities of these gene promoters in transgenic poplars. In one month, transgenic plantlets grown in medium showed intensive GUS staining signals that were visible in the leaves and apical buds, suggesting substantial expression activities of these gene promoters in plantlets predominantly undergoing primary growth. In contrast, for three-month-old transgenic poplars in the greenhouse with predominantly developed secondary stem tissues, the promoters of seven of nine candidate genes, including PtrMYB103, PtrIRX12, and PtrMAP70, showed secondary xylem fiber-dominant GUS signals with considerable spatial specificity. Overall, this study presents xylem fiber-dominant promoters that are well-suited for specifically expressing genes of interest in wood fibers for forest tree breeding. Full article

Understanding the heterogeneity of Rheumatoid Arthritis (RA) and identifying therapeutic targets remain challenging using traditional bulk transcriptomics alone, as it lacks the spatial and protein-level resolution needed to fully capture disease and tissue complexities. In this study, we applied Laser Capture Microdissection (LCM) coupled with mass spectrometry-based proteomics to analyze histopathological niches of the RA synovium, enabling the identification of protein expression profiles of the diseased synovial lining and sublining microenvironments compared to their healthy counterparts. In this respect, key pathogenetic RA proteins like membrane proteins (TYROBP, AOC3, SLC16A3, TCIRG1, and NCEH1), and extracellular matrix (ECM) proteins (PLOD2, OGN, and LUM) showed different expression patterns in diseased synovium compartments. To enhance our understanding of cellular dynamics within the dissected regions, we further integrated the proteomic dataset with single-cell RNA sequencing (scRNA-seq), and deduced cell type enrichment, including T cells, fibroblasts, NK cells, myeloid cells, B cells, and synovial endothelial cells. By combining high-resolution spatial proteomics and transcriptomic analyses, we provide novel insights into the molecular mechanisms driving RA, and highlight potential protein targets for therapeutic intervention. This integrative approach offers a more comprehensive view of RA synovial pathology, and mitigates the limitations of traditional bulk transcriptomics in target discovery. Full article

Background: Tomatoes are self-pollinating plants, and successful fruit set depends on the production of functional pollen within the same flower. Our previous studies have shown that the ‘Black Vernissage’ tomato variety exhibits greater resilience to heat stress in terms of pollen productivity compared to the ‘Micro-Tom’ variety. Pollen productivity is determined by meiotic activity during microsporogenesis and the development of free microspores during gametogenesis. This study focused on identifying heat stress (HS)-induced proteomes in pollen mother cells (PMCs) and microspores. Methods: Tomato plants were grown under two temperature conditions: 26 °C (non-heat-treated control) and 37 °C (heat-treated). Homogeneous cell samples of meiotic PMCs (prior to the tetrad stage) and free microspores were collected using laser capture microdissection (LCM). The heat-induced proteomes were identified using tandem mass tag (TMT)–quantitative proteomics analysis. Results: The enrichment of the meiotic cell cycle in PMCs and the pre-mitotic process in free microspores confirmed the correlation between proteome expression and developmental stage. Under HS, PMCs in both tomato varieties were enriched with heat shock proteins (HSPs). However, the ‘Black Vernissage’ variety exhibited a greater diversity of HSP species and a higher level of enrichment compared to the ‘Micro-Tom’ variety. Additionally, several proteins involved in gene expression and protein translation were downregulated in PMCs and microspores of both varieties. In the PMC proteomes, the relative abundance of proteins showed no significant differences between the two varieties under normal conditions, with very few exceptions. However, HS induced significant differential expression both within and between the varieties. More importantly, these heat-induced differentially abundant proteins (DAPs) in PMCs are directly involved in meiotic cell division, including the meiosis-specific protein ASY3 (Solyc01g079080), the cell division protein kinase 2 (Solyc11g070140), COP9 signalosome complex subunit 1 (Solyc01g091650), the kinetochore protein ndc80 (Solyc01g104570), MORC family CW-type zinc finger 3 (Solyc02g084700), and several HSPs that function in protecting the fidelity of the meiotic processes, including the DNAJ chaperone (Solyc04g009770, Solyc05g055160), chaperone protein htpG (Solyc04g081570), and class I and class II HSPs. In the microspores, most of the HS-induced DAPs were consistently observed across both varieties, with only a few proteins showing significant differences between them under heat stress. These HS-induced DAPs include proteases, antioxidant proteins, and proteins related to cell wall remodeling and the generation of pollen exine. Conclusions: HS induced more dynamic proteomic changes in meiotic PMCs compared to microspores, and the inter-varietal differences in the PMC proteomes align with the effects of HS on pollen productivity observed in the two varieties. This research highlights the importance of the cell-type-specific proteomics approach in identifying the molecular mechanisms that are critical for the pollen developmental process under elevated temperature conditions. Full article

Podocyte injury is a hallmark of both focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD), ultimately reflected in foot process effacement and proteinuria. Triggers and pathogenic pathways leading to podocyte cytoskeleton rearrangements are, however, incompletely explained. Here, we aimed to contribute to the understanding of these pathways using tissue bottom-up proteomic profiling of laser-capture microdissected glomeruli from MCD and FSGS. Forty-six differentially expressed proteins were identified between the two groups (p < 0.05, |FC| ≥ 1.2). Pathway analysis showed that 16 out of 46 proteins were associated with the immune system, with E2 ubiquitin-conjugating enzyme (UBE2K) and complement factor H-related protein-1 (CFHR1) yielding the highest fold change in FSGS compared to MCD. The two target proteins were further validated through immunohistochemistry, confirming the podocyte localization of UBE2K and endothelial staining of CFHR. Additionally, several other differentially expressed proteins were linked to the cytoskeleton structure and its regulation. Our results point to the possibility that complement dysregulation may be the source of cytoskeleton rearrangement in FSGS. Full article

In celiac disease (CeD), interleukin 15 (IL-15) affects the epithelial barrier by acting on intraepithelial lymphocytes, promoting interferon γ (IFN-γ) production and inducing strong cytotoxic activity as well as eliciting apoptotic death of enterocytes by the Fas/Fas ligand system. This study investigates the effects of a monoclonal antibody neutralizing the effects of IL-15 (aIL-15) on tissue-damaging immune response in untreated CeD patients by using an organ culture system. Jejunal biopsies from 10 untreated CeD patients were cultured ex vivo with or without aIL-15. Epithelial expressions of CD95/Fas, HLA-E and perforin were analyzed by immunohistochemistry. Apoptosis was detected in the epithelium by using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Additionally, the surface epithelium compartment of ex vivo cultured biopsy samples was isolated by laser capture microdissection (LCM). RNA from each LCM sample was extracted and the relative expression of IFN-γ was evaluated by quantitative reverse transcriptase-PCR (qRT-PCR). Biopsies cultured with the aIL-15 antibody showed a reduction in Fas, HLA-E and perforin epithelial expression, as well as a decrease in epithelial TUNEL+ cells compared to biopsies cultured without the aIL-15 antibody. Moreover, downregulation of epithelial IFN-γ expression was recorded in biopsies incubated with aIL-15, compared to those cultured without aIL-15. Our findings suggest that neutralizing the effects of IL-15 in ex vivo cultured untreated CeD intestinal mucosa could block apoptosis by downregulating Fas and HLA-E expression and the release of cytotoxic proteins, such as perforin. Furthermore, it can dampen the hyperactive immune response by reducing IFN-γ expression. More generally, our study provides new evidence for the effects of anti-IL-15 neutralizing monoclonal antibodies in preventing or repairing epithelial damage and further supports the concept that IL-15 is a meaningful therapeutic target in CeD, or inflammatory diseases associated with the upregulation of IL-15. Full article

Proteomics accelerates diagnosis and research of muscular diseases by enabling the robust analysis of proteins relevant for the manifestation of neuromuscular diseases in the following aspects: (i) evaluation of the effect of genetic variants on the corresponding protein, (ii) prediction of the underlying genetic defect based on the proteomic signature of muscle biopsies, (iii) analysis of pathophysiologies underlying different entities of muscular diseases, key for the definition of new intervention concepts, and (iv) patient stratification according to biochemical fingerprints as well as (v) monitoring the success of therapeutic interventions. This review presents—also through exemplary case studies—the various advantages of mass proteomics in the investigation of genetic muscle diseases, discusses technical limitations, and provides an outlook on possible future application concepts. Hence, proteomics is an excellent large-scale analytical tool for the diagnostic workup of (hereditary) muscle diseases and warrants systematic profiling of underlying pathophysiological processes. The steady development may allow to overcome existing limitations including a quenched dynamic range and quantification of different protein isoforms. Future directions may include targeted proteomics in diagnostic settings using not only muscle biopsies but also liquid biopsies to address the need for minimally invasive procedures. Full article

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterized by repetitive alveolar injuries with excessive deposition of extracellular matrix (ECM) proteins. A crucial need in understanding IPF pathogenesis is identifying cell types associated with histopathological regions, particularly local fibrosis centers known as fibroblast foci. To address this, we integrated published spatial transcriptomics and single-cell RNA sequencing (scRNA-seq) transcriptomics and adopted the Query method and the Overlap method to determine cell type enrichments in histopathological regions. Distinct fibroblast cell types are highly associated with fibroblast foci, and transitional alveolar type 2 and aberrant KRT5-/KRT17+ (KRT: keratin) epithelial cells are associated with morphologically normal alveoli in human IPF lungs. Furthermore, we employed laser capture microdissection-directed mass spectrometry to profile proteins. By comparing with another published similar dataset, common differentially expressed proteins and enriched pathways related to ECM structure organization and collagen processing were identified in fibroblast foci. Importantly, cell type enrichment results from innovative spatial proteomics and scRNA-seq data integration accord with those from spatial transcriptomics and scRNA-seq data integration, supporting the capability and versatility of the entire approach. In summary, we integrated spatial multi-omics with scRNA-seq data to identify disease-associated cell types and potential targets for novel therapies in IPF intervention. The approach can be further applied to other disease areas characterized by spatial heterogeneity. Full article

Background: This study explores the potential for hidden variations within seemingly uniform regions of growth hormone-secreting pituitary neuroendocrine tumors (GH-PitNETs). We employed archived tissue samples using Laser Capture Microdissection Sequencing (LCM-RNAseq) to probe the molecular landscape of these tumors at a deeper level. Methods: A customized protocol was developed to extract, process, and sequence small amounts of RNA from formalin-fixed, paraffin-embedded (FFPE) tissues derived from five patients with GH-secreting PitNETs and long-term follow-up (≥10 years). This approach ensured precise isolation of starting material of enough quality for subsequent sequencing. Results: The LCM-RNAseq analysis revealed a surprising level of diversity within seemingly homogeneous tumor regions. Interestingly, the 30 most highly expressed genes included the well-known long noncoding RNA (lncRNA) MALAT1. We further validated the levels of MALAT1 and of other tumor-associated lncRNAs using digital droplet PCR. Conclusions: This study demonstrates the potential of LCM-RNAseq to unlock hidden molecular diversity within archived pituitary tumor samples. By focusing on specific cell populations, we identified lncRNAs expressed at different levels within the tumors, potentially offering new insights into the complex biology of GH-secreting PitNETs. This evidence prompts further research into the role of lncRNAs in pituitary neuroendocrine tumor aggressiveness and personalized treatment strategies. Full article

Inflammatory breast cancer (IBC) is characterized by numerous tumor emboli within lymphatics. In a recent study, we observed tumor embolic budding both in vitro and in vivo within lymphovascular spaces and proposed this to account for the plethora of tumor emboli seen in IBC. These observations did not address, however, how lymphovascular invasion is initiated or the mechanisms involved. In the present study, using the well-characterized patient-derived xenograft (PDX), Mary-X, which exhibited florid lymphovascular invasion (LVI) in athymic mice (LVI) as defined by E-cadherin-positive tumor emboli within lymphatic channels distinguished by podoplanin and LYVE1 membrane and Prox1 nuclear immunoreactivities and spontaneous spheroidgenesis in vitro and human cases of IBC which showed similar LVI, we compared laser-captured microdissected emboli from Mary-X and from the cases of human IBC to non-embolic areas. Mary-X and IBC emboli expressed high levels of E-cadherin and no evidence of epithelial–mesenchymal transition (EMT). Mary-X spheroids expressed high levels of VEGF, especially VEGF-C, and stimulated both vascular and lymphatic endothelial haptotaxis. We then transplanted Mary-X serially into green, cyano, red, and nestin-green fluorescing protein (GFP-, CFP-, RFP-, and nestin-GFP) transgenic reporter mice in various combinations. Multicolor murine imaging studies indicated that reporter-labeled stroma initially encircled clumps of tumor cells and then served as a scaffold that supported nestin-GFP-labeled endothelial haptotaxis resulting in encircling lymphangiogenesis, confirmed by dual LYVE1 immunofluorescence. The present studies demonstrate a possible mechanism of a critical step of the tumor emboli formation of IBC. Full article

The neuromuscular junction (NMJ) is the site where the motor neuron innervates skeletal muscle, enabling muscular contraction. Congenital myasthenic syndromes (CMS) arise when mutations in any of the approximately 35 known causative genes cause impaired neuromuscular transmission at the NMJ, resulting in fatigable muscle weakness. A subset of five of these CMS-causative genes are associated with protein glycosylation. Glutamine-fructose-6-phosphate transaminase 1 (Gfpt1) is the rate-limiting enzyme within the hexosamine biosynthetic pathway (HBP), a metabolic pathway that produces the precursors for glycosylation. We hypothesized that deficiency in Gfpt1 expression results in aberrant or reduced glycosylation, impairing the proper assembly and stability of key NMJ-associated proteins. Using both in vitro and in vivo Gfpt1-deficient models, we determined that the acetylcholine receptor delta subunit (AChRδ) has reduced expression and is hypo-glycosylated. Using laser capture microdissection, NMJs were harvested from Gfpt1 knockout mouse muscle. A lower-molecular-weight species of AChRδ was identified at the NMJ that was not detected in controls. Furthermore, Gfpt1-deficient muscle lysates showed impairment in protein O-GlcNAcylation and sialylation, suggesting that multiple glycan chains are impacted. Other key NMJ-associated proteins, in addition to AChRδ, may also be differentially glycosylated in Gfpt1-deficient muscle. Full article

One of the main hallmarks of aging is aging-associated inflammation, also known as inflammaging. In this study, by comparing plasma and kidney proteome profiling of young and old mice using LC–MS profiling, we discovered that immunoglobulins are the proteins that exhibit the highest increase with age. This observation seems to have been disregarded because conventional proteome profiling experiments typically overlook the expression of high-abundance proteins or employ depletion methods to remove them before LC–MS analysis. We show that proteome profiling of immunoglobulins will likely be a useful biomarker of aging. Spatial profiling using immunofluorescence staining of kidney sections indicates that the main increases in immunoglobulins with age are localized in the glomeruli of the kidney. Using laser capture microdissection coupled with LC–MS, we show an increase in multiple immune-related proteins in glomeruli from aged mice. Increased deposition of immunoglobulins, immune complexes, and complement proteins in the kidney glomeruli may be a factor leading to reduced filtering capacity of the kidney with age. Therapeutic strategies to reduce the deposition of immunoglobulins in the kidney may be an attractive strategy for healthy aging. Full article

The mechanism underlying podocyte dysfunction in minimal change disease (MCD) remains unknown. This study aimed to shed light on the potential pathophysiology of MCD using glomerular proteomic analysis. Shotgun proteomics using label-free quantitative mass spectrometry was performed on formalin-fixed, paraffin-embedded (FFPE) renal biopsies from two groups of samples: control (CTR) and MCD. Glomeruli were excised from FFPE renal biopsies using laser capture microdissection (LCM), and a single-pot solid-phase-enhanced sample preparation (SP3) digestion method was used to improve yield and protein identifications. Principal component analysis (PCA) revealed a distinct separation between the CTR and MCD groups. Forty-eight proteins with different abundance between the two groups (p-value ≤ 0.05 and |FC| ≥ 1.5) were identified. These may represent differences in podocyte structure, as well as changes in endothelial or mesangial cells and extracellular matrix, and some were indeed found in several of these structures. However, most differentially expressed proteins were linked to the podocyte cytoskeleton and its dynamics. Some of these proteins are known to be involved in focal adhesion (NID1 and ITGA3) or slit diaphragm signaling (ANXA2, TJP1 and MYO1C), while others are structural components of the actin and microtubule cytoskeleton of podocytes (ACTR3 and NES). This study suggests the potential of mass spectrometry-based shotgun proteomic analysis with LCM glomeruli to yield valuable insights into the pathogenesis of podocytopathies like MCD. The most significantly dysregulated proteins in MCD could be attributable to cytoskeleton dysfunction or may be a compensatory response to cytoskeleton malfunction caused by various triggers. Full article

Mycoplasma hyopneumoniae (Mhyo) is the causative agent of porcine enzootic pneumonia (EP), as well as one of the main pathogens involved in the porcine respiratory disease complex. The host–pathogen interaction between Mhyo and infected pigs is complex and not completely understood; however, improving the understanding of these intricacies is essential for the development of effective control strategies of EP. In order to improve our knowledge about this interaction, laser-capture microdissection was used to collect bronchi, bronchi-associated lymphoid tissue, and lung parenchyma from animals infected with different strains of Mhyo, and mRNA expression levels of different molecules involved in Mhyo infection (ICAM1, IL-8, IL-10, IL-23, IFN-α, IFN-γ, TGF-β, and TNF-α) were analyzed by qPCR. In addition, the quantification of Mhyo load in the different lung compartments and the scoring of macroscopic and microscopic lung lesions were also performed. Strain-associated differences in virulence were observed, as well as the presence of significant differences in expression levels of cytokines among lung compartments. IL-8 and IL-10 presented the highest upregulation, with limited differences between strains and lung compartments. IFN-α was strongly downregulated in BALT, implying a relevant role for this cytokine in the immunomodulation associated with Mhyo infections. IL-23 was also upregulated in all lung compartments, suggesting the potential involvement of a Th17-mediated immune response in Mhyo infections. Our findings highlight the relevance of Th1 and Th2 immune response in cases of EP, shedding light on the gene expression levels of key cytokines in the lung of pigs at a microscopic level. Full article

Age-associated deep-subcortical white matter lesions (DSCLs) are an independent risk factor for dementia, displaying high levels of CD68⁺ microglia. This study aimed to characterize the transcriptomic profile of microglia in DSCLs and surrounding radiologically normal-appearing white matter (NAWM) compared to non-lesional control white matter. CD68⁺ microglia were isolated from white matter groups (n = 4 cases per group) from the Cognitive Function and Ageing Study neuropathology cohort using immuno-laser capture microdissection. Microarray gene expression profiling, but not RNA-sequencing, was found to be compatible with immuno-LCM-ed post-mortem material in the CFAS cohort and identified significantly differentially expressed genes (DEGs). Functional grouping and pathway analysis were assessed using the Database for Annotation Visualization and Integrated Discovery (DAVID) software, and immunohistochemistry was performed to validate gene expression changes at the protein level. Transcriptomic profiling of microglia in DSCLs compared to non-lesional control white matter identified 181 significant DEGs (93 upregulated and 88 downregulated). Functional clustering analysis in DAVID revealed dysregulation of haptoglobin–haemoglobin binding (Enrichment score 2.5, p = 0.017), confirmed using CD163 immunostaining, suggesting a neuroprotective microglial response to blood–brain barrier dysfunction in DSCLs. In NAWM versus control white matter, microglia exhibited 347 DEGs (209 upregulated, 138 downregulated), with significant dysregulation of protein de-ubiquitination (Enrichment score 5.14, p < 0.001), implying an inability to maintain protein homeostasis in NAWM that may contribute to lesion spread. These findings enhance understanding of microglial transcriptomic changes in ageing white matter pathology, highlighting a neuroprotective adaptation in DSCLs microglia and a potentially lesion-promoting phenotype in NAWM microglia. Full article