Recently, the myelin proteolipid protein gene (
) was shown to be expressed in the glia of the enteric nervous system (ENS) in mouse. However, beyond this, not much is known about its expression in ...the intestine. To address this matter, we investigated
expression at the mRNA and protein levels in the intestine of mice at different ages (postnatal days 2, 9, 21, and 88). In this study, we show that
expression preferentially occurs during early postnatal development, primarily as the DM20 isoform. Western blot analysis indicated that DM20 migrated according to its formula weight when isolated from the intestine. However, mobilities of both PLP and DM20 were faster than expected when procured from the brain. The 6.2hPLP(+)Z/FL transgene, which uses the first half of the human
gene to drive expression of a
reporter gene, recapitulated the developmental pattern observed with the native gene in the intestine, indicating that it can be used as a proxy for
gene expression. As such, the relative levels of β-galactosidase (β-gal) activity emanating from the 6.2hPLP(+)Z/FL transgene suggest that
expression is highest in the duodenum, and decreases successively along the segments, toward the colon. Moreover, removal of the wmN1 enhancer region from the transgene (located within
intron 1) resulted in a dramatic reduction in both transgene mRNA levels and β-gal activity in the intestine, throughout development, suggesting that this region contains a regulatory element crucial for
expression. This is consistent with earlier studies in both the central and peripheral nervous systems, indicating that it may be a common (if not universal) means by which
gene expression is governed.
Much of what is known about the mechanisms that control the developmental expression of the myelin proteolipid protein gene (
) has been attained through use of transgenic animal models. In this ...study, we analyzed expression of related transgenes which utilize
genomic DNA from either human or mouse to drive expression of a
reporter. Human
(
) sequence span either the proximal 6.2 or 2.7 kb of 5'-flanking DNA to an internal site in Exon 2, while those from mouse comprise the proximal 2.3 kb of 5'-flanking DNA to an analogous site in Exon 2. Transgenes with
sequence were named, in part, to the amount of upstream sequence they have 6.2hPLP(+)Z/FL and 2.7hPLP(+)Z. The transgene containing mouse sequence is referred to here as mPLP(+)Z, to denote the species origin of
DNA. Mice which harbor the 6.2hPLP(+)Z/FL transgene were used as a model system to investigate the developmental expression of splice variants that incorporate supplementary exons from what is classically defined as
intron 1. While expression of the splice variants were detected in brain through RT-PCR analysis, they are present at much lower levels relative to the archetypal (classic) transcript. Additionally, we show that mice which harbor the 6.2hPLP(+)Z/FL transgene demonstrate wide-ranging expression throughout brain at P2, whereas expression of mPLP(+)Z is quite limited at this age. Therefore, we generated new transgenic mouse lines with the 2.7hPLP(+)Z transgene, which contains
sequence orthologous to just that in mPLP(+)Z. Of the seven lines analyzed, six showed higher levels of 2.7hPLP(+)Z expression in brain at P21 compared to P2; the other line expressed the transgene, only weakly, at either age. This trend, coupled with the robust expression observed for 6.2hPLP(+)Z/FL at P2, suggests that the distal 3.5 kb of 5'-flanking
DNA specific to 6.2hPLP(+)Z/FL contains regulatory element(s) important for promoting early postnatal expression in brain.
Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin (FXN). Most FRDA patients are homozygous for large expansions of GAA ...repeats in intron 1 of FXN, while some are compound heterozygotes with an expanded GAA tract in one allele and a missense or nonsense mutation in the other. A missense mutation, changing a glycine to valine at position 130 (G130V), is prevalent among the clinical variants. We and others have demonstrated that levels of mature FXN protein in FRDA G130V samples are reduced below those detected in samples harboring homozygous repeat expansions. Little is known regarding expression and function of endogenous FXN-G130V protein due to lack of reagents and models that can distinguish the mutant FXN protein from the wild-type FXN produced from the GAA-expanded allele. We aimed to determine the effect of the G130V (murine G127V) mutation on Fxn expression and to define its multi-system impact in vivo. We used CRISPR/Cas9 to introduce the G127V missense mutation in the Fxn coding sequence and generated homozygous mice (FxnG127V/G127V). We also introduced the G127V mutation into a GAA repeat expansion FRDA mouse model (FxnGAA230/KO; KIKO) to generate a compound heterozygous strain (FxnG127V/GAA230). We performed neurobehavioral tests on cohorts of WT and Fxn mutant animals at three-month intervals for one year, and collected tissue samples to analyze molecular changes during that time. The endogenous Fxn G127V protein is detected at much lower levels in all tissues analyzed from FxnG127V/G127V mice compared to age and sex-matched WT mice without differences in Fxn transcript levels. FxnG127V/G127V mice are significantly smaller than WT counterparts, but perform similarly in most neurobehavioral tasks. RNA sequencing analysis revealed reduced expression of genes in oxidative phosphorylation and protein synthesis, underscoring the metabolic consequences in our mouse model expressing extremely low levels of Fxn. Results of these studies provide insight into the unique pathogenic mechanism of the FXN G130V mechanism and the tolerable limit of Fxn/FXN expression in vivo.
•Extremely low Fxn levels are detected in CNS and heart tissues of Fxn G127V mice.•Fxn G127V mice are small and develop a hunched posture.•Endurance and activity are reduced in adult Fxn G127V mice.•Motor coordination is not affected in adult Fxn G127V mice.
The recent identification of profilin1 mutations in 25 familial ALS cases has linked altered function of this cytoskeleton-regulating protein to the pathogenesis of motor neuron disease. To ...investigate the pathological role of mutant profilin1 in motor neuron disease, we generated transgenic lines of mice expressing human profilin1 with a mutation at position 118 (hPFN1G118V). One of the mouse lines expressing high levels of mutant human PFN1 protein in the brain and spinal cord exhibited many key clinical and pathological features consistent with human ALS disease. These include loss of lower (ventral horn) and upper motor neurons (corticospinal motor neurons in layer V), mutant profilin1 aggregation, abnormally ubiquitinated proteins, reduced choline acetyltransferase (ChAT) enzyme expression, fragmented mitochondria, glial cell activation, muscle atrophy, weight loss, and reduced survival. Our investigations of actin dynamics and axonal integrity suggest that mutant PFN1 protein is associated with an abnormally low filamentous/globular (F/G)-actin ratio that may be the underlying cause of severe damage to ventral root axons resulting in a Wallerian-like degeneration. These observations indicate that our novel profilin1 mutant mouse line may provide a new ALS model with the opportunity to gain unique perspectives into mechanisms of neurodegeneration that contribute to ALS pathogenesis.
Friedreich's ataxia is caused by large homozygous, intronic expansions of GAA repeats in the frataxin (FXN) gene, resulting in severe downregulation of its expression. Pathogenic repeats are located ...in intron one, hence patients express unaffected FXN protein, albeit in low quantities. Although FRDA symptoms typically afflict the nervous system, hypertrophic cardiomyopathy is the predominant cause of death. Our studies were conducted using cardiomyocytes differentiated from induced pluripotent stem cells derived from control individuals, FRDA patients, and isogenic cells corrected by zinc finger nucleases-mediated excision of pathogenic expanded GAA repeats. This correction of the FXN gene removed the primary trigger of the transcription defect, upregulated frataxin expression, reduced pathological lipid accumulation observed in patient cardiomyocytes, and reversed gene expression signatures of FRDA cardiomyocytes. Transcriptome analyses revealed hypertrophy-specific expression signatures unique to FRDA cardiomyocytes, and emphasized similarities between unaffected and ZFN-corrected FRDA cardiomyocytes. Thus, the iPSC-derived FRDA cardiomyocytes exhibit various molecular defects characteristic for cellular models of cardiomyopathy that can be corrected by genome editing of the expanded GAA repeats. These results underscore the utility of genome editing in generating isogenic cellular models of FRDA and the potential of this approach as a future therapy for this disease.
•Decreased frataxin expression in FRDA iPSC derived cardiomyocytes•Lipid droplet accumulation in FRDA cardiomyocytets•Cardiac hypertrophy expression signature FRDA cardiomyocytets•Frataxin deficiency alleviated by excision of expanded GAA repeats.•Reduced lipid droplet accumulation and reversal of gene expression changes upon correction of frataxin gene.
BackgroundThe utilization of Chimeric Antigen Receptor T cells (CAR-Ts) has demonstrated exceptional efficacy in the treatment of cancer, revolutionizing the field of drug development through the use ...of genetically modified T cells as a therapeutic approach. However, the development of these therapies presents distinct challenges due to the inherent variability among individuals. The precise mechanisms that drive clinical outcomes remain inadequately understood. In this study, we exploit the variability observed in healthy donors and employ multi-omics analysis to discover proteomic and genomic indicators linked to enhanced functionality of CAR-T cells.MethodsHuman CD8+ T cells were isolated from unique healthy donors Peripheral Blood Mononuclear Cells (PBMC) and simultaneously stimulated using anti-CD3/28 and transduced with lentiviral particles containing CD19 CAR (CD3z, 28z, or BBz) P2A GFP. 7 days post transduction CAR T cells were sorted to >95% purity and cocultured with the NALM6 cells at various ratios. Live cell imaging was used to count target cells over a 48h period and determine a cytotoxicity score. Purified naïve CD8+ T cells and resultant CAR T cells were subjected to extensive DNA Methylation Array and Proteomic analysis. Multi-omic analysis on the top and bottom 25% performing CAR T cells was conducted.ResultsFunctional analysis revealed a wide range of CAR T cell variability that does not correlate with age (28z, BBz, CD3z respectively R2 =0.01578, 0.08983, 0.06378, p-value= 0.6194, 0.2425, 0.3281). However, we did find a significant positive correlation in DNA methylation informed Age(mAge) and with expression of inhibitory receptors during T cell activation, specifically CLTA-4 (R2 = 0.33, p-value= 0.025) and TIGIT (R2 = 0.29, p-value= 0.036). Further we found a negative correlation of CAR T cell cytotoxicity and the expression of the exhaustion-associated transcription factor TOX2 (28z, BBz, CD3z respectively R2 = 0.4271, 0.2776, 0.2847, p-value= 0.0033, 0.0298, 0.0274). Full exploratory proteomic analysis or naïve CD8+ T cells and CAR T cells is presented comparing the top and bottom 25% performing CAR T cells.ConclusionsA large amount of variability exists in healthy donor CAR T cells that cannot be explained by chronological age. Here we leverage this variability to identify new proteomic and genomic biomarkers associated with CAR T cell function. These findings have the potential to inform CAR T cell monitoring and engineering strategies.
Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that leads to the loss of motor neurons. The molecular mechanisms of motor neuron degeneration are largely unknown ...and there are currently no effective therapies to treat this disease. In this work, we report whole transcriptome profiling of spinal cords of mutant transgenic hPFN1
G118V
mice and their wildtype transgenic hPFN1
WT
controls at a pre-symptomatic stage and at the end-stage of disease. Analyses revealed that end-stage hPFN1
G118V
mice had 890 differentially expressed genes (747 up-regulated, 143 down-regulated) when compared to pre-symptomatic hPFN1
G118V
mice, and they had 836 differentially expressed genes (742 up-regulated, 94 down-regulated) when compared to age-matched hPFN1
WT
controls. Pre-symptomatic hPFN1
G118V
mice were not significantly different from age-matched hPFN1
WT
controls. Ingenuity Pathway Analysis identified inflammatory pathways significantly activated in end-stage hPFN1
G118V
samples, suggesting an excess of glial activation at end-stage disease, possibly due to an increase in glial composition within the spinal cord during disease progression. In conclusion, our RNA-Seq data identified molecules and pathways involved in the mechanisms of neurodegeneration that could potentially serve as therapeutic targets for ALS.
Breast cancer remains one of the leading cancers among women. Cancer stem cells (CSCs) are tumor-initiating cells which drive progression, metastasis, and reoccurrence of the disease. CSCs are ...resistant to conventional chemo- and radio-therapies and their ability to survive such treatment enables tumor reestablishment. Metastasis is the main cause of mortality in women with breast cancer, thus advances in treatment will depend on therapeutic strategies targeting CSCs. Salinomycin (SAL) is a naturally occurring polyether ionophore antibiotic known for its anticancer activity towards several types of tumor cells. In the present work, a library of 17 C1-single and C1/C20-double modified SAL analogs was screened to identify compounds with improved activity against breast CSCs. Six single- and two double-modified analogs were more potent (IC50 range of 1.1 ± 0.1–1.4 ± 0.2 µM) toward the breast cancer cell line MDA-MB-231 compared to SAL (IC50 of 4.9 ± 1.6 µM). Double-modified compound 17 was found to be more efficacious than SAL against the majority of cancer cell lines in the NCI-60 Human Tumor Cell Line Panel. Compound 17 was more potent than SAL in inhibiting cell migration and cell renewal properties of MDA-MB-231 cells, as well as inducing selective loss of the CD44+/CD24/low stem-cell-like subpopulation in both monolayer (2D) and organoid (3D) culture. The present findings highlight the therapeutic potential of SAL analogs towards breast CSCs and identify select compounds that merit further study and clinical development.
Display omitted
•Single- and double-modified salinomycin analogs were more potent towards MDA-MB-231 breast cancer cells than salinomycin•Double modified analog 17 induced DNA fragmentation and apoptotic cell death in MDA-MB-231 cell monolayer and organoids•Analog 17 reduced colony formation potential and migration of MDA-MB-231 cells to the greater extent than salinomycin•Analog 17 more effectively than salinomycin reduced CD44+/CD24- cancer stem-like cells in monolayer and organoid cultures
BackgroundT cells are subjected to a variety of genotoxic events in the tumor microenvironment including replication stress, off-target toxicity of chemotherapy and radiation therapy, and ...mitochondrial-derived reactive oxygen species.1 Our preliminary data suggests that accumulation of DNA damage accelerates T cell exhaustion. Using an antibody-stimulation model of T cell exhaustion, we performed proteomic analyses to compare regulation of the DNA damage response in acutely stimulated T cells versus chronically stimulated T cells.2 MethodsHuman CD8+ T cells were isolated from three healthy donors and stimulated using CD2/3/28 beads either once (acute stimulation), or every 2 days for eight days (chronic stimulation). Samples were taken at days 4, 6, and 8 for proteomic analysis and at day 8 for phospho-proteomic analysis through the IDEA National Resource for Quantitative Proteomics. To validate exhaustion, flow cytometry for exhaustion markers was performed at days 2, 4, 6, and 8, and qPCR for T cell phenotype markers was performed at day 8.ResultsFlow cytometry, qPCR, and proteomic analysis confirmed an increase in exhaustion markers after chronic stimulation. In our proteomic analysis, we observed downregulation of pro-apoptosis factors including PYCARD, Bax, and several Caspases. Mismatch repair proteins including ERCC4, MLH1, and PMS2 were upregulated in exhausted T cells. Interestingly, we observed a downregulation of ATM, a kinase upstream in the DNA damage response to double-stranded breaks, while it’s downstream repair factor 53BP1 is upregulated. Phospho-TMT analysis revealed that 53BP1 is inactivated through phosphorylation in chronically stimulated T cells.3 Overall, we identified 654 proteins and 1176 phospho-sites which were differentially regulated, log2FC >1 or <-1 with an adjusted p-value < 0.05.ConclusionsT cells must be able to overcome DNA damage to continue to proliferate and perform its cytotoxic functions. We found that DNA damage contributes to T cell exhaustion. To combat this, exhausted T cells alter their proteome to promote DNA repair and downregulate apoptotic mechanisms, but ultimately the T cell still becomes dysfunctional. These results highlight repair pathways which could be exploited in future T cell therapies.ReferencesNE Scharping, et al. ‘Mitochondrial stress induced by continuous stimulation under hypoxia rapidly drives T cell exhaustion’, Nat Immunol, Feb. 2021;22(2)205–215, doi: 10.1038/s41590–020-00834–9.LS Dunsford, RH Thoirs, E Rathbone, A Patakas, ‘A Human In Vitro T Cell Exhaustion Model for Assessing Immuno-Oncology Therapies’, in Methods in Pharmacology and Toxicology, Humana Press Inc., 2020, pp. 89–101. doi: 10.1007/978–1-0716–0171-6_6.P von Morgen, T Lidak, Z Horejsi, L Macurek, ‘Nuclear localisation of 53BP1 is regulated by phosphorylation of the nuclear localisation signal’, Biol Cell, Jun. 2018;110(6)137–146, doi: 10.1111/boc.201700067.
BackgroundUnleashing the immune anti-tumor response through immune checkpoint blockade (ICB) therapy has been successful in combating solid-tumor malignancies, including metastatic melanoma. When ...successful, the anti-tumor response is potent; however, half of melanoma patients fail to respond. ICB responsiveness is dictated by the immune milieu and antigenicity of the tumor microenvironment (TME), with therapy-resistant melanomas exhibiting reduced expression of major histocompatibility complex class I (MHC-I) and increased levels of endoplasmic reticulum (ER) stress. Though the TME is known to be immunosuppressive, the specific mechanisms governing metabolic stress and antigen presentation in the context of ICB response have not been well characterized.MethodsTo further study the regulation of MHC-I, we cultured melanoma in conditions of prolonged metabolic stress, forcing cells to adapt to the absence of glucose. Proteomic profiling indicates this model establishes a reversible, adaptive phenotype reminiscent of the changes seen in published ICB-responder datasets. Quantification of tumor immunogenicity was achieved by RNA signatures, flow cytometry, and immunoblotting. Functional assessment of immune-mediated killing was achieved through co-culture killing assays with B16-OVA cells and activated OT-1 CD8+ T-cells. Chromatin immunoprecipitation (ChIP) was used to quantify chromatin modification, globally and at specific gene loci. To assess the role and functionality of specific epigenetic regulators on ICB response, mutant cell lines were established through lentiviral transduction of B16 melanoma cell lines.ResultsMelanoma metabolically adapted to glucose-free media had increases in MHC-I antigen presentation. Remodeling the energy metabolism globally restored MHC-I genes and significantly increases tumor sensitivity to T-cells, independent of IFN-γ. Proteomic analysis exhibited significant dysregulation in histone modifiers, specifically the loss EZH2 (Enhancer of Zeste Homolog 2), a histone methyltransferase characterized by transcriptional repression. As confirmed by ChIP-Sequencing and ChIP-PCR, the catalytic mark of EZH2, H3K27me3, is reduced in metabolically reprogrammed melanoma at gene loci specific to MHC-I antigen presentation. Additionally, EZH2 mutant melanoma cell lines demonstrate differential sensitivity to activated CD8+ T-cells, further implicating EZH2 in the determination of ICB response.ConclusionsThe melanoma response to ICB is influenced by the metabolic state of the tumor. EZH2, a potent regulator of gene expression, is critical to the interferon-independent mechanisms of antigen presentation and immune escape, which dictate the ICB response. Conditions of increased mitochondrial respiration and ER stress significantly decrease EZH2 levels and presence of H3K27me3 at the promoter regions of MHC-I antigen-presenting genes. This demonstrates the clear potential for metabolic and EZH2 status as a prognostic indicator for ICB-responsiveness in metastatic melanoma
PDF
