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Insights into RNA-mediated pathology in new mouse models of Huntington's disease
AbstractHuntington's disease (HD) is a neurodegenerative polyglutamine (polyQ) disease resulting from the expansion of CAG repeats located in the ORF of the huntingtin gene (HTT). The extent to which mutant mRNA‐driven disruptions contribute to HD pathogenesis, particularly in comparison to the dominant mechanisms related to the gain‐of‐function effects of the mutant polyQ protein, is still debatable. To evaluate this contribution in vivo, we generated two mouse models through a knock‐in strategy at the Rosa26 locus. These models expressed distinct variants of human mutant HTT cDNA fragment: a translated variant (HD/100Q model, serving as a reference) and a nontranslated variant (HD/100CAG model). The cohorts of animals were subjected to a broad spectrum of molecular, behavioral, and cognitive analysis for 21 months. Behavioral testing revealed alterations in both models, with the HD/100Q model exhibiting late disease phenotype. The rotarod, static rod, and open‐field tests showed some motor deficits in HD/100CAG and HD/100Q model mice during the light phase, while ActiMot indicated hyperkinesis during the dark phase. Both models also exhibited certain gene deregulations in the striatum that are related to disrupted pathways and phenotype alterations observed in HD. In conclusion, we provide in vivo evidence for a minor contributory role of mutant RNA in HD pathogenesis. The separated effects resulting from the presence of mutant RNA in the HD/100CAG model led to less severe but, to some extent, similar types of impairments as in the HD/100Q model. Increased anxiety was one of the most substantial effects caused by mutant HTT RNA.
Diet-induced hyperhomocysteinemia causes sex-dependent deficiencies in offspring musculature and brain function
Hyperhomocysteinemia (HHcy), characterized by elevated homocysteine (Hcy) levels, is a known risk factor for cardiovascular, renal, and neurological diseases, as well as pregnancy complications. Our study aimed to investigate whether HHcy induced by a high-methionine (high-Met) diet exacerbates cognitive and behavioral deficits in offspring and leads to other breeding problems. Dietary HHcy was induced four weeks before mating and continued throughout gestation and post-delivery. A battery of behavioral tests was conducted on offspring between postnatal days (PNDs) 5 and 30 to assess motor function/activity and cognition. The results were correlated with brain morphometric measurements and quantitative analysis of mammalian target of rapamycin (mTOR)/autophagy markers. The high-Met diet significantly increased parental and offspring urinary tHcy levels and influenced offspring behavior in a sex-dependent manner. Female offspring exhibited impaired cognition, potentially related to morphometric changes observed exclusively in HHcy females. Male HHcy pups demonstrated muscle weakness, evidenced by slower surface righting, reduced hind limb suspension (HLS) hanging time, weaker grip strength, and decreased activity in the beaker test. Western blot analyses indicated the downregulation of autophagy and the upregulation of mTOR activity in HHcy cortexes. HHcy also led to breeding impairments, including reduced breeding rate, in-utero fetal death, lower pups’ body weight, and increased mortality, likely attributed to placental dysfunction associated with HHcy. In conclusion, a high-Met diet impairs memory and cognition in female juveniles and weakens muscle strength in male pups. These effects may stem from abnormal placental function affecting early neurogenesis, the dysregulation of autophagy-related pathways in the cortex, or epigenetic mechanisms of gene regulation triggered by HHcy during embryonic development.
Deletion of the Homocysteine Thiolactone Detoxifying Enzyme Bleomycin Hydrolase, in Mice, Causes Memory and Neurological Deficits and Worsens Alzheimer’s Disease-Related Behavioral and Biochemical Traits in the 5xFAD Model of Alzheimer’s Disease
Background: Bleomycin hydrolase (BLMH), a homocysteine (Hcy)-thiolactone detoxifying enzyme, is attenuated in Alzheimer’s disease (AD) brains. Blmh loss causes astrogliosis in mice while the loss of histone demethylase Phf8, which controls mTOR signaling, causes neuropathy in mice and humans. Objective: To examine how Blmh gene deletion affects the Phf8/H4K20me1/mTOR/autophagy pathway, amyloid-β (Aβ) accumulation, and cognitive/neuromotor performance in mice. Methods: We generated a new mouse model of AD, the Blmh-/-5xFAD mouse. Behavioral assessments were conducted by cognitive/neuromotor testing. Blmh and Phf8 genes were silenced in mouse neuroblastoma N2a-APPswe cells by RNA interference. mTOR- and autophagy-related proteins, and AβPP were quantified by western blotting and the corresponding mRNAs by RT-qPCR. Aβ was quantified by western blotting (brains) and by confocal microscopy (cells). Results: Behavioral testing showed cognitive/neuromotor deficits in Blmh-/- and Blmh-/-5xFAD mice. Phf8 was transcriptionally downregulated in Blmh-/- and Blmh-/-5xFAD brains. H4K20me1, mTOR, phospho-mTOR, and AβPP were upregulated while autophagy markers Becn1, Atg5, and Atg7 were downregulated in Blmh-/- and Blmh-/-5xFAD brains. Aβ was elevated in Blmh-/-5xFAD brains. These biochemical changes were recapitulated in Blmh-silenced N2a-APPswe cells, which also showed increased H4K20me1-mTOR promoter binding and impaired autophagy flux (Lc3-I, Lc3-II, p62). Phf8-silencing or treatments with Hcy-thiolactone or N-Hcy-protein, metabolites elevated in Blmh-/- mice, induced biochemical changes in N2a-APPswe cells like those induced by the Blmh-silencing. However, Phf8-silencing elevated Aβ without affecting AβPP. Conclusions: Our findings show that Blmh interacts with AβPP and the Phf8/H4K20me1/mTOR/autophagy pathway, and that disruption of those interactions causes Aβ accumulation and cognitive/neuromotor deficits.
Mapping the molecular signature of ABA-regulated gene expression in germinating barley embryos
Abstract Background Abscisic acid (ABA) regulates key plant processes, including seed germination, dormancy, and abiotic stress responses. While its physiological role in germination is well-documented, the molecular mechanisms are still poorly understood. To address this, we analyzed transcriptomic and metabolomic changes in ABA-treated germinating barley (Hordeum vulgare) embryos. To map ABA-responsive gene expression across embryonic tissues, we employed the Visium Spatial Transcriptomics (10× Genomics). This approach, which remains technically challenging to be applied in plant tissues, enabled the precise localization of gene expression across six embryo regions, offering insights into tissue-specific expression patterns that cannot be resolved by traditional RNA-seq. Results Transcriptomic analysis indicated that ABA acts primarily as a germination repressor. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses linked ABA-inhibited genes to energy metabolism, lignin biosynthesis, cell wall organization, and photosynthesis, while induced genes were associated with environmental adaptation and phytohormone signaling. Differentially expressed genes (DEGs) correlated with metabolites involved in phytohormone pathways, including gibberellins, jasmonates, brassinosteroids, salicylic acid, auxins, and ABA metabolism. Comparisons with developing seed transcriptomes suggested an ABA-associated gene expression signature in embryos. Spatial transcriptomics technique made possible the precise identification of ABA-induced transcriptional changes within distinct embryonic tissues. Conclusions Integrating transcriptomics, metabolomics and spatial transcriptomics defined the molecular signature of ABA-induced modulation of phytohormonal crosstalk, energy metabolism, and tissue-specific gene activity in germinating seeds. The successful use of spatial transcriptomics adds a novel layer of resolution for understanding tissue-specific ABA responses during barley seed germination. These findings offer new insights into the ABA role in seed germination and potential strategies for enhancing crop resilience.
Effects of Tcte1 knockout on energy chain transportation and spermatogenesis: implications for male infertility
Abstract STUDY QUESTION Is the Tcte1 mutation causative for male infertility? SUMMARY ANSWER Our collected data underline the complex and devastating effect of the single-gene mutation on the testicular molecular network, leading to male reproductive failure. WHAT IS KNOWN ALREADY Recent data have revealed mutations in genes related to axonemal dynein arms as causative for morphology and motility abnormalities in spermatozoa of infertile males, including dysplasia of fibrous sheath (DFS) and multiple morphological abnormalities in the sperm flagella (MMAF). The nexin–dynein regulatory complex (N-DRC) coordinates the dynein arm activity and is built from the DRC1–DRC7 proteins. DRC5 (TCTE1), one of the N-DRC elements, has already been reported as a candidate for abnormal sperm flagella beating; however, only in a restricted manner with no clear explanation of respective observations. STUDY DESIGN, SIZE, DURATION Using the CRISPR/Cas9 genome editing technique, a mouse Tcte1 gene knockout line was created on the basis of the C57Bl/6J strain. The mouse reproductive potential, semen characteristics, testicular gene expression levels, sperm ATP, and testis apoptosis level measurements were then assessed, followed by visualization of N-DRC proteins in sperm, and protein modeling in silico. Also, a pilot genomic sequencing study of samples from human infertile males (n = 248) was applied for screening of TCTE1 variants. PARTICIPANTS/MATERIALS, SETTING, METHODS To check the reproductive potential of KO mice, adult animals were crossed for delivery of three litters per caged pair, but for no longer than for 6 months, in various combinations of zygosity. All experiments were performed for wild-type (WT, control group), heterozygous Tcte1+/− and homozygous Tcte1−/− male mice. Gross anatomy was performed on testis and epididymis samples, followed by semen analysis. Sequencing of RNA (RNAseq; Illumina) was done for mice testis tissues. STRING interactions were checked for protein–protein interactions, based on changed expression levels of corresponding genes identified in the mouse testis RNAseq experiments. Immunofluorescence in situ staining was performed to detect the N-DRC complex proteins: Tcte1 (Drc5), Drc7, Fbxl13 (Drc6), and Eps8l1 (Drc3) in mouse spermatozoa. To determine the amount of ATP in spermatozoa, the luminescence level was measured. In addition, immunofluorescence in situ staining was performed to check the level of apoptosis via caspase 3 visualization on mouse testis samples. DNA from whole blood samples of infertile males (n = 137 with non-obstructive azoospermia or cryptozoospermia, n = 111 samples with a spectrum of oligoasthenoteratozoospermia, including n = 47 with asthenozoospermia) was extracted to perform genomic sequencing (WGS, WES, or Sanger). Protein prediction modeling of human-identified variants and the exon 3 structure deleted in the mouse knockout was also performed. MAIN RESULTS AND THE ROLE OF CHANCE No progeny at all was found for the homozygous males which were revealed to have oligoasthenoteratozoospermia, while heterozygous animals were fertile but manifested oligozoospermia, suggesting haploinsufficiency. RNA-sequencing of the testicular tissue showed the influence of Tcte1 mutations on the expression pattern of 21 genes responsible for mitochondrial ATP processing or linked with apoptosis or spermatogenesis. In Tcte1−/− males, the protein was revealed in only residual amounts in the sperm head nucleus and was not transported to the sperm flagella, as were other N-DRC components. Decreased ATP levels (2.4-fold lower) were found in the spermatozoa of homozygous mice, together with disturbed tail:midpiece ratios, leading to abnormal sperm tail beating. Casp3-positive signals (indicating apoptosis) were observed in spermatogonia only, at a similar level in all three mouse genotypes. Mutation screening of human infertile males revealed one novel and five ultra-rare heterogeneous variants (predicted as disease-causing) in 6.05% of the patients studied. Protein prediction modeling of identified variants revealed changes in the protein surface charge potential, leading to disruption in helix flexibility or its dynamics, thus suggesting disrupted interactions of TCTE1 with its binding partners located within the axoneme. LARGE SCALE DATA All data generated or analyzed during this study are included in this published article and its supplementary information files. RNAseq data are available in the GEO database (https://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE207805. The results described in the publication are based on whole-genome or exome sequencing data which includes sensitive information in the form of patient-specific germline variants. Information regarding such variants must not be shared publicly following European Union legislation, therefore access to raw data that support the findings of this study are available from the corresponding author upon reasonable request. LIMITATIONS, REASONS FOR CAUTION In the study, the in vitro fertilization performance of sperm from homozygous male mice was not checked. WIDER IMPLICATIONS OF THE FINDINGS This study contains novel and comprehensive data concerning the role of TCTE1 in male infertility. The TCTE1 gene is the next one that should be added to the ‘male infertility list’ because of its crucial role in spermatogenesis and proper sperm functioning. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by National Science Centre in Poland, grants no.: 2015/17/B/NZ2/01157 and 2020/37/B/NZ5/00549 (to M.K.), 2017/26/D/NZ5/00789 (to A.M.), and HD096723, GM127569-03, NIH SAP #4100085736 PA DoH (to A.N.Y.). The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Is a rare CXCL8 gene variant a new possible cause or course factor of inflammatory bowel disease?
IntroductionThe pathogenesis of inflammatory bowel diseases (IBD) involves genetic, environmental, immunological, and microbial factors; however, it remains unclear. Pro-inflammatory interleukin 8 (IL-8), encoded by the CXCL8 gene, assumes a crucial chemotactic role in leukocyte migration.MethodsThis study aimed to investigate whether an association exists between IBD and two CXCL8 variants, namely, c.-251A>T (rs4073) and c.91G>T (rs188378669), and IL-8 concentration. We analyzed the distribution of both variants among 353 Polish IBD patients and 200 population subjects using pyrosequencing, competitive allele-specific PCR and Sanger sequencing.ResultsThe c.91T stop-gained allele was significantly more frequent in IBD patients (2.12%) than in controls (0.25%) (p = 0.0121), while the c.-251T allele frequencies were similar (54% vs. 51.5%, p = 0.4955). Serum IL-8 concentrations, measured using ELISA, were higher in IBD patients with the c.91 GG genotype compared to healthy controls (mean, 70.02 vs. 51.5 pg/ml, p<0.01) and patients with c.91 GT (mean, 61.73 pg/ml). Moreover, clinical data indicated that carriers of the c.91T variant need more often corticosteroids and surgical treatment of the disease than GG homozygous IBD patients.ConclusionThis suggest that the CXCL8 c.91T allele may influence IBD manifestation and the course of the disorders in Polish patients, potentially serving as a novel target for future studies and therapeutic approaches.