Search results

The age-dependent development of abnormal cardiac metabolism in the peroxisome proliferator-activated receptor α-knockout mouse.

BACKGROUND AND AIMS: Peroxisome proliferator-activated receptor α (PPARα) is crucial for regulating cardiac β-oxidation in the heart, liver, and kidney. Ageing can induce cardiac metabolic alterations, but the role of PPARα has not been extensively characterised. The aim of this research was to investigate the role of PPARα in the aged heart. METHODS: Hyperpolarized [1-13C]pyruvate was used to evaluate in vivo cardiac carbohydrate metabolism in fed and fasted young (3 months) and old (20-22 months) PPARα knockout (KO) mice versus controls. Cine MRI assessed cardiac structural and functional changes. Cardiac tissue analysis included qRT-PCR and Western blotting for Pparα, medium chain acyl-CoA dehydrenase (MCAD), uncoupling protein (UCP) 3, glucose transporter (GLUT) 4 and PDH kinase (PDK) 1,2, and 4 expression. RESULTS: PPARα-KO hearts from both young and old mice showed significantly reduced Pparα mRNA and a 58-59 % decrease in MCAD protein levels compared to controls. Cardiac PDH flux was similar in young control and PPARα-KO mice but 96 % higher in old PPARα-KO mice. Differences between genotypes were consistent in fed and fasted states, with reduced PDH flux when fasted. Increased PDH flux was accompanied by a 179 % rise in myocardial GLUT4 protein. No differences in PDK 1, 2, or 4 protein levels were observed between fed groups, indicating the increased PDH flux in aged PPARα-KO mice was not due to changes in PDH phosphorylation. CONCLUSIONS: Aged PPARα-KO mice demonstrated higher cardiac PDH flux compared to controls, facilitated by increased myocardial GLUT4 protein levels, leading to enhanced glucose uptake and glycolysis.

Hypothesis-driven genome-wide association studies provide novel insights into genetics of reading disabilities.

Reading Disability (RD) is often characterized by difficulties in the phonology of the language. While the molecular mechanisms underlying it are largely undetermined, loci are being revealed by genome-wide association studies (GWAS). In a previous GWAS for word reading (Price, 2020), we observed that top single-nucleotide polymorphisms (SNPs) were located near to or in genes involved in neuronal migration/axon guidance (NM/AG) or loci implicated in autism spectrum disorder (ASD). A prominent theory of RD etiology posits that it involves disturbed neuronal migration, while potential links between RD-ASD have not been extensively investigated. To improve power to identify associated loci, we up-weighted variants involved in NM/AG or ASD, separately, and performed a new Hypothesis-Driven (HD)-GWAS. The approach was applied to a Toronto RD sample and a meta-analysis of the GenLang Consortium. For the Toronto sample (n = 624), no SNPs reached significance; however, by gene-set analysis, the joint contribution of ASD-related genes passed the threshold (p~1.45 × 10-2, threshold = 2.5 × 10-2). For the GenLang Cohort (n = 26,558), SNPs in DOCK7 and CDH4 showed significant association for the NM/AG hypothesis (sFDR q = 1.02 × 10-2). To make the GenLang dataset more similar to Toronto, we repeated the analysis restricting to samples selected for reading/language deficits (n = 4152). In this GenLang selected subset, we found significant association for a locus intergenic between BTG3-C21orf91 for both hypotheses (sFDR q 

Correlation between the number of interstitial neurons of the white matter and number of neurons within cortical layers: Histological analyses in postnatal macaque.

We have examined the number and distribution of NeuN-immunoreactive cortical white matter interstitial cells (WMICs) and compared them to the neurons in layers 1-6 across the overlying cortex in coronal sections from postnatal macaques. The data have been gathered from over 300 selected regions at gyral crowns, at sulci, and at linear regions of the cortex where we also determined cortical layer thicknesses: standard thicknesses and tangential thicknesses. Cortical thicknesses and cell numbers showed variability according to gyral, linear, or sulcal regions. In spite of these variations, our standardized cell numbers in layers 1 to 6b and interstitial cells underlying layer 6b-white matter boundary have shown a consistent correlation between the number of WMICs and the number of layer 5 and 6a cortical neurons on all cortical regions studied: for each WMIC, there are on the order of five cortical neurons in layer 5 and approximately three cortical neurons in layer 6a, irrespective of the origins of the selected cortical area or whether they are from gyral, linear, or sulcal regions. We propose that the number of interstitial neurons in the postnatal macaque cortex is correlated to the density of neurons within layers 5 and 6a and, from a clinical perspective, the change in density or distribution of interstitial neurons in schizophrenia or epilepsy may in fact be linked to the number of layers 5 and 6a neurons.

Utrophin correlates with disease severity in Duchenne muscular dystrophy.

This month in Med, the description of an unusually severely affected DMD patient suffering from a large deletion in the dystrophin gene confirms that absence of utrophin worsens the dystrophy and supports the concept that utrophin upregulation ameliorates the pathology. This study may guide the development of dystrophin-based gene therapies.

Early deficits in an in vitro striatal microcircuit model carrying the Parkinson's GBA-N370S mutation.

Understanding medium spiny neuron (MSN) physiology is essential to understand motor impairments in Parkinson's disease (PD) given the architecture of the basal ganglia. Here, we developed a custom three-chambered microfluidic platform and established a cortico-striato-nigral microcircuit partially recapitulating the striatal presynaptic landscape in vitro using induced pluripotent stem cell (iPSC)-derived neurons. We found that, cortical glutamatergic projections facilitated MSN synaptic activity, and dopaminergic transmission enhanced maturation of MSNs in vitro. Replacement of wild-type iPSC-derived dopamine neurons (iPSC-DaNs) in the striatal microcircuit with those carrying the PD-related GBA-N370S mutation led to a depolarisation of resting membrane potential and an increase in rheobase in iPSC-MSNs, as well as a reduction in both voltage-gated sodium and potassium currents. Such deficits were resolved in late microcircuit cultures, and could be reversed in younger cultures with antagonism of protein kinase A activity in iPSC-MSNs. Taken together, our results highlight the unique utility of modelling striatal neurons in a modular physiological circuit to reveal mechanistic insights into GBA1 mutations in PD.

Histone transcription regulator Slm9 is required for cytoophidium biogenesis.

The cytoophidium, a subcellular structure composed of CTP synthase, can be observed during the division of Schizosaccharomyces pombe. Cytoophidium formation changes periodically with the cell cycle of yeast cells. Here, we find that histone chaperone Slm9 is required for the integrity of cytoophidia in fission yeast. When the slm9 gene is knocked out, we observe that morphological characteristics, the abundance of cytoophidia and the division of the yeast cells are significantly affected. Fragmented cytoophidia occur in slm9 mutant cells, a phenomenon rarely observed in wild-type cells. Our study reveals a potential link between a chromosomal regulatory factor and cytoophidium biogenesis.

eCRUIS captures RNA-protein interaction in vitro and in vivo.

As an information bridge between DNA and protein, RNA regulates cellular processes and gene expression in various ways. From its synthesis to degradation, RNA is associated with a range of RNA-binding proteins. Therefore, it is necessary to develop innovative methods to study the interaction between RNA and proteins. Previously, we developed an RNA-centric method, called CRISPR-based RNA-United Interacting System (CRUIS), to capture RNA-protein interaction in cells. On this basis, here we develop an enhanced CRUIS (eCRUIS) by combining the power of dCas13d and the engineered promiscuous ligase TurboID. The current version allows us to rapidly label RNA-binding proteins on the target RNA within 30 minutes, potentially for in vivo use. By introducing bait-assay with exogenous RNA, we confirm that eCRUIS can effectively label RNA-binding proteins on bait RNA in a short time. eCRUIS provides a broader range of in vitro and in vivo applications for studying RNA-protein interactions.

The Impact of Developmental and Metabolic Cues on Cytoophidium Formation.

The cytoophidium, composed mainly of CTP synthase (CTPS), is a newly discovered dynamic filamentous structure in various organisms such as archaea, bacteria, and humans. These filamentous structures represent a fascinating example of intracellular compartmentation and dynamic regulation of metabolic enzymes. Currently, cytoophidia have been proven to be tightly regulated and highly dynamic, responding rapidly to developmental and metabolic cues and playing a critical role in maintaining cellular homeostasis. In this review, we would like to discuss in detail the characteristics, mechanisms, functions, and potential applications of this conservative but promising organelle.

Differential Cytoophidium Assembly between Saccharomyces cerevisiae and Schizosaccharomyces pombe.

The de novo synthesis of cytidine 5'-triphosphate (CTP) is catalyzed by the enzyme CTP synthase (CTPS), which is known to form cytoophidia across all three domains of life. In this study, we use the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe as model organisms to compare cytoophidium assembly under external environmental and intracellular CTPS alterations. We observe that under low and high temperature conditions, cytoophidia in fission yeast gradually disassemble, while cytoophidia in budding yeast remain unaffected. The effect of pH changes on cytoophidia maintenance in the two yeast species is different. When cultured in the yeast-saturated cultured medium, cytoophidia in fission yeast disassemble, while cytoophidia in budding yeast gradually form. Overexpression of CTPS results in the presence and maintenance of cytoophidia in both yeast species from the log phase to the stationary phase. In summary, our results demonstrate differential cytoophidium assembly between Saccharomyces cerevisiae and Schizosaccharomyces pombe, the two most studied yeast species.

Structural Basis of Bifunctional CTP/dCTP Synthase.

The final step in the de novo synthesis of cytidine 5'-triphosphate (CTP) is catalyzed by CTP synthase (CTPS), which can form cytoophidia in all three domains of life. Recently, we have discovered that CTPS binds to ribonucleotides (NTPs) to form filaments, and have successfully resolved the structures of Drosophila melanogaster CTPS bound with NTPs. Previous biochemical studies have shown that CTPS can bind to deoxyribonucleotides (dNTPs) to produce 2'-deoxycytidine-5'-triphosphate (dCTP). However, the structural basis of CTPS binding to dNTPs is still unclear. In this study, we find that Drosophila CTPS can also form filaments with dNTPs. Using cryo-electron microscopy, we are able to resolve the structure of Drosophila melanogaster CTPS bound to dNTPs with a resolution of up to 2.7 Å. By combining these structural findings with biochemical analysis, we compare the binding and reaction characteristics of NTPs and dNTPs with CTPS. Our results indicate that the same enzyme can act bifunctionally as CTP/dCTP synthase in vitro, and provide a structural basis for these activities.

The IMPDH cytoophidium couples metabolism and fetal development in mice.

The cytoophidium is an evolutionarily conserved subcellular structure formed by filamentous polymers of metabolic enzymes. In vertebrates, inosine monophosphate dehydrogenase (IMPDH), which catalyses the rate-limiting step in guanosine triphosphate (GTP) biosynthesis, is one of the best-known cytoophidium-forming enzymes. Formation of the cytoophidium has been proposed to alleviate the inhibition of IMPDH, thereby facilitating GTP production to support the rapid proliferation of certain cell types such as lymphocytes, cancer cells and pluripotent stem cells (PSCs). However, past studies lacked appropriate models to elucidate the significance of IMPDH cytoophidium under normal physiological conditions. In this study, we demonstrate that the presence of IMPDH cytoophidium in mouse PSCs correlates with their metabolic status rather than pluripotency. By introducing IMPDH2 Y12C point mutation through genome editing, we established mouse embryonic stem cell (ESC) lines incapable of forming IMPDH polymers and the cytoophidium. Our data indicate an important role of IMPDH cytoophidium in sustaining a positive feedback loop that couples nucleotide biosynthesis with upstream metabolic pathways. Additionally, we find that IMPDH2 Y12C mutation leads to decreased cell proliferation and increased DNA damage in teratomas, as well as impaired embryo development following blastocoel injection. Further analysis shows that IMPDH cytoophidium assembly in mouse embryonic development begins after implantation and gradually increases throughout fetal development. These findings provide insights into the regulation of IMPDH polymerisation in embryogenesis and its significance in coordinating cell metabolism and development.

Cytoophidia Influence Cell Cycle and Size in Schizosaccharomyces pombe.

Cytidine triphosphate synthase (CTPS) forms cytoophidia in all three domains of life. Here we focus on the function of cytoophidia in cell proliferation using Schizosaccharomyces pombe as a model system. We find that converting His359 of CTPS into Ala359 leads to cytoophidium disassembly. By reducing the level of CTPS protein or specific mutation, the loss of cytoophidia prolongs the G2 phase and expands cell size. In addition, the loss-filament mutant of CTPS leads to a decrease in the expression of genes related to G2/M transition and cell growth, including histone chaperone slm9. The overexpression of slm9 alleviates the G2 phase elongation and cell size enlargement induced by CTPS loss-filament mutants. Overall, our results connect cytoophidia with cell cycle and cell size control in Schizosaccharomyces pombe.

Dynamic Arabidopsis P5CS filament facilitates substrate channelling.

In plants, the rapid accumulation of proline is a common response to combat abiotic stress1-7. Delta-1-pyrroline-5-carboxylate synthase (P5CS) is a rate-limiting enzyme in proline synthesis, catalysing the initial two-step conversion from glutamate to proline8. Here we determine the first structure of plant P5CS. Our results show that Arabidopsis thaliana P5CS1 (AtP5CS1) and P5CS2 (AtP5CS2) can form enzymatic filaments in a substrate-sensitive manner. The destruction of AtP5CS filaments by mutagenesis leads to a significant reduction in enzymatic activity. Furthermore, separate activity tests on two domains reveal that filament-based substrate channelling is essential for maintaining the high catalytic efficiency of AtP5CS. Our study demonstrates the unique mechanism for the efficient catalysis of AtP5CS, shedding light on the intricate mechanisms underlying plant proline metabolism and stress response.

Filamentation and inhibition of prokaryotic CTP synthase with ligands.

Cytidine triphosphate synthase (CTPS) plays a pivotal role in the de novo synthesis of cytidine triphosphate (CTP), a fundamental building block for RNA and DNA that is essential for life. CTPS is capable of directly binding to all four nucleotide triphosphates: adenine triphosphate, uridine triphosphate, CTP, and guanidine triphosphate. Furthermore, CTPS can form cytoophidia in vivo and metabolic filaments in vitro, undergoing regulation at multiple levels. CTPS is considered a potential therapeutic target for combating invasions or infections by viral or prokaryotic pathogens. Utilizing cryo-electron microscopy, we determined the structure of Escherichia coli CTPS (ecCTPS) filament in complex with CTP, nicotinamide adenine dinucleotide (NADH), and the covalent inhibitor 6-diazo-5-oxo- l-norleucine (DON), achieving a resolution of 2.9 Å. We constructed a phylogenetic tree based on differences in filament-forming interfaces and designed a variant to validate our hypothesis, providing an evolutionary perspective on CTPS filament formation. Our computational analysis revealed a solvent-accessible ammonia tunnel upon DON binding. Through comparative structural analysis, we discern a distinct mode of CTP binding of ecCTPS that differs from eukaryotic counterparts. Combining biochemical assays and structural analysis, we determined and validated the synergistic inhibitory effects of CTP with NADH or adenine on CTPS. Our results expand our comprehension of the diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.

Dynamic Cytoophidia during Late-Stage Drosophila Oogenesis.

CTP synthase (CTPS) catalyzes the final step of de novo synthesis of CTP. CTPS was first discovered to form filamentous structures termed cytoophidia in Drosophila ovarian cells. Subsequent studies have shown that cytoophidia are widely present in cells of three life domains. In the Drosophila ovary model, our previous studies mainly focused on the early and middle stages, with less involvement in the later stages. In this work, we focus on the later stages of female germline cells in Drosophila. We use live-cell imaging to capture the continuous dynamics of cytoophidia in Stages 10-12. We notice the heterogeneity of cytoophidia in the two types of germline cells (nurse cells and oocytes), manifested in significant differences in morphology, distribution, and dynamics. Surprisingly, we also find that neighboring nurse cells in the same egg chamber exhibit multiple dynamic patterns of cytoophidia over time. Although the described dynamics may be influenced by the in vitro incubation conditions, our observation provides an initial understanding of the dynamics of cytoophidia during late-stage Drosophila oogenesis.

Developing Device of Death Operation (DODO) to Detect Apoptosis in 2D and 3D Cultures.

The real-time detection of intracellular biological processes by encoded sensors has broad application prospects. Here, we developed a degron-based modular reporting system, the Device of Death Operation (DODO), that can monitor various biological processes. The DODO system consists of a "reporter", an "inductor", and a "degron". After zymogen activation and cleavage, the degron will be released from the "reporter", which eventually leads to the stabilization of the "reporter", and can be detected. By replacing different "inductors" and "reporters", a series of biological processes can be reported through various signals. The system can effectively report the existence of TEV protease. To prove this concept, we successfully applied the DODO system to report apoptosis in 2D and 3D cultures. In addition, the reporter based on degron will help to design protease reporters other than caspase.

Architecture of CTPS filament networks revealed by cryo-electron tomography.

The cytoophidium is a novel type of membraneless organelle, first observed in the ovaries of Drosophila using fluorescence microscopy. In vitro, purified Drosophila melanogaster CTPS (dmCTPS) can form metabolic filaments under the presence of either substrates or products, and their structures that have been analyzed using cryo-electron microscopy (cryo-EM). These dmCTPS filaments are considered the fundamental units of cytoophidia. However, due to the resolution gap between light and electron microscopy, the precise assembly pattern of cytoophidia remains unclear. In this study, we find that dmCTPS filaments can spontaneously assemble in vitro, forming network structures that reach micron-scale dimensions. Using cryo-electron tomography (cryo-ET), we reconstruct the network structures formed by dmCTPS filaments under substrate or product binding conditions and elucidate their assembly process. The dmCTPS filaments initially form structural bundles, which then further assemble into larger networks. By identifying, tracking, and statistically analyzing the filaments, we observed distinct characteristics of the structural bundles formed under different conditions. This study provides the first systematic analysis of dmCTPS filament networks, offering new insights into the relationship between cytoophidia and metabolic filaments.

Characterization of Lymphatic Vasculature Using Whole-Mount Immunostaining of Mouse Embryonic Dorsal Skin.

Understanding the development of the lymphatic vasculature is essential to the understanding of how these vessels function in health and disease. High-resolution imaging of histological techniques such as immunostaining of sectioned tissue provides a snapshot into lymphatic vessel morphogenesis, patterning, and organization. Whole-mount staining of embryonic dermal vasculature allows for a deeper analysis and characterization of the developing lymphatic vascular network.

Histological Examination of Mitochondrial Morphology in a Parkinson's Disease Model.

Mitochondria play a central role in the energy metabolism of cells, and their function is especially important for neurons due to their high energy demand. Therefore, mitochondrial dysfunction is a pathological hallmark of various neurological disorders, including Parkinson's disease. The shape and organization of the mitochondrial network is highly plastic, which allows the cell to respond to environmental cues and needs, and the structure of mitochondria is also tightly linked to their health. Here, we present a protocol to study mitochondrial morphology in situ based on immunostaining of the mitochondrial protein VDAC1 and subsequent image analysis. This tool could be particularly useful for the study of neurodegenerative disorders because it can detect subtle differences in mitochondrial counts and shape induced by aggregates of α-synuclein, an aggregation-prone protein heavily involved in the pathology of Parkinson's disease. This method allows one to report that substantia nigra pars compacta dopaminergic neurons harboring pS129 lesions show mitochondrial fragmentation (as suggested by their reduced Aspect Ratio, AR) compared to their healthy neighboring neurons in a pre-formed fibril intracranial injection Parkinson model.

Advanced Methods In Parkinson's Disease Research.