The effects of increased Plp1 gene dosage on expression and processing of myelin proteins

Mutations in proteolipid protein 1 (PLP1), an X-linked gene causes Pelizaeus-Merzbacher disease (PMD) in humans. The most frequent cause of PMD is the duplication of PLP1, which encodes the major myelin membrane protein of the human CNS. The #66 transgenic mice with extra copies of the wild type Plp1 gene are a valid model of PMD caused by increased gene dosage (Readhead et al., 1994). These mice develop dysmyelinating or demyelinating phenotypes dependant on the gene dosage. This study investigated the effects of both low and high increased Plp1 gene dosage on various different selected aspects of myelin, including morphology, message and protein levels of PLP/DM20 and other representative myelin proteins and PLP/DM20 dynamics. Early in development mice with low increased gene dosage (hemizygous) are indistinguishable at the protein and myelin levels from their wild type littermates. During myelination these animals display elevated levels of PLP/DM20 in the oligodendrocyte cell body and alterations in other myelin protein levels and to the structure of myelin but these are transitory effects. These transient changes suggest the oligodendrocytes at low gene dosage retain the ability to regulate expression, production and incorporation of proteins into myelin thus maintaining the normal process of myelination. At high increased gene dosage (homozygous), oligodendrocytes in culture, pre and early myelinating oligodendrocytes in vivo and oligodendrocytes in vivo during peak myelination all exhibit elevated levels of PLP/DM20 in the their cell bodies. The protein is sequestered into autophagic vacuoles and late endosomes/lysosomes (LE/Ls), while the levels in myelin are reduced compared to wild type and hemizygous cells. Synthesis, partitioning with lipids and incorporation of PLP/DM20 are all also affected in the homozygous animals. The increased Plp1 gene dosage does affect other myelin proteins, in particular MBP, which showed a consistent and dramatic reduction in oligodendrocytes and myelin. These results indicate the heterogeneity of phenotypes and underlying changes caused by low and high increased Plp1 gene dosage. The cause of the dysmyelination observed in #66 mice and patients with PMD does not appear to be due to one single change in myelinogenesis. Each alteration observed in #66 transgenic mice could be a contributing factor. Importantly, the perturbation of MBP expression, in the light of this gene’s pivotal role in myelination, highlights that the relationship between Plp1 and Mbp expression is implicated in the pathogenesis of dysmyelination

MANAGEMENT OF GESTATIONAL DIABETES MELLITUS: AN INSIGHT INTO EVIDENCE-BASED PRACTICE AMONG POSTGRADUATE TRAINEES OF OBSTETRICS AND MEDICINE DISCIPLINES

OBJECTIVE: To explore the discrepancies regarding screening, diagnosis & management of gestational diabetes mellitus (GDM) among postgraduate trainees of Obstetrics & Gynaecology (OBG) and Medicine disciplines. METHODS: This multicentre cross-sectional study was conducted from 1st to 31st August 2022. The questionnaire regarding screening, diagnosis, management of GDM, & postnatal follow-up with neonatal care were distributed among postgraduate trainees of medicine/OBG through google-form/hardcopies. Data was analysed through SPSS-22 RESULTS: Out of 236 trainees, 184 (78%) were following national institute of clinical excellence (NICE) guidelines for management of GDM. Majority of medicine (n=87/120 (72.5%) & OBG (n=76/116; 65.5%) trainees failed to identify the correct cut-off of oral glucose tolerance test for GDM. A big chunk of both OBG (n=93/116; 80.2%) & Medicine (n=96/120; 80%) trainees were unable to differentiate pre-existing diabetes mellitus from GDM. The clinical knowledge about carbohydrate diet (n=119/236; 50.4%), calories intake (103/236; 43.6%) & low glycaemic index (138/236; 58.5%) was poor among trainees of both specialities. Surprisingly, the medicine trainee’s knowledge about insulin types, dose & tocolytic agent was not evidence-based. The practicing knowledge of both specialities was poor about identification of neonatal hypoglycemia (n=30/236; 12.7%) & its management (n=47; 19.9%). Trainees of both specialities had poor knowledge about postnatal follow-up (n=64/236; 27.1%) of GDM patients. CONCLUSION: GDM is a common domain for OBG & medicine disciplines with no consensus guidelines for its uniform management. This study has identified some basic gaps in the clinical practice of future consultants regarding GDM management, urging the need of combined local guidelines

Reversed argininosuccinate lyase activity in fumarate hydratase-deficient cancer cells.

BACKGROUND: Loss of function of fumarate hydratase (FH), the mitochondrial tumor suppressor and tricarboxylic acid (TCA) cycle enzyme, is associated with a highly malignant form of papillary and collecting duct renal cell cancer. The accumulation of fumarate in these cells has been linked to the tumorigenic process. However, little is known about the overall effects of the loss of FH on cellular metabolism. METHODS: We performed comprehensive metabolomic analyses of urine from Fh1-deficient mice and stable isotopologue tracing from human and mouse FH-deficient cell lines to investigate the biochemical signature of the loss of FH. RESULTS: The metabolomics analysis revealed that the urea cycle metabolite argininosuccinate is a common metabolic biomarker of FH deficiency. Argininosuccinate was found to be produced from arginine and fumarate by the reverse activity of the urea cycle enzyme argininosuccinate lyase (ASL), making these cells auxotrophic for arginine. Depleting arginine from the growth media by the addition of pegylated arginine deiminase (ADI-PEG 20) decreased the production of argininosuccinate in FH-deficient cells and reduced cell survival and proliferation. CONCLUSIONS: These results unravel a previously unidentified correlation between fumarate accumulation and the urea cycle enzyme ASL in FH-deficient cells. The finding that FH-deficient cells become auxotrophic for arginine opens a new therapeutic perspective for the cure of hereditary leiomyomatosis and renal cell cancer (HLRCC).RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

mTORC2 signaling drives the development and progression of pancreatic cancer

mTOR signaling controls several critical cellular functions and is deregulated in many cancers, including pancreatic cancer. To date, most efforts have focused on inhibiting the mTORC1 complex. However, clinical trials of mTORC1 inhibitors in pancreatic cancer have failed, raising questions about this therapeutic approach. We employed a genetic approach to delete the obligate mTORC2 subunit Rictor and identified the critical times during which tumorigenesis requires mTORC2 signaling. Rictor deletion resulted in profoundly delayed tumorigenesis. Whereas previous studies showed most pancreatic tumors were insensitive to rapamycin, treatment with a dual mTORC1/2 inhibitor strongly suppressed tumorigenesis. In late-stage tumor-bearing mice, combined mTORC1/2 and PI3K inhibition significantly increased survival. Thus, targeting mTOR may be a potential therapeutic strategy in pancreatic cancer

Macropinocytosis renders a subset of pancreatic tumor cells resistant to mTOR inhibition

Pancreatic ductal adenocarcinoma (PDAC) features a near-universal mutation in KRAS. Additionally, the tumor suppressor PTEN is lost in ∼10% of patients, and in mouse models, this dramatically accelerates tumor progression. While oncogenic KRAS and phosphatidylinositol 3-kinase (PI3K) cause divergent metabolic phenotypes individually, how they synergize to promote tumor metabolic alterations and dependencies remains unknown. We show that in KRAS-driven murine PDAC cells, loss of Pten strongly enhances both mTOR signaling and macropinocytosis. Protein scavenging alleviates sensitivity to mTOR inhibition by rescuing AKT phosphorylation at serine 473 and consequently cell proliferation. Combined inhibition of mTOR and lysosomal processing of internalized protein eliminates the macropinocytosis-mediated resistance. Our results indicate that mTORC2, rather than mTORC1, is an important regulator of protein scavenging and that protein-mediated resistance could explain the lack of effectiveness of mTOR inhibitors in certain genetic backgrounds. Concurrent inhibition of mTOR and protein scavenging might be a valuable therapeutic approach

Mitotic stress is an integral part of the oncogene-induced senescence program that promotes multinucleation and cell cycle arrest

Oncogene-induced senescence (OIS) is a tumor suppression mechanism that blocks cell proliferation in response to oncogenic signaling. OIS is frequently accompanied by multinucleation; however, the origin of this is unknown. Here, we show that multinucleate OIS cells originate mostly from failed mitosis. Prior to senescence, mutant H-RasV12 activation in primary human fibroblasts compromised mitosis, concordant with abnormal expression of mitotic genes functionally linked to the observed mitotic spindle and chromatin defects. Simultaneously, H-RasV12 activation enhanced survival of cells with damaged mitoses, culminating in extended mitotic arrest and aberrant exit from mitosis via mitotic slippage. ERK-dependent transcriptional upregulation of Mcl1 was, at least in part, responsible for enhanced survival and slippage of cells with mitotic defects. Importantly, mitotic slippage and oncogene signaling cooperatively induced senescence and key senescence effectors p21 and p16. In summary, activated Ras coordinately triggers mitotic disruption and enhanced cell survival to promote formation of multinucleate senescent cells

Mutant p53R270H drives altered metabolism and increased invasion in pancreatic ductal adenocarcinoma

Pancreatic cancer is characterized by nearly universal activating mutations in KRAS. Among other somatic mutations, TP53 is mutated in more than 75% of human pancreatic tumors. Genetically engineered mice have proven instrumental in studies of the contribution of individual genes to carcinogenesis. Oncogenic Kras mutations occur early during pancreatic carcinogenesis and are considered an initiating event. In contrast, mutations in p53 occur later during tumor progression. In our model, we recapitulated the order of mutations of the human disease, with p53 mutation following expression of oncogenic Kras. Further, using an inducible and reversible expression allele for mutant p53, we inactivated its expression at different stages of carcinogenesis. Notably, the function of mutant p53 changes at different stages of carcinogenesis. Our work establishes a requirement for mutant p53 for the formation and maintenance of pancreatic cancer precursor lesions. In tumors, mutant p53 becomes dispensable for growth. However, it maintains the altered metabolism that characterizes pancreatic cancer and mediates its malignant potential. Further, mutant p53 promotes epithelial-mesenchymal transition (EMT) and cancer cell invasion. This work generates new mouse models that mimic human pancreatic cancer and expands our understanding of the role of p53 mutation, common in the majority of human malignancies

Targeting the LOX/hypoxia axis reverses many of the features that make pancreatic cancer deadly: inhibition of LOX abrogates metastasis and enhances drug efficacy

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer‐related mortality. Despite significant advances made in the treatment of other cancers, current chemotherapies offer little survival benefit in this disease. Pancreaticoduodenectomy offers patients the possibility of a cure, but most will die of recurrent or metastatic disease. Hence, preventing metastatic disease in these patients would be of significant benefit. Using principal component analysis (PCA), we identified a LOX/hypoxia signature associated with poor patient survival in resectable patients. We found that LOX expression is upregulated in metastatic tumors from Pdx1‐Cre KrasG12D/+ Trp53R172H/+ (KPC) mice and that inhibition of LOX in these mice suppressed metastasis. Mechanistically, LOX inhibition suppressed both migration and invasion of KPC cells. LOX inhibition also synergized with gemcitabine to kill tumors and significantly prolonged tumor‐free survival in KPC mice with early‐stage tumors. This was associated with stromal alterations, including increased vasculature and decreased fibrillar collagen, and increased infiltration of macrophages and neutrophils into tumors. Therefore, LOX inhibition is able to reverse many of the features that make PDAC inherently refractory to conventional therapies and targeting LOX could improve outcome in surgically resectable disease

Optimisation of sample preparation from primary mouse tissue to maintain RNA integrity for methods examining translational control

The protein output of different mRNAs can vary by two orders of magnitude; therefore, it is critical to understand the processes that control gene expression operating at the level of translation. Translatome-wide techniques, such as polysome profiling and ribosome profiling, are key methods for determining the translation rates occurring on specific mRNAs. These techniques are now widely used in cell lines; however, they are underutilised in tissues and cancer models. Ribonuclease (RNase) expression is often found to be higher in complex primary tissues in comparison to cell lines. Methods used to preserve RNA during lysis often use denaturing conditions, which need to be avoided when maintaining the interaction and position of the ribosome with the mRNA is required. Here, we detail the cell lysis conditions that produce high-quality RNA from several different tissues covering a range of endogenous RNase expression levels. We highlight the importance of RNA integrity for accurate determination of the global translation status of the cell as determined by polysome gradients and discuss key aspects to optimise for accurate assessment of the translatome from primary mouse tissue

Targeted irradiation in an autochthonous mouse model of pancreatic cancer

The value of radiotherapy in the treatment of pancreatic cancer has been the subject of much debate but limited preclinical research. We hypothesise that the poor translation of radiation research into clinical trials of radiotherapy in pancreatic cancer is due, in part, to inadequate preclinical study models. Here, we have developed and refined methods for targeted irradiation in autochthonous mouse models of pancreatic cancer, using a small animal radiotherapy research platform. We tested and optimised strategies for administration of contrast agents, iohexol and the liver imaging agent Fenestra LC, to enable the use of computed tomography imaging in tumour localisation. We demonstrate accurate tumour-targeting, negligible off-target effects, and therapeutic efficacy, depending on dose, number of fractions and tumour size, and provide proof-of-concept that precise radiation can be delivered effectively to mouse pancreatic tumours with a clinically relevant microenvironment. This advance will allow investigation of the radiation response in murine pancreatic cancer, discovery of mechanisms and biomarkers of radiosensitivity or resistance, and development of radiosensitising strategies to inform clinical trials for precision radiotherapy in this disease