Defective membrane remodeling in neuromuscular diseases: insights from animal models

Proteins involved in membrane remodeling play an essential role in a plethora of cell functions including endocytosis and intracellular transport. Defects in several of them lead to human diseases. Myotubularins, amphiphysins, and dynamins are all proteins implicated in membrane trafficking and/or remodeling. Mutations in myotubularin, amphiphysin 2 (BIN1), and dynamin 2 lead to different forms of centronuclear myopathy, while mutations in myotubularin-related proteins cause Charcot-Marie-Tooth neuropathies. In addition to centronuclear myopathy, dynamin 2 is also mutated in a dominant form of Charcot-Marie-Tooth neuropathy. While several proteins from these different families are implicated in similar diseases, mutations in close homologues or in the same protein in the case of dynamin 2 lead to diseases affecting different tissues. This suggests (1) a common molecular pathway underlying these different neuromuscular diseases, and (2) tissue-specific regulation of these proteins. This review discusses the pathophysiology of the related neuromuscular diseases on the basis of animal models developed for proteins of the myotubularin, amphiphysin, and dynamin families. A better understanding of the common mechanisms between these neuromuscular disorders will lead to more specific health care and therapeutic approaches.     

Trans-4-hydroxy-2-hexenal is a neurotoxic product of docosahexaenoic (22:6; n-3) acid oxidation

Lipid peroxidation of docosahexaenoic (22:6; n-3) acid (DHA) is elevated in the CNS in patients with Alzheimer's disease and in animal models of seizure and ethanol withdrawal. One product of DHA oxidation is trans -4-hydroxy-2-hexenal (HHE), a six carbon analog of the n-6 fatty acid derived trans -4-hydroxy-2-nonenal (HNE). In this work, we studied the neurotoxic potential of HHE. HHE and HNE were toxic to primary cultures of cerebral cortical neurons with LD₅₀'s of 23 and 18 μmol/L, respectively. Toxicity was prevented by the addition of thiol scavengers. HHE and HNE depleted neuronal GSH content identically with depletion observed with 10 μmol/L of either compound. Using an antibody raised against HHE–protein adducts, we show that HHE modified specific proteins of 75, 50, and 45 kDa in concentration- and time-dependent manners. The time-dependent formation of HHE differed from that of F₄-neuroprostanes following in vitro DHA oxidation likely as a result of the different oxidation pathways involved. Using purified mitochondrial aldehyde dehydrogenase ALDH5A, we found that HHE was oxidized 6.5-fold less efficiently than HNE. Our data demonstrate that HHE and HNE have similarities but also differences in their neurotoxic mechanisms and metabolism.     

Transparent adult zebrafish as a tool for in vivo transplantation analysis

The zebrafish is a useful model for understanding normal and cancer stem cells, but analysis has been limited to embryogenesis due to the opacity of the adult fish. To address this, we have created a transparent adult zebrafish in which we transplanted either hematopoietic stem/progenitor cells or tumor cells. In a hematopoiesis radiation recovery assay, transplantation of GFP-labeled marrow cells allowed for striking in vivo visual assessment of engraftment from 2 hr-5 weeks posttransplant. Using FACS analysis, both transparent and wild-type fish had equal engraftment, but this could only be visualized in the transparent recipient. In a tumor engraftment model, transplantation of RAS-melanoma cells allowed for visualization of tumor engraftment, proliferation, and distant metastases in as little as 5 days, which is not seen in wild-type recipients until 3 to 4 weeks. This transparent adult zebrafish serves as the ideal combination of both sensitivity and resolution for in vivo stem cell analyses.     

MCL-1 inhibits BAX in the absence of MCL-1/BAX Interaction

The BCL-2 family of proteins plays a major role in the control of apoptosis as the primary regulator of mitochondrial permeability. The pro-apoptotic BCL-2 homologues BAX and BAK are activated following the induction of apoptosis and induce cytochrome c release from mitochondria. A second class of BCL-2 homologues, the BH3-only proteins, is required for the activation of BAX and BAK. The activity of both BAX/BAK and BH3-only proteins is opposed by anti-apoptotic BCL-2 homologues such as BCL-2 and MCL-1. Here we show that anti-apoptotic MCL-1 inhibits the function of BAX downstream of its initial activation and translocation to mitochondria. Although MCL-1 interacted with BAK and inhibited its activation, the activity of MCL-1 against BAX was independent of an interaction between the two proteins. However, the anti-apoptotic function of MCL-1 required the presence of BAX. These results suggest that the pro-survival activity of MCL-1 proceeds via inhibition of BAX function at mitochondria, downstream of its activation and translocation to this organelle.     

Generation and characterization of two novel mouse models exhibiting the phenotypes of the metabolic syndrome: Apob48-/-Lepob/ob mice devoid of ApoE or Ldlr

The metabolic syndrome is a group of disorders including obesity, insulin resistance, atherogenic dyslipidemia, hyperglycemia, and hypertension. To date, few animal models have been described to recapitulate the phenotypes of the syndrome. In this study, we generated and characterized two lines of triple-knockout mice that are deficient in either apolipoprotein E (Apoe(-/-)) or low-density lipoprotein receptor (Ldlr(-/-)) and express no leptin (Lep(ob/ob)) or apolipoprotein B-48 but exclusively apolipoprotein B-100 (Apob(100/100)). These two lines are referred to as Apoe triple-knockout-Apoe 3KO (Apoe(-/-)Apob(100/100)Lep(ob/ob)) and Ldlr triple-knockout-Ldlr 3KO (Ldlr(-/-)Apob(100/100)Lep(ob/ob)) mice. Both lines develop obesity, hyperinsulinemia, hyperlipidemia, hypertension, and atherosclerosis. However, only Apoe 3KO mice are hyperglycemic and glucose intolerant and are more obese than Ldlr 3KO mice. To evaluate the utility of these lines as pharmacological models, we treated both with leptin and found that leptin therapy ameliorated most metabolic derangements. Leptin was more effective in improving glucose tolerance in Ldlr 3KO than Apoe 3KO animals. The reduction of plasma cholesterol by leptin in Ldlr 3KO mice can be accounted for by its suppressive effect on food intake. However, in Apoe 3KO mice, leptin further reduced plasma cholesterol independently of its effect on food intake, and this improvement correlated with a smaller plaque lesion area. These effects suggest a direct role of leptin in modulating VLDL levels and, likewise, the lesion areas in VLDL-enriched animals. These two lines of mice represent new models with features of the metabolic syndrome and will be useful in testing therapies targeted for combating the human condition.     

Erratum to Production of diploid and triploid offspring by inbreeding of the triploid planarian <Emphasis Type="Italic">Dugesia ryukyuensis</Emphasis>

Triploidy has generally been considered to be an evolutionary dead end due to problems of chromosomal pairing and segregation during meiosis. Thus, the formation of tetraploids and diploids from triploid types is a rare phenomenon. In the present study, we demonstrated that inbreeding of the triploid planarian Dugesia ryukyuensis resulted in both diploid and triploid offspring in nature. In the triploids of D. ryukyuensis, chiasmata between homologous chromosomes were observed in both female and male germ lines. This result suggests that both diploid and triploid offspring of this species are produced bisexually by zygotic fusion between sperm and eggs. Hence, this phenomenon may be a novel mechanism in planarian for escaping the triploid state.     

An optimized fast-performance liquid chromatography method for analyzing lipoprotein profiles using microliter volumes of serum

Plasma or serum lipoprotein analysis is commonly carried out with a conventional size-exclusion fast-performance liquid chromatography method that requires large sample volumes (1-2 ml). To determine lipoprotein profiles of mice with this method, plasma or serum samples have to be pooled from a group of animals, which often requires sacrificing animals. Here we report an optimized anion-exchange chromatography method with simplified cholesterol collection and detection system. After 5-10 μl serum was injected for anion-exchange chromatography, a stepwise gradient was applied and fractions were collected on a 96-well plate. Cholesterol content in each well was measured using a fluorescence-based detection method. With this method, distinct lipoprotein peaks corresponding to high-density lipoprotein, low-density lipoprotein, and very-low-density lipoprotein, can be easily separated and identified with excellent resolution. The entire high-performance liquid chromatography run takes about 30 min and the results are reproducible with a low variability. The small sample size allows analyzing the lipoprotein profile in a given mouse at a given time point with nonterminal bleeding. The method is simple to set up with commercially available parts and convenient to run.