No abstract

SUMMARY Neuronal microtubules support intracellular transport, facilitate axon growth, and form a basis for neuronal morphology. While microtubules in non-neuronal cells are depolymerized by cold, Ca2+ or antimitotic drugs, neuronal microtubules are unusually stable. Such stability is important for normal axon growth and maintenance, while hyperstability may compromise neuronal function in aging and degeneration. Though mechanisms for stability were unclear, studies suggested that stable microtubules contain biochemically distinct tubulins that are more basic than conventional tubulins. Transglutaminase-catalyzed posttranslational incorporation of polyamines is one of the few modifications of intracellular proteins that add positive charges. Here we show that neuronal tubulin can be polyaminated by transglutaminase. Endogenous brain transglutaminase-catalyzed polyaminated tubulins have the biochemical characteristics of neuronal stable microtubules. Inhibiting polyamine synthesis or transglutaminase activity significantly decreases microtubule stability in vitro and in vivo. Together, this suggests that transglutaminase-catalyzed polyamination of tubulins stabilizes neuronal microtubules essential for unique neuronal structures and functions.

FXR1 is one of the two known homologues of FMR1. FXR1 shares a high degree of sequence homology with FMR1 and also encodes two KH domains and an RGG domain, conferring RNA-binding capabilities. In comparison with FMRP, very little is known about the function of FXR1P in vivo. Mouse knockout (KO) models exist for both Fmr1 and Fxr2. To study the function of Fxr1 in vivo, we generated an Fxr1 KO mouse model. Homozygous Fxr1 KO neonates die shortly after birth most likely due to cardiac or respiratory failure. Histochemical analyses carried out on both skeletal and cardiac muscles show a disruption of cellular architecture and structure in E19 Fxr1 neonates compared with wild-type (WT) littermates. In WT E19 skeletal and cardiac muscles, Fxr1p is localized to the costameric regions within the muscles. In E19 Fxr1 KO littermates, in addition to the absence of Fxr1p, costameric proteins vinculin, dystrophin and alpha-actinin were found to be delocalized. A second mouse model (Fxr1 + neo), which expresses strongly reduced levels of Fxr1p relative to WT littermates, does not display the neonatal lethal phenotype seen in the Fxr1 KOs but does display a strongly reduced limb musculature and has a reduced life span of approximately 18 weeks. The results presented here point towards a role for Fxr1p in muscle mRNA transport/translation control similar to that seen for Fmrp in neuronal cells.

Although traditional roles ascribed to myelinating glial cells are structural and supportive, the importance of compact myelin for proper functioning of the nervous system can be inferred from mutations in myelin proteins and neuropathologies associated with loss of myelin. Myelinating Schwann cells are known to affect local properties of peripheral axons (de Waegh et al., 1992), but little is known about effects of oligodendrocytes on CNS axons. The shiverer mutant mouse has a deletion in the myelin basic protein gene that eliminates compact myelin in the CNS. In shiverer mice, both local axonal features like phosphorylation of cytoskeletal proteins and neuronal perikaryon functions like cytoskeletal gene expression are altered. This leads to changes in the organization and composition of the axonal cytoskeleton in shiverer unmyelinated axons relative to age-matched wild-type myelinated fibers, although connectivity and patterns of neuronal activity are comparable. Remarkably, transgenic shiverer mice with thin myelin sheaths display an intermediate phenotype indicating that CNS neurons are sensitive to myelin sheath thickness. These results indicate that formation of a normal compact myelin sheath is required for normal maturation of the neuronal cytoskeleton in large CNS neurons.

Angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) are secreted proteins that regulate triglyceride (TG) metabolism in part by inhibiting lipoprotein lipase (LPL).Recently, we showed that treatment of wild-type mice with monoclonal antibody (mAb) 14D12, specific for ANGPTL4, recapitulated the Angptl4 knock-out (؊/؊) mouse phenotype of reduced serum TG levels. In the present study, we mapped the region of mouse ANGPTL4 recognized by mAb 14D12 to amino acids Gln 29 -His 53 , which we designate as specific epitope 1 (SE1). The 14D12 mAb prevented binding of ANGPTL4 with LPL, consistent with its ability to neutralize the LPL-inhibitory activity of ANGPTL4. Alignment of all angiopoietin family members revealed that a sequence similar to ANGPTL4 SE1 was present only in ANGPTL3, corresponding to amino acids Glu 32 -His 55 . We produced a mouse mAb against this SE1-like region in ANGPTL3. This mAb, designated 5.50.3, inhibited the binding of ANGPTL3 to LPL and neutralized ANGPTL3-mediated inhibition of LPL activity in vitro. Treatment of wild-type as well as hyperlipidemic mice with mAb 5.50.3 resulted in reduced serum TG levels, recapitulating the lipid phenotype found in Angptl3 ؊/؊ mice. These results show that the SE1 region of ANGPTL3 and ANGPTL4 functions as a domain important for binding LPL and inhibiting its activity in vitro and in vivo. Moreover, these results demonstrate that therapeutic antibodies that neutralize ANGPTL4 and ANGPTL3 may be useful for treatment of some forms of hyperlipidemia.Lipoprotein lipase (LPL) 5 plays a pivotal role in lipid metabolism by catalyzing the hydrolysis of plasma triglycerides (TGs).LPL is likely to be regulated by mechanisms that depend on nutritional status and on the tissue in which it is expressed (1-3). Two secreted proteins, angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4), play important roles in the regulation of LPL activity (4, 5). ANGPTL3 and ANGPTL4 consist of a signal peptide, an N-terminal segment containing coiled-coil domains, and a C-terminal fibrinogen-like domain. The N-terminal segment as well as full-length ANGPTL3 and ANGPTL4 have been shown to inhibit LPL activity, and deletion of the N-terminal segment of ANGPTL3 and ANGPTL4 resulted in total loss of LPL-inhibiting activity (6, 7). These observations clearly indicate that the N-terminal region of ANGPTL4 contains the functional domain that inhibits LPL and affects plasma lipid levels. The coiled-coil domains have been proposed to be responsible for oligomerization (8); however, it is not known whether the coiled-coil domains directly mediate the inhibition of LPL activity.To define the physiological role of ANGPTL4 more clearly, we characterized the pharmacological consequences of ANGPTL4 inhibition in mice treated with the ANGPTL4-neutralizing monoclonal antibody (mAb) 14D12 (9). Injection of mAb 14D12 significantly lowered fasting TG levels in C57BL/6J mice relative to levels in C57BL/6J mice treated with an isotypematched anti-KLH control (KLH) mAb (9). These reduced TG...