A mutant ataxin-3 fragment results from processing at a site N-terminal to amino acid 190 in brain of Machado-Joseph disease-like transgenic mice.

Machado-Joseph disease also called spinocerebellar ataxia type 3 (MJD/SCA3) is a hereditary and neurodegenerative movement disorder caused by ataxin-3 with a polyglutamine expansion (mutant ataxin-3). Neuronal loss in MJD/SCA3 is associated with a mutant ataxin-3 toxic fragment. Defining mutant ataxin-3 proteolytic site(s) could facilitate the identification of the corresponding enzyme(s). Previously, we reported a mutant ataxin-3 mjd1a fragment in the brain of transgenic mice (Q71) that contained epitopes C-terminal to amino acid 220. In this study, we generated and characterized neuroblastoma cells and transgenic mice expressing mutant ataxin-3 mjd1a lacking amino acids 190-220 (deltaQ71). Less deltaQ71 than Q71 fragments were detected in the cell but not mouse model. The transgenic mice developed an MJD/SCA3-like phenotype and their brain homogenates had a fragment containing epitopes C-terminal to amino acid 220. Our results support the toxic fragment hypothesis and narrow the mutant ataxin-3 cleavage site to the N-terminus of amino acid 190.

A mutant ataxin-3 putative-cleavage fragment in brains of Machado-Joseph disease patients and transgenic mice is cytotoxic above a critical concentration.

Machado-Joseph disease (MJD) is an inherited neurodegenerative disorder caused by ataxin-3 with a polyglutamine expansion. It is proposed that a toxic cleavage fragment of mutant ataxin-3 alternatively spliced isoform mjd1a triggers neurodegeneration, although this fragment has not yet been detected in the brains of MJD patients or in animal models. We have now generated transgenic mice expressing human mutant (Q71) or normal (Q20) ataxin-3 mjd1a under the control of the mouse prion promoter. Q71 transgenic mice expressing mutant ataxin-3 mjd1a above a critical level developed a phenotype similar to MJD including progressive postural instability, gait and limb ataxia, weight loss, premature death, neuronal intranuclear inclusions, and decreased tyrosine hydroxylase-positive neurons in the substantia nigra (determined by unbiased stereology). Q20 transgenic mice had normal behavior and pathology. Brains from sick Q71 transgenic mice contained an abundant mutant ataxin-3 mjd1a putative-cleavage fragment (Fragment), which was scarce in normal Q71 transgenic mice. Reactivity of the Fragment with a panel of antibodies and comigration with truncations of mutant ataxin-3 revealed that it contained residues C terminal to amino acid 221 to include the polyglutamine expansion. A similar portion of mutant ataxin-3 mjd1a expressed in transfected neuroblastoma cells was toxic above a critical concentration. The Fragment was more abundant in two affected brain regions of MJD patients. Thus, we have developed a murine model for mutant ataxin-3 mjd1a toxicity and identified a putative-cleavage fragment of the disease protein in the brains of these transgenic mice and MJD patients that is cytotoxic above a critical concentration.

37-kDa laminin receptor precursor modulates cytotoxic necrotizing factor 1-mediated RhoA activation and bacterial uptake.

Cytotoxic necrotizing factor 1 (CNF1) is a bacterial toxin known to activate Rho GTPases and induce host cell cytoskeleton rearrangements. The constitutive activation of Rho GTPases by CNF1 is shown to enhance bacterial uptake in epithelial cells and human brain microvascular endothelial cells. However, it is unknown how exogenous CNF1 exhibits such phenotypes in eukaryotic cells. Here, we identified 37-kDa laminin receptor precursor (LRP) as the receptor for CNF1 from screening the cDNA library of human brain microvascular endothelial cells by the yeast two-hybrid system using the N-terminal domain of CNF1 as bait. CNF1-mediated RhoA activation and bacterial uptake were inhibited by exogenous LRP or LRP antisense oligodeoxynucleotides, whereas they were increased in LRP-overexpressing cells. These findings indicate that the CNF1 interaction with LRP is the initial step required for CNF1-mediated RhoA activation and bacterial uptake in eukaryotic cells.

Effects of lipoprotein lipase on uptake and transcytosis of low density lipoprotein (LDL) and LDL-associated alpha-tocopherol in a porcine in vitro blood-brain barrier model.

During the present study the contribution of lipoprotein lipase (LPL) to low density lipoprotein (LDL) holoparticle and LDL-lipid (alpha-tocopherol (alphaTocH)) turnover in primary porcine brain capillary endothelial cells (BCECs) was investigated. The addition of increasing LPL concentrations to BCECs resulted in up to 11-fold higher LDL holoparticle cell association. LPL contributed to LDL holoparticle turnover, an effect that was substantially increased in response to LDL-receptor up-regulation. The addition of LPL increased selective uptake of LDL-associated alphaTocH in BCECs up to 5-fold. LPL-dependent selective alphaTocH uptake was unaffected by the lipase inhibitor tetrahydrolipstatin but was substantially inhibited in cells where proteoglycan sulfation was inhibited by treatment with NaClO(3). Thus, selective uptake of LDL-associated alphaTocH requires interaction of LPL with heparan-sulfate proteoglycans. Although high level adenoviral overexpression of scavenger receptor BI (SR-BI) in BCECs resulted in a 2-fold increase of selective LDL-alphaTocH uptake, SR-BI did not act in a cooperative manner with LPL. Although the addition of LPL to BCEC Transwell cultures significantly increased LDL holoparticle cell association and selective uptake of LDL-associated alphaTocH, holoparticle transcytosis across this porcine blood-brain barrier (BBB) model was unaffected by the presence of LPL. An important observation during transcytosis experiments was a substantial alphaTocH depletion of LDL particles that were resecreted into the basolateral compartment. The relevance of LPL-dependent alphaTocH uptake across the BBB was confirmed in LPL-deficient mice. The absence of LPL resulted in significantly lower cerebral alphaTocH concentrations than observed in control animals.