Faculty Bibliography

BACKGROUND AND PURPOSE: No treatments have been identified to lower the risk of intracerebral hemorrhage due to cerebral amyloid angiopathy (CAA). A potential approach to prevention is the use of agents that interfere with the pathogenic cascade initiated by the beta-amyloid peptide (Abeta). Tramiprosate (3-amino-1-propanesulfonic acid) is a candidate molecule shown in preclinical studies to reduce CAA in a transgenic mouse model. METHODS: We performed a 5-center phase 2 double-blinded trial to evaluate the safety, tolerability, and pharmacokinetics of tramiprosate in subjects with lobar intracerebral hemorrhage. Twenty-four subjects age > or =55 years with possible or probable CAA were randomized to receive 12 weeks of tramiprosate at 1 of 3 oral doses (50, 100, or 150 mg twice daily). Subjects were followed for clinical adverse effects, laboratory, vital sign, electrocardiogram, cognitive, or functional changes, appearance of new symptomatic or asymptomatic hemorrhages, and pharmacokinetic parameters. RESULTS: Enrolled subjects were younger (mean age 70.8+/-5.4, range 61 to 78) and had more advanced baseline disease (measured by number of previous hemorrhages) than consecutive subjects in a CAA natural history cohort. No concerning safety issues were encountered with treatment. Nausea and vomiting were the most common adverse events and were more frequent at high doses. Nine subjects had new symptomatic or asymptomatic hemorrhages during treatment; all occurred in subjects with advanced baseline disease, with no apparent effect of drug dosing assignment. CONCLUSIONS: These data suggest that tramiprosate can be given safely to subjects with suspected CAA and support future efficacy trials.

PURPOSE OF REVIEW: Amyloid deposits are a defining feature of several age-related and debilitating diseases. Their widespread presence in atherosclerotic plaques suggests a potential role in lesion development. This review discusses the proteins known to accumulate in atheroma and examines the evidence that amyloid-like structures activate macrophage signaling pathways linked to inflammation and prothrombotic potential. RECENT FINDINGS: Numerous proteins that accumulate in atherosclerotic plaques form amyloid fibrils in vivo, including apolipoproteins, beta-amyloid, and alpha1-antitrypsin. In addition, oxidation or enzymatic modification of low-density lipoproteins induces a structural reorganization of the particle, including the acquisition of amyloid-like properties. Similarly, glycation of serum albumin, as observed in diabetes, is accompanied by the formation of aggregates with all the hallmarks of amyloid. Several receptors implicated in atherogenesis modulate the fate of amyloid fibrils by mediating their clearance (scavenger receptors A and B-I), activating inflammatory signaling cascades (receptor for advanced glycation endproducts), or both (CD36). Finally, recent studies indicate that amyloid deposition accelerates diet-induced atherosclerosis in mice. SUMMARY: Given the substantial evidence that amyloid fibrils or preamyloidogenic species are cytotoxic, the aberrant deposition of amyloid in the intima may be pathologically important in vascular inflammation and the promotion of atherosclerosis

Sonic hedgehog has received an enormous amount of attention since its role as a morphogen that directs ventral patterning in the spinal cord was discovered a decade ago. Since that time, a bewildering array of information has been generated concerning both the components of the hedgehog signalling pathway and the remarkable number of contexts in which it functions. Nowhere is this more evident than in the nervous system, where hedgehog signalling has been implicated in events as disparate as axonal guidance and stem cell maintenance. Here we review our present knowledge of the hedgehog signalling pathway and speculate about areas in which further insights into this versatile pathway might be forthcoming

Alpha-internexin, a neuronal intermediate filament protein implicated in neurodegenerative disease, coexists with the neurofilament (NF) triplet proteins (NF-L, NF-M, and NF-H) but has an unknown function. The earlier peak expression of alpha-internexin than the triplet during brain development and its ability to form homopolymers, unlike the triplet, which are obligate heteropolymers, have supported a widely held view that alpha-internexin and neurofilament triplet form separate filament systems. Here, we demonstrate, however, that despite a postnatal decline in expression, alpha-internexin is as abundant as the triplet in the adult CNS and exists in a relatively fixed stoichiometry with these subunits. Alpha-internexin exhibits transport and turnover rates identical to those of triplet proteins in optic axons and colocalizes with NF-M on single neurofilaments by immunogold electron microscopy. Alpha-internexin also coassembles with all three neurofilament proteins into a single network of filaments in quadruple-transfected SW13vim(-) cells. Genetically deleting NF-M alone or together with NF-H in mice dramatically reduces alpha-internexin transport and content in axons throughout the CNS. Moreover, deleting alpha-internexin potentiates the effects of NF-M deletion on NF-H and NF-L transport. Finally, overexpressing a NF-H-LacZ fusion protein in mice induces alpha-internexin and neurofilament triplet to aggregate in neuronal perikarya and greatly reduces their transport and content selectively in axons. Our data show that alpha-internexin and the neurofilament proteins are functionally interdependent. The results strongly support the view that alpha-internexin is a fourth subunit of neurofilaments in the adult CNS, providing a basis for its close relationship with neurofilaments in CNS diseases associated with neurofilament accumulation

Increasing evidence implicates the interaction of the EphB4 receptor with its preferred ligand, ephrinB2, in pathological forms of angiogenesis and in tumorigenesis. To identify the molecular determinants of the unique specificity of EphB4 for ephrinB2, we determined the crystal structure of the ligand binding domain of EphB4 in complex with the extracellular domain of ephrinB2. This structural analysis suggested that one amino acid, Leu-95, plays a particularly important role in defining the structural features that confer the ligand selectivity of EphB4. Indeed, all other Eph receptors, which promiscuously bind many ephrins, have a conserved arginine at the position corresponding to Leu-95 of EphB4. We have also found that amino acid changes in the EphB4 ligand binding cavity, designed based on comparison with the crystal structure of the more promiscuous EphB2 receptor, yield EphB4 variants with altered binding affinity for ephrinB2 and an antagonistic peptide. Isothermal titration calorimetry experiments with an EphB4 Leu-95 to arginine mutant confirmed the importance of this amino acid in conferring high affinity binding to both ephrinB2 and the antagonistic peptide ligand. Isothermal titration calorimetry measurements also revealed an interesting thermodynamic discrepancy between ephrinB2 binding, which is an entropically driven process, and peptide binding, which is an enthalpically driven process. These results provide critical information on the EphB4*ephrinB2 protein interfaces and their mode of interaction, which will facilitate development of small molecule compounds inhibiting the EphB4*ephrinB2 interaction as novel cancer therapeutics.

The tight junctions of the endothelial and epithelial regions of the blood-brain barrier are regulated by interactions with the neighboring tissue. We examined how the neural retina regulates the assembly of tight junctions in the retinal pigment epithelium (RPE). The proteins JAM-A, AF-6, PAR-3 and PAR-6 have been implicated in the assembly of other epithelial tight junctions. Using chick embryos and primary cell culture, we examined gene expression of these proteins during embryonic development, and whether retinal secretions regulate their expression. Three highly conserved RNA splice sites of AF-6 were identified in chick ocular tissues, but only two were expressed in RPE. JAM-A and AF-6 were expressed at relatively high levels early in development when adherens junctions form, but before tight junctions form. Expression of JAM-A and the AF-6 isoforms actually decreased when tight junctions were forming and expanding. The expression of PAR-3 and PAR-6 was constant. Despite the expression of these proteins in vitro (along with claudins, occludin, ZO-1 and ZO-2), the tight junctional networks that form were discontinuous (Rahner, C., Fukuhara, M., Peng, S., Kojima, S., Rizzolo, L.J., 2004. The apical and basal environments of the retinal pigment epithelium regulate the maturation of tight junctions during development. J. Cell Sci. 117, 3307-3318). The expression of these assembly proteins was unaffected by a retinal conditioned medium that induced the completion of tight junction formation. These data indicate that the early expression of the assembly proteins corresponds to the initial establishment of the adherens and tight junctions, but secretory products of the neural retina must induce the expression of additional proteins to complete the maturation process.

Protein misfolding caused by exposure to arsenite is associated with transcriptional activation of the AIRAP gene. We report here that AIRAP is an arsenite-inducible subunit of the proteasome's 19S cap that binds near PSMD2 at the 19S base. Compared to the wild-type, knockout mouse cells or C. elegans lacking AIRAP accumulate more polyubiquitylated proteins and exhibit higher levels of stress when exposed to arsenite, and proteasomes isolated from arsenite-treated AIRAP knockout cells are relatively impaired in substrate degradation in vitro. AIRAP's association with the 19S cap reverses the stabilizing affect of ATP on the 26S proteasome during particle purification, and AIRAP-containing proteasomes, though constituted of 19S and 20S subunits, acquire features of hybrid proteasomes with both 19S and 11S regulatory caps. These features include enhanced cleavage of peptide substrates and suggest that AIRAP adapts the cell's core protein degradation machinery to counteract proteotoxicity induced by an environmental toxin

Cotranslational translocation of apoB100 across the endoplasmic reticulum (ER) membrane is inefficient, resulting in exposure of nascent apoB on the cytosolic surface of the ER. This predisposes apoB100 to ubiquitinylation and targeting for proteasomal degradation. It has been suggested that pause transfer sequences (PTS) present throughout apoB cause inefficient translocation. On the other hand, our previous study demonstrated that the translocation efficiency of apoB100 is dependent on the presence of a beta-sheet domain between 29 and 34% of full-length apoB100 (Liang, J.-S., Wu, X., Jiang, H., Zhou, M., Yang, H., Angkeow, P., Huang, L.-S., Sturley, S. L., and Ginsberg, H. N. (1998) J. Biol. Chem. 273, 35216-35221); this region of apoB has no PTS. However, the effects of the beta-sheet domain may require the presence of PTS elsewhere in the N-terminal region of apoB100. To further investigate the roles of PTS and beta-sheet domains in the translocation of apoB100 across the ER, we transfected McArdle RH7777, HepG2, or Chinese hamster ovary cells with human albumin (ALB)/human apoB chimeric cDNA constructs: ALB/B12-17 (two PTS but no beta-sheet), ALB/B29-34 (beta-sheet but no PTS), ALB/B36-41 (two PTS and a beta-sheet), and ALB/B49-54 (neither PTS nor a beta-sheet). ALB/ALB1-40 served as a control. Compared with ALB/ALB1-40, secretion rates of ALB/B12-17, ALB/B29-34, and ALB/B36-41 were reduced. Secretion of ALB/B49-54 was similar to that of ALB/ALB1-40. However, only ALB/B29-34 and ALB/B36-41 had increased proteinase K sensitivity, ubiquitinylation, and increased physical interaction with Sec61alpha. These results indicate that the translocation efficiency of apoB100 is determined mainly by the presence of beta-sheet domains. PTS do not appear to affect translocation, but may affect secretion by other mechanisms.