In mammals, an adequate supply of thyroid hormones is essential for normal growth and neurological development. The biosynthesis of thyroid hormones involves an iodinated precursor protein, thyroglobulin, which may be considered an extreme example of a pro-hormone. Thyroglobulin is a dimeric glycoprotein of relative molecular mass (Mr) 660,000 (660K), which is secreted by the thyrocyte and stored in the lumen of the thyroid follicle. The hormonogenic reaction is extracellular, and involves iodination of tyrosyl residues of thyroglobulin and the intramolecular coupling of a subset of these into thyroxine (T4) and triiodothyronine (T3), which remain part of the polypeptide chain. Secretion of hormones results from the endocytosis of thyroglobulin followed by its complete hydrolysis in lysosomes. Considering that the maximum yield of hormones is approximately 6-8 per 660K protein, the whole process is apparently wasteful. However, the efficiency of thyroglobulin as a thyroid hormone precursor is extremely high when the supply of iodine is short; in such conditions, almost all the iodine incorporated is found in iodothyronine. Hence it is suggested that the thyroglobulin structure has evolved to allow for the preferential and efficient iodination and coupling of the hormonogenic tyrosines. Here we report the complete primary structure of bovine thyroglobulin, derived from the sequence of its 8,431-base-pair complementary DNA. The 2,769-amino-acid sequence is characterized by a pattern of imperfect repeats derived from three cysteine-rich motifs. Four hormonogenic tyrosines have been precisely localized near the amino and carboxyl ends of the protein.

The ␤-amyloid peptide (A␤) present in the senile plaques of Alzheimer's disease derives from the cleavage of a membrane protein, named APP, driven by two enzymes, known as ␤-and ␥-secretases. The mechanisms regulating this cleavage are not understood. We have developed an experimental system to identify possible extracellular signals able to trigger the cleavage of an APPGal4 fusion protein, which is detected by measuring the expression of the CAT gene transcribed under the control of the Gal4 transcription factor, which is released from the membrane upon the cleavage of APP-Gal4. By using this assay, we purified a protein contained in the C6 cellconditioned medium, which activates the cleavage of APP-Gal4 and which we demonstrated to be PDGF-BB. The APP-Gal4 processing induced by PDGF is dependent on the ␥-secretase activity, being abolished by an inhibitor of this enzyme, and is the consequence of the activation of a pathway downstream of the PDGF-receptor, which includes the non-receptor tyrosine kinase Src and the small G-protein Rac1. These findings are confirmed by the observation that a constitutively active form of Src increases A␤ generation and that, in cells stably expressing APP, the generation of A␤ is strongly decreased by the Src tyrosine kinase inhibitor PP2. ␤-Amyloid (A␤)1 deposition in the so-called amyloid plaques is one of the main features of Alzheimer's pathology. ␤-Amyloid consists of ϳ4-kDa peptides derived from the proteolytic processing of a membrane protein named amyloid precursor protein (APP). This amyloidogenic processing is driven by two enzyme activities, ␤-site APP cleaving enzyme (BACE) and ␥-secretase. BACE cleaves APP at 28 residues from the boundary between the extracellular/intraluminal domain of APP and the transmembrane domain of the protein (for a review see Ref. 1), releasing a large soluble protein, including nearly all the extracellular/intraluminal part of APP, and a short transmembrane peptide, including the 99 C-terminal residues of APP. This transmembrane C99 stub is a substrate for the ␥-secretase activity, which cleaves it, in a presenilin-dependent fashion, within the membrane ␣-helix, giving rise to the A␤ peptide 40 -42 amino acids long and to a peptide named APP intracellular domain (AID), which includes the small C-terminal cytosolic domain of APP (for a review see Refs. 2 and 3).The functions of APP and its proteolytic processing are still unknown. However, although the functions of the APP ectodomain remain elusive, there is increasing evidence that its cytodomain is the center of a complex network of interactions with several proteins, involved in vesicle transport and in signal transduction. In fact, it was demonstrated that APP cytoplasmic domain interacts with kinesin light chain and contributes to vesicles transport (4), thus suggesting that APP cleavage could regulate the transport of vesicles in the axons. On the other hand, the APP cytodomain binds several PTB domain-containing pro-

The aberrant metabolism of ␤-amyloid precursor protein (APP) and the progressive deposition of its derived fragment ␤-amyloid peptide are early and constant pathological hallmarks of Alzheimer's disease. Because APP is able to function as a cell surface receptor, we investigated here whether a disruption of the normal function of APP may contribute to the pathogenic mechanisms in Alzheimer's disease. To this aim, we generated a specific chicken polyclonal antibody directed against the extracellular domain of APP, which is common with the ␤-amyloid precursor-like protein type 2. Exposure of cultured cortical neurons to this antibody (APP-Ab) induced cell death preceded by neurite degeneration, oxidative stress, and nuclear condensation. Interestingly, caspase-3-like protease was not activated in this neurotoxic action suggesting a different mode of cell death than classical apoptosis. Further analysis of the molecular mechanisms revealed a calpain-and calcineurindependent proteolysis of the neuroprotective calcium/ calmodulin-dependent protein kinase IV and its nuclear target protein cAMP responsive element binding protein. These effects were abolished by the G protein inhibitor pertussis toxin, strongly suggesting that APP binding operates via a GTPase-dependent pathway to cause neuronal death. Alzheimer's disease (AD)1 is a devastating neurodegenerative disorder characterized by deposition of ␤-amyloid (A␤) plaques, accumulation of intracellular neurofibrillary tangles, and neuronal cell loss (1). A␤ peptide is generated by proteolysis of the amyloid precursor protein (APP), which is the product of a gene located on human chromosome 21 and on mouse chromosome 16. APP is expressed in most mammalian tissues (2, 3) and is present in at least 10 different spliced variants (4). In the brain, the major isoform generating A␤ is a peptide consisting of 695 residues that contains a single transmembrane domain (5). Although the physiological role of APP is still unclear, several studies have suggested that it is implicated in important physiological functions of neurons, such as neurite outgrowth, synaptogenesis, cell substrate adhesion and neuronal survival (for review see Ref. 6).Up to now, most of the research has been focused on the toxicity of A␤ peptide related to AD. A␤ has been reported to exert a variety of toxic effects on neurons both in vitro (7,8) and in vivo (9). However, it has been reported that mice overproducing A␤1-42 extracellularly showed no neuronal loss (10), thus suggesting that A␤ peptide seems not to be the only constituent in neurotoxicity associated to AD. Previous studies have demonstrated that overexpression of full-length APP induced degeneration of postmitotic neurons derived from embryonal carcinoma cells (11). Moreover, intracellular accumulation of wild-type APP in the rat hippocampus caused a specific type of neuronal degeneration in vivo in the absence of extracellular A␤ deposition (12), and viral vector-mediated overexpression of wild-type APP induced apoptosis-like death of neurons...

The serotonin 5-hydroxytryptamine (5-HT 4 ) receptor is of potential interest for the treatment of AlzheimerÕs disease because it increases memory and learning. In this study, we investigated the effect of zinc metalloprotease inhibitors on the amyloid precursor protein (APP) processing induced by the serotonin 5-HT 4 receptor in vitro. We show that secretion of the nonamyloidogenic form of APP, sAPPa induced by the 5-HT 4(e) receptor isoform was not due to a general boost of the constitutive secretory pathway but rather to its specific effect on a-secretase activity. Although the h5-HT 4(e) receptor increased IP3 production, inhibition of PKC did not modify its effect on sAPPa secretion. In addition, we found that a secretase activity is regulated by the cAMP-regulated guanine nucleotide exchange factor, Epac and the small GTPase Rac.

Increased production and deposition of the 40 -42-amino acid ␤-amyloid peptide (A␤) is believed to be central to the pathogenesis of Alzheimer's disease. A␤ is derived from the amyloid precursor protein (APP), but the mechanisms that regulate APP processing to produce A␤ are not fully understood. X11␣ (also known as munc-18-interacting protein-1 (Mint1)) is a neuronal adaptor protein that binds APP and modulates APP processing in transfected non-neuronal cells. To investigate the in vivo effect of X11␣ on A␤ production in the brain, we created transgenic mice that overexpress X11␣ and crossed these with transgenics harboring a familial Alzheimer's disease mutant APP that produces increased levels of A␤ (APPswe Tg2576 mice). Analyses of A␤ levels in the offspring generated from two separate X11␣ founder mice revealed a significant, approximate 20% decrease in A␤(1-40) in double transgenic mice expressing APPswe/X11␣ compared with APPswe mice. At a key time point in A␤ plaque deposition (8 months old), the number of A␤ plaques was also deceased in APPswe/X11␣ mice. Thus, we report here the first demonstration that X11␣ inhibits A␤ production and deposition in vivo in the brain.One pathological hallmark of Alzheimer's disease is deposition of the 40 -42-amino acid ␤-amyloid peptide (A␤) 1 within the brains of affected individuals. A␤ is derived by proteolytic cleavage from the amyloid precursor protein (APP). APP is a type-1 membrane-spanning protein that contains a large ectodomain and a smaller, intracellular endodomain, and A␤ is derived from sequences that encompass parts of both ecto-and trans-membrane domains. Aberrant APP processing/metabolism leading to increased production and deposition of A␤ is believed to be central to the pathogenesis of Alzheimer's disease (for reviews see Refs. 1 and 2).A number of proteins have now been shown to interact with the APP endodomain, including a variety of phosphotyrosinebinding domain proteins (3, 4). These phosphotyrosine-binding domain proteins comprise the Fe65 and X11 families. Disabled and JNK-interacting proteins and their interactions are mediated via the phosphotyrosine-binding domains and sequences surrounding the YENPTY motif in APP (5-18).One such protein is the neuronal adaptor X11␣, which contains a single, centrally located phosphotyrosine-binding domain through which it binds APP. However, X11␣ also contains a number of other protein-protein interaction domains and via these domains mediates the assembly of multi-protein complexes. Aside from APP family members, X11␣-binding partners identified to date include CASK, munc-18, spinophilin/ neurabin, neurexins, the N-type Ca 2ϩ channel pore-forming ␣ 1B subunit, the kinesin superfamily motor protein KIF17, presenilin 1, and the copper chaperone for superoxide dismutase-1 (19 -30).The binding of X11␣ to APP has now been shown to inhibit A␤ production in transfected non-neuronal cells, although the mechanisms that underlie this effect are not known (16,(31)(32)(33). However, whether it fulfills similar functio...