Amylin, a pancreatic peptide, and amyloid-beta peptides (Aβ), a major component of Alzheimer's disease (AD) brain, share similar β-sheet secondary structures, but it is not known whether pancreatic amylin affects amyloid pathogenesis in the AD brain. Using AD mouse models, we investigated the effects of amylin and its clinical analog, pramlintide, on AD pathogenesis. Surprisingly, chronic intraperitoneal (i.p.) injection of AD animals with either amylin or pramlintide reduces the amyloid burden as well as lowers the concentrations of Aβ in the brain. These treatments significantly improve their learning and memory assessed by two behavioral tests, Y maze and Morris water maze. Both amylin and pramlintide treatments increase the concentrations of Aβ1-42 in cerebral spinal fluid (CSF). A single i.p. injection of either peptide also induces a surge of Aβ in the serum, the magnitude of which is proportionate to the amount of Aβ in brain tissue. One intracerebroventricular injection of amylin induces a more significant surge in serum Aβ than one i.p. injection of the peptide. In 330 human plasma samples, a positive association between amylin and Aβ1-42 as well as Aβ1-40 is found only in patients with AD or amnestic mild cognitive impairment. As amylin readily crosses the blood–brain barrier, our study demonstrates that peripheral amylin's action on the central nervous system results in translocation of Aβ from the brain into the CSF and blood that could be an explanation for a positive relationship between amylin and Aβ in blood. As naturally occurring amylin may play a role in regulating Aβ in brain, amylin class peptides may provide a new avenue for both treatment and diagnosis of AD.

Synaptotagmin-7 is a candidate Ca 2؉ sensor for exocytosis that is at least partly localized to synapses. Similar to synaptotagmin-1, which functions as a Ca 2؉ sensor for fast synaptic vesicle (SV) exocytosis, synaptotagmin-7 contains C2A and C2B domains that exhibit Ca 2؉ -dependent phospholipid binding. However, synaptotagmin-7 cannot replace synaptotagmin-1 as a Ca 2؉ sensor for fast SV exocytosis, raising questions about the physiological significance of its Ca 2؉ -binding properties. Here, we examine how synaptotagmin-7 binds Ca 2؉ and test whether this Ca 2؉ binding regulates Ca 2؉ -triggered SV exocytosis. We show that the synaptotagmin-7 C 2A domain exhibits a Ca 2؉ -binding mode similar to that of the synaptotagmin-1 C2A domain, suggesting that the synaptotagmin-1 and -7 C 2 domains generally employ comparable Ca 2؉ -binding mechanisms. We then generated mutant mice that lack synaptotagmin-7 or contain point mutations inactivating Ca 2؉ binding either to both C2 domains of synaptotagmin-7 or only to its C2B domain. Synaptotagmin-7-mutant mice were viable and fertile. Inactivation of Ca 2؉ binding to both C2 domains caused an Ϸ70% reduction in synaptotagmin-7 levels, whereas inactivation of Ca 2؉ binding to only the C2B domain did not alter synaptotagmin-7 levels. The synaptotagmin-7 deletion did not change fast synchronous release, slow asynchronous release, or short-term synaptic plasticity of release of neurotransmitters. Thus, our results show that Ca 2؉ binding to the synaptotagmin-7 C2 domains is physiologically important for stabilizing synaptotagmin-7, but that Ca 2؉ binding by synaptotagmin-7 likely does not regulate SV exocytosis, consistent with a role for synaptotagmin-7 in other forms of Ca 2؉ -dependent synaptic exocytosis.asynchronous release ͉ calcium-binding protein ͉ neurotransmitter release ͉ synaptic plasticity

Amyloidogenic processing of the amyloid precursor protein (APP) generates a large secreted ectodomain fragment (APPsβ), β-amyloid (Aβ) peptides, and an APP intracellular domain (AICD). Whereas Aβ is viewed as critical for Alzheimer's disease pathogenesis, the role of other APP processing products remains enigmatic. Of interest, the AICD has been implicated in transcriptional regulation, and N-terminal cleavage of APPsβ has been suggested to produce an active fragment that may mediate axonal pruning and neuronal cell death. We previously reported that mice deficient in APP and APP-like protein 2 ( APLP2 ) exhibit early postnatal lethality and neuromuscular synapse defects, whereas mice with neuronal conditional deletion of APP and APLP2 are viable. Using transcriptional profiling, we now identify transthyretin ( TTR ) and Klotho as APP/APLP2-dependent genes whose expression is decreased in loss-of-function states but increased in gain-of-function states. Significantly, by creating an APP knockin allele that expresses only APPsβ protein, we demonstrate that APPsβ is not normally cleaved in vivo and is fully capable of mediating the APP-dependent regulation of TTR and Klotho gene expression. Despite being an active regulator of gene expression, APPsβ did not rescue the lethality and neuromuscular synapse defects of APP and APLP2 double-KO animals. Our studies identify TTR and Klotho as physiological targets of APP that are regulated by soluble APPsβ independent of developmental APP functions. This unexpected APP-mediated signaling pathway may play an important role in maintaining TTR and Klotho levels and their respective functions in Aβ sequestration and aging.

Sec1͞Munc18-like (SM) proteins functionally interact with soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) in membrane fusion, but the mechanisms of these interactions differ. In vertebrates, SM proteins that mediate exocytosis (Munc18-1, 18-2, and 18c) bind to the closed conformation of syntaxins 1-4, which requires the N-terminal H abc domains and SNARE motifs of these syntaxins. In contrast, SM proteins that mediate Golgi and endoplasmic reticulum fusion (Sly1 and Vps45) bind only to short N-terminal sequences of syntaxins 5, 16, or 18, independently of their H abc domains and SNARE motifs. We now show that Munc18-1, Sly1, and Vps45 interact with cognate syntaxins via similar, autonomously folded N-terminal domains, but the syntaxin 5-binding surface of the Sly1 N-terminal domain is opposite to the syntaxin 1-binding surface of the Munc18-1 Nterminal domain. In transfected cells, the N-terminal domain of Sly1 specifically disrupts the structure of the Golgi complex, supporting the notion that the interaction of Sly1 with syntaxin 5 is essential for fusion. These data, together with previous results, suggest that a relatively small N-terminal domain of SM proteins is dedicated to mechanistically distinct interactions with SNAREs, leaving the remaining large parts of SM proteins free to execute their as yet unknown function as effector domains.SM proteins ͉ syntaxins ͉ Sly1 ͉ Vps45p I ntracellular membrane fusion involves several families of conserved proteins, including soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) and Sec1͞Munc18-like (SM) proteins (1). SNAREs are localized on opposing membranes before fusion and form complexes with each other to force the membranes into close apposition during fusion (2, 3). SM proteins are cytosolic proteins of Ϸ600-700 residues that directly or indirectly bind to SNAREs. The entire sequences of SM proteins are homologous to each other, whereas SNAREs only share a single 70-residue homologous sequence, the so-called SNARE motif, that is instrumental in the formation of SNARE complexes (reviewed in refs. 1 and 4). In contrast to the many SNARE isoforms in vertebrates and yeast, only seven SM protein isoforms are expressed in vertebrates (Munc18-1, 18-2, 18c, Sly1, Vps45, Vps33a, and Vps33b), and only four SM protein isoforms in yeast (Sec1p, Sly1p, Vps45p, and Vps33p). SNAREs and SM proteins often function in multiple fusion reactions, suggesting that they are not responsible for the specificity of fusion (reviewed in ref. 5). At least in synaptic vesicle exocytosis and yeast vacuole fusion, deletion of the respective SM proteins (Munc18-1 and Vps33p) has a more severe effect on fusion (6, 7) than deletion of corresponding SNARE proteins [e.g., deletion of synaptobrevin 2 or SNAP-25 at the synapse, or deletion of Vam3p and Vam7p at the vacuole (8-10)]. Thus, the function of SM proteins in fusion likely extends beyond SNAREs, but their precise role and the significance of their binding to SNAREs remain unclear.The m...

The amyloid precursor protein (APP) has been under intensive study in recent years, mainly due to its critical role in the pathogenesis of Alzheimer's disease (AD). β-Amyloid (Aβ) peptides generated from APP proteolytic cleavage can aggregate, leading to plaque formation in human AD brains. Point mutations of APP affecting Aβ production are found to be causal for hereditary early onset familial AD. It is very likely that elucidating the physiological properties of APP will greatly facilitate the understanding of its role in AD pathogenesis. A number of APP loss-and gainof-function models have been established in model organisms including Caenorhabditis elegans, Drosophila, zebrafish and mouse. These in vivo models provide us valuable insights into APP physiological functions. In addition, several knock-in mouse models expressing mutant APP at a physiological level are available to allow us to study AD pathogenesis without APP overexpression. This article will review the current physiological and pathophysiological animal models of APP.

Squamous cell carcinoma of the esophagus (ESCC), a major subtype of esophageal carcinoma, is one of the aggressive cancers with worst prognosis in the world. The dismal outcome of ESCC is attributed to multiple reasons including its aggressive nature, largely unknown molecular mechanism of its progression, and the lack of biomarkers for early detection and effective prediction of its clinical behavior. To identify proteins with prognostic and/or predictive value, we applied a proteomics strategy to quantify proteins differentially expressed in ESCC using matched samples of carcinoma and adjacent normal epithelial cells. The analysis led to identification of 28 proteins aberrantly expressed in cancer cells with changes of at least three-fold in ESCC relative to normal squamous epithelial cells. These changes represent functional alterations of essential proteins for normal cellular physiology, accounting for many cellular changes involved in development of ESCC, including cell transformation, loss of differentiation, tumor growth, apoptosis, tumor invasion, and cell metabolism. The differentially expressed proteins shed new insights on the mechanism of tumorigenesis and provide candidate biomarkers for early detection of ESCC.

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