Faculty Bibliography

Using transgenic mice expressing human cystatin C (encoded by CST3), we show that cystatin C binds soluble amyloid-beta peptide and inhibits cerebral amyloid deposition in amyloid-beta precursor protein (APP) transgenic mice. Cystatin C expression twice that of the endogenous mouse cystatin C was sufficient to substantially diminish amyloid-beta deposition. Thus, cystatin C has a protective role in Alzheimer's disease pathogenesis, and modulation of cystatin C concentrations may have therapeutic implications for the disease

Epidemiological studies support an association between vascular risk factors, including hypercholesterolemia, and Alzheimer's disease (AD). Recently, there has been much interest in the possibility that hypercholesterolemia might directly promote beta-amyloid (Abeta) production. Indeed, in vitro studies have shown that increasing cellular cholesterol levels enhances Abeta production. However, studies in AD transgenic mouse models have not consistently found that elevated plasma cholesterol leads to increased Abeta production or deposition in vivo. In this study, we determined whether elevated peripheral cholesterol influences Abeta production in mice with a null mutation of the low-density lipoprotein receptor (LDLR). We show that dramatically elevated plasma cholesterol levels, whether induced by high cholesterol, high fat, or high fat/high cholesterol diets, did not affect either levels of brain Abeta40, Abeta42, or APP, or the Abeta42/40 or APP-CTF/APP ratios, nor substantially alter brain cholesterol levels. ApoE protein levels in brain were, however, elevated, in LDLR-/- mice by post-transcriptional mechanisms. Collectively, these studies argue that plasma cholesterol levels do not normally regulate production of brain Abeta

Studies in continuously cultured cells have established that familial Alzheimer's disease (FAD) mutant presenilin 1 (PS1) delays exit of the amyloid precursor protein (APP) from the trans-Golgi network (TGN). Here we report the first description of PS1-regulated APP trafficking in cerebral neurons in culture and in vivo. Using neurons from transgenic mice or a cell-free APP transport vesicle biogenesis system derived from the TGN of those neurons, we demonstrated that knocking-in an FAD-associated mutant PS1 transgene was associated with delayed kinetics of APP arrival at the cell surface. Apparently, this delay was at least partially attributable to impaired exit of APP from the TGN, which was documented in the cell-free APP transport vesicle biogenesis assay. To extend the study to APP and carboxyl terminal fragment (CTF) trafficking to cerebral neurons in vivo, we performed subcellular fractionation of brains from APP transgenic mice, some of which carried a second transgene encoding an FAD-associated mutant form of PS1. The presence of the FAD mutant PS1 was associated with a slight shift in the subcellular localization of both holoAPP and APP CTFs toward iodixanol density gradient fractions that were enriched in a marker for the TGN. In a parallel set of experiments, we used an APP : furin chimeric protein strategy to test the effect of artificially forcing TGN concentration of an APP : furin chimera that could be a substrate for beta- and gamma-cleavage. This chimeric substrate generated excess Abeta42 when compared with wildtype APP. These data indicate that the presence of an FAD-associated mutant human PS1 transgene is associated with redistribution of the APP and APP CTFs in brain neurons toward TGN-enriched fractions. The chimera experiment suggests that TGN-enrichment of a beta-/gamma-secretase substrate may play an integral role in the action of mutant PS1 to elevate brain levels of Abeta42

The sequential processing of the APP (amyloid precursor protein) by the beta- and gamma-secretase and generation of the Abeta (amyloid-beta) peptide is a primary pathological factor in AD (Alzheimer's disease). Regulation of the processing or turnover of these proteins represents potential targets for the development of AD therapies. Sumoylation is a process by which SUMOs (small ubiquitin-like modifiers) are covalently conjugated to target proteins, resulting in a number of functional consequences. These include regulation of protein-protein interactions, intracellular trafficking and protein stability, which all have the potential to impact on several aspects of the amyloidogenic pathway. The present study examines the effects of overexpression and knockdown of the major SUMO isoforms (SUMO1, 2 and 3) on APP processing and the production of Abeta peptides. SUMO3 overexpression significantly increased Abeta40 and Abeta42 secretion, which was accompanied by an increase in full-length APP and its C-terminal fragments. These effects of SUMO3 were independent of its covalent attachment or chain formation, as mutants lacking the motifs responsible for SUMO chain formation or SUMO conjugation led to similar changes in Abeta. SUMO3 overexpression also up-regulated the expression of the transmembrane protease BACE (beta-amyloid-cleaving enzyme), but failed to affect levels of several other unrelated proteins. Suppression of SUMO1 or combined SUMO2+3 by RNA interference did not affect APP levels or Abeta production. These findings confirm a specific effect of SUMO3 overexpression on APP processing and the production of Abeta peptides but also suggest that endogenous sumoylation is not essential and likely plays an indirect role in modulating the amyloid processing pathway

BACKGROUND: Amyloid-beta peptide species ending at positions 40 and 42 (Abeta40, Abeta42) are generated by the proteolytic processing of the Alzheimer's amyloid precursor protein (APP). Abeta peptides accumulate in the brain early in the course of Alzheimer's disease (AD), especially Abeta42. The cytoplasmic domain of APP regulates intracellular trafficking and metabolism of APP and its carboxyl-terminal fragments (CTFalpha, CTFbeta). The role of protein phosphorylation in general, and that of the phosphorylation state of APP at threonine-668 (Thr668) in particular, has been investigated in detail by several laboratories (including our own). Some investigators have recently proposed that the phosphorylation state of Thr668 plays a pivotal role in governing brain Abeta levels, prompting the current study. METHODOLOGY: In order to evaluate whether the phosphorylation state of Thr668 controlled brain Abeta levels, we studied the levels and subcellular distributions of holoAPP, sAPPalpha, sAPPbeta, CTFalpha, CTFbeta, Abeta40 and Abeta42 in brains from 'knock-in' mice in which a non-phosphorylatable alanyl residue had been substituted at position 668, replacing the threonyl residue present in the wild-type protein. CONCLUSIONS: The levels and subcellular distributions of holoAPP, sAPPalpha, sAPPbeta, CTFalpha, CTFbeta, Abeta40 and Abeta42 in the brains of Thr668Ala mutant mice were identical to those observed in wild-type mice. These results indicate that, despite speculation to the contrary, the phosphorylation state of APP at Thr668 does not play an obvious role in governing the physiological levels of brain Abeta40 or Abeta42 in vivo

Alzheimer's disease (AD) is characterized by progressive neurodegeneration and cerebral accumulation of the beta-amyloid peptide (Abeta), but it is unknown what makes neurons susceptible to degeneration. We report that the TGF-beta type II receptor (TbetaRII) is mainly expressed by neurons, and that TbetaRII levels are reduced in human AD brain and correlate with pathological hallmarks of the disease. Reducing neuronal TGF-beta signaling in mice resulted in age-dependent neurodegeneration and promoted Abeta accumulation and dendritic loss in a mouse model of AD. In cultured cells, reduced TGF-beta signaling caused neuronal degeneration and resulted in increased levels of secreted Abeta and beta-secretase-cleaved soluble amyloid precursor protein. These results show that reduced neuronal TGF-beta signaling increases age-dependent neurodegeneration and AD-like disease in vivo. Increasing neuronal TGF-beta signaling may thus reduce neurodegeneration and be beneficial in AD.

Protein aggregation is an established pathogenic mechanism in Alzheimer's disease, but little is known about the initiation of this process in vivo. Intracerebral injection of dilute, amyloid-beta (Abeta)-containing brain extracts from humans with Alzheimer's disease or beta-amyloid precursor protein (APP) transgenic mice induced cerebral beta-amyloidosis and associated pathology in APP transgenic mice in a time- and concentration-dependent manner. The seeding activity of brain extracts was reduced or abolished by Abeta immunodepletion, protein denaturation, or by Abeta immunization of the host. The phenotype of the exogenously induced amyloidosis depended on both the host and the source of the agent, suggesting the existence of polymorphic Abeta strains with varying biological activities reminiscent of prion strains

We have generated a novel transgenic mouse model on a C57BL/6J genetic background that coexpresses KM670/671NL mutated amyloid precursor protein and L166P mutated presenilin 1 under the control of a neuron-specific Thy1 promoter element (APPPS1 mice). Cerebral amyloidosis starts at 6-8 weeks and the ratio of human amyloid (A)beta42 to Abeta40 is 1.5 and 5 in pre-depositing and amyloid-depositing mice, respectively. Consistent with this ratio, extensive congophilic parenchymal amyloid but minimal amyloid angiopathy is observed. Amyloid-associated pathologies include dystrophic synaptic boutons, hyperphosphorylated tau-positive neuritic structures and robust gliosis, with neocortical microglia number increasing threefold from 1 to 8 months of age. Global neocortical neuron loss is not apparent up to 8 months of age, but local neuron loss in the dentate gyrus is observed. Because of the early onset of amyloid lesions, the defined genetic background of the model and the facile breeding characteristics, APPPS1 mice are well suited for studying therapeutic strategies and the pathomechanism of amyloidosis by cross-breeding to other genetically engineered mouse models