Person:

Tanzi, Rudolph

Background: The amyloid β-protein (Aβ) is believed to be the key mediator of Alzheimer's disease (AD) pathology. Aβ is most often characterized as an incidental catabolic byproduct that lacks a normal physiological role. However, Aβ has been shown to be a specific ligand for a number of different receptors and other molecules, transported by complex trafficking pathways, modulated in response to a variety of environmental stressors, and able to induce pro-inflammatory activities. Methodology/Principal Findings: Here, we provide data supporting an in vivo function for Aβ as an antimicrobial peptide (AMP). Experiments used established in vitro assays to compare antimicrobial activities of Aβ and LL-37, an archetypical human AMP. Findings reveal that Aβ exerts antimicrobial activity against eight common and clinically relevant microorganisms with a potency equivalent to, and in some cases greater than, LL-37. Furthermore, we show that AD whole brain homogenates have significantly higher antimicrobial activity than aged matched non-AD samples and that AMP action correlates with tissue Aβ levels. Consistent with Aβ-mediated activity, the increased antimicrobial action was ablated by immunodepletion of AD brain homogenates with anti-Aβ antibodies. Conclusions/Significance: Our findings suggest Aβ is a hitherto unrecognized AMP that may normally function in the innate immune system. This finding stands in stark contrast to current models of Aβ-mediated pathology and has important implications for ongoing and future AD treatment strategies.

Alzheimer’s disease (AD) is the most common form of dementia, characterized by two pathological hallmarks: β-amyloid plaques and neurofibrillary tangles1. The amyloid hypothesis of AD posits that excessive accumulation of β-amyloid peptide (Aβ) leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau2,3. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. AD mouse models with familial AD (FAD) mutations exhibited Aβ-induced synaptic and memory deficits but they were not able to fully recapitulate other key pathological events of AD including clear neurofibrillary tangle pathology4,5. AD patient-derived human neurons showed elevated levels of toxic Aβ species and phosphor-tau (p-tau) but they also could not replicate β-amyloid plaques or neurofibrillary tangles6-11. Here we show that FAD mutations in the amyloid-β precursor protein (APP) and presenilin (PS) 1 genes are able to induce robust extracellular deposition of Aβ, including β-amyloid plaques, in a human neural stem cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of p-tau in the soma and neurites. Immunoelectron microscopy also demonstrated the presence of filamentous tau, only in detergent-resistant fractions from 3D-cultured cells expressing FAD mutations. Inhibition of Aβ generation with β- or γ-secretase inhibitors not only decreased Aβ pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated Aβ-mediated tau phosphorylation. In summary, we have successfully recapitulated Aβ and tau pathology in a single 3D human neural cell culture system for the first time. Our unique strategy for recapitulating AD pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models for other neurodegenerative disorders.

Background: Accumulation of the β-amyloid peptide (Aβ) is a major pathological hallmark of Alzheimer’s disease (AD). Recent studies have shown that synaptic Aβ toxicity may directly impair synaptic function. However, proteins regulating Aβ generation at the synapse have not been characterized. Here, we sought to identify synaptic proteins that interact with the extracellular domain of APP and regulate Aβ generation. Results: Affinity purification-coupled mass spectrometry identified members of the Synaptotagmin (Syt) family as novel interacting proteins with the APP ectodomain in mouse brains. Syt-1, −2 and −9 interacted with APP in cells and in mouse brains in vivo. Using a GST pull-down approach, we have further demonstrated that the Syt interaction site lies in the 108 amino acids linker region between the E1 and KPI domains of APP. Stable overexpression of Syt-1 or Syt-9 with APP in CHO and rat pheochromocytoma cells (PC12) significantly increased APP-CTF and sAPP levels, with a 2 to 3 fold increase in secreted Aβ levels in PC12 cells. Moreover, using a stable knockdown approach to reduce the expression of endogenous Syt-1 in PC12 cells, we have observed a ~ 50 % reduction in secreted Aβ generation. APP processing also decreased in these cells, shown by lower CTF levels. Lentiviral-mediated knock down of endogenous Syt-1 in mouse primary neurons also led to a significant reduction in both Aβ40 and Aβ42 generation. As secreted sAPPβ levels were significantly reduced in PC12 cells lacking Syt-1 expression, our results suggest that Syt-1 regulates Aβ generation by modulating BACE1-mediated cleavage of APP. Conclusion: Altogether, our data identify the synaptic vesicle proteins Syt-1 and 9 as novel APP-interacting proteins that promote Aβ generation and thus may play an important role in the pathogenesis of AD. Electronic supplementary material The online version of this article (doi:10.1186/s13024-015-0028-5) contains supplementary material, which is available to authorized users.

Post-operative cognitive dysfunction (POCD) is associated with increased cost of care, morbidity, and mortality. However, its pathogenesis remains largely to be determined. Specifically, it is unknown why elderly patients are more likely to develop POCD and whether POCD is dependent on general anesthesia. We therefore set out to investigate the effects of peripheral surgery on the cognition and Alzheimer-related neuropathology in mice with different ages. Abdominal surgery under local anesthesia was established in the mice. The surgery induced post-operative elevation in brain β-amyloid (Aβ) levels and cognitive impairment in the 18 month-old wild-type and 9 month-old Alzheimer's disease transgenic mice, but not the 9 month-old wild-type mice. The Aβ accumulation likely resulted from elevation of beta-site amyloid precursor protein cleaving enzyme and phosphorylated eukaryotic translation initiation factor 2α. γ-Secretase inhibitor compound E ameliorated the surgery-induced brain Aβ accumulation and cognitive impairment in the 18 month-old mice. These data suggested that the peripheral surgery was able to induce cognitive impairment independent of general anesthesia, and that the combination of peripheral surgery with aging- or Alzheimer gene mutation-associated Aβ accumulation was needed for the POCD to occur. These findings would likely promote more research to investigate the pathogenesis of POCD.

Amyloid-β-protein (Aβ), the key component of senile plaques in Alzheimer's disease (AD) brain, is produced from amyloid precursor protein (APP) by cleavage of β-secretase and then γ-secretase. APP adaptor proteins with phosphotyrosine-binding (PTB) domains, including Dab (gene: DAB) and Numb (gene: NUMB), can bind to and interact with the conserved YENPTY-motif in the APP C-terminus. Here we describe, for the first time, the effects of RNAi knock-down of Dab and Numb expression on APP processing and Aβ production. RNAi knock-down of Dab and Numb in H4 human neuroglioma cells stably transfected to express either FL-APP (H4-FL-APP cells) or APP-C99 (H4-APP-C99 cells) increased levels of APP-C-terminal fragments (APP-CTFs) and lowered Aβ levels in both cell lines by inhibiting γ-secretase cleavage of APP. Finally, RNAi knock-down of APP also reduced levels of Numb in H4-APP cells. These findings suggest that pharmacologically blocking interaction of APP with Dab and Numb may provide novel therapeutic strategies of AD. The notion of attenuating γ-secretase cleavage of APP via the APP adaptor proteins, Dab and Numb, is particularly attractive with regard to therapeutic potential, given that side effects of γ-secretase inhibition owing to impaired proteolysis of other γ-secretase substrates, e.g. Notch, might be avoided.

Post-operative cognitive dysfunction is associated with morbidity and mortality. However, its neuropathogenesis remains largely to be determined. Neuroinflammation and accumulation of β-amyloid (Aβ) have been reported to contribute to cognitive dysfunction in humans and cognitive impairment in animals. Our recent studies have established a pre-clinical model in mice, and have found that the peripheral surgical wounding without the influence of general anesthesia induces an age-dependent Aβ accumulation and cognitive impairment in mice. We therefore set out to assess the effects of peripheral surgical wounding, in the absence of general anesthesia, on neuroinflammation in mice with different ages. Abdominal surgery under local anesthesia was established in 9 and 18 month-old mice. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), Iba1 positive cells (the marker of microglia activation), CD33, and cognitive function in mice were determined. The peripheral surgical wounding increased the levels of TNF-α, IL-6, and Iba1 positive cells in the hippocampus of both 9 and 18 month-old mice, and age potentiated these effects. The peripheral surgical wounding increased the levels of CD33 in the hippocampus of 18, but not 9, month-old mice. Finally, anti-inflammatory drug ibuprofen ameliorated the peripheral surgical wounding-induced cognitive impairment in 18 month-old mice. These data suggested that the peripheral surgical wounding could induce an age-dependent neuroinflammation and elevation of CD33 levels in the hippocampus of mice, which could lead to cognitive impairment in aged mice. Pending further studies, anti-inflammatory therapies may reduce the risk of postoperative cognitive dysfunction in elderly patients.

Pancreatitis is a complex, progressively destructive inflammatory disorder. Alcohol was long thought to be the primary causative agent, but genetic contributions have been of interest since the discovery that rare PRSS1, CFTR, and SPINK1 variants were associated with pancreatitis risk. We now report two significant genome-wide associations identified and replicated at PRSS1-PRSS2 (1×10-12) and x-linked CLDN2 (p < 1×10-21) through a two-stage genome-wide study (Stage 1, 676 cases and 4507 controls; Stage 2, 910 cases and 4170 controls). The PRSS1 variant affects susceptibility by altering expression of the primary trypsinogen gene. The CLDN2 risk allele is associated with atypical localization of claudin-2 in pancreatic acinar cells. The homozygous (or hemizygous male) CLDN2 genotype confers the greatest risk, and its alleles interact with alcohol consumption to amplify risk. These results could partially explain the high frequency of alcohol-related pancreatitis in men – male hemizygous frequency is 0.26, female homozygote is 0.07.

Down syndrome (DS, trisomy 21) is the most common chromosomal disorder and the leading genetic cause of intellectual disability in humans. In DS, triplication of chromosome 21 invariably includes the APP gene (21q21) encoding the Alzheimer's disease (AD) amyloid precursor protein (APP). Triplication of the APP gene accelerates APP expression leading to cerebral accumulation of APP-derived amyloid-(\beta) peptides (A(\beta)), early-onset AD neuropathology, and age-dependent cognitive sequelae. The DS phenotype complex also includes distinctive early-onset cerulean cataracts of unknown etiology. Previously, we reported increased A(\beta) accumulation, co-localizing amyloid pathology, and disease-linked supranuclear cataracts in the ocular lenses of subjects with AD. Here, we investigate the hypothesis that related AD-linked A(\beta) pathology underlies the distinctive lens phenotype associated with DS. Ophthalmological examinations of DS subjects were correlated with phenotypic, histochemical, and biochemical analyses of lenses obtained from DS, AD, and normal control subjects. Evaluation of DS lenses revealed a characteristic pattern of supranuclear opacification accompanied by accelerated supranuclear A(\beta) accumulation, co-localizing amyloid pathology, and fiber cell cytoplasmic A(\beta) aggregates ((\sim)5 to 50 nm) identical to the lens pathology identified in AD. Peptide sequencing, immunoblot analysis, and ELISA confirmed the identity and increased accumulation of A(\beta) in DS lenses. Incubation of synthetic A(\beta) with human lens protein promoted protein aggregation, amyloid formation, and light scattering that recapitulated the molecular pathology and clinical features observed in DS lenses. These results establish the genetic etiology of the distinctive lens phenotype in DS and identify the molecular origin and pathogenic mechanism by which lens pathology is expressed in this common chromosomal disorder. Moreover, these findings confirm increased A(\beta) accumulation as a key pathogenic determinant linking lens and brain pathology in both DS and AD.