Faculty Bibliography

Aims Acetylcholinesterase (AChE) exists as different splicing variants with particular regional, cellular, and subcellular locations that may reflect differential physiological roles. So we aimed to study the expression of AChE variants in Alzheimer's disease (AD) brain. Method We have analyzed protein levels of AChE variants in postmortem cerebral cortex from AD patients by Western blot using specific anti-AChE antibodies. Levels of AChE transcripts were also analysed by qRT-PCR. Further, we investigated expression levels of the anchoring AChE subunit proline-rich membrane anchor (PRiMA-1), limiting factor for correct localization of cholinergic AChE at plasma membrane. In addition we analysed expression levels of AChE variants in cell cultures after PRiMA overexpression. Changes in AChE promoter were also evaluated by Luciferase assays. Results We found similar protein and mRNA levels of the major cholinergic "tailed"-variant (AChE-T) and the anchorage subunit PRiMA-1 in cortex from AD patients and non-demented controls. Interestingly, we observed an increment in protein and transcript levels of the non-cholinergic "readthrought" AChE (AChE-R) subunits in cortex of AD patients compared to controls. Moreover an increase in N-extended variants of AChE, which were assigned to N-AChE-R variants, was detected in AD cortex. We further observed that PRiMA 1 could regulate the expression of AChE-T variants without effect in AChE-R forms. Conclusion Our findings reveal previously unknown expression patterns of AChE variants in AD cortex likely reflecting specific roles and/or differential regulation for each variant in AD, which may have strong implications for the re-evaluation of AChE inhibitors as therapeutic agents in dementia

Individuals with Down syndrome (DS) exhibit intellectual disability and develop Alzheimer's disease-like neuropathology during the third decade of life. The Ts65Dn mouse model of DS exhibits key features of both disorders, including impairments in learning, attention and memory, as well as atrophy of basal forebrain cholinergic neurons (BFCNs). The present study evaluated attentional function in relation to BFCN morphology in young (3 months) and middle-aged (12 months) Ts65Dn mice and disomic (2N) controls. Ts65Dn mice exhibited attentional dysfunction at both ages, with greater impairment in older trisomics. Density of BFCNs was significantly lower for Ts65Dn mice independent of age, which may contribute to attentional dysfunction since BFCN density was positively associated with performance on an attention task. BFCN volume decreased with age in 2N but not Ts65Dn mice. Paradoxically, BFCN volume was greater in older trisomic mice, suggestive of a compensatory response. In sum, attentional dysfunction occurred in both young and middle-aged Ts65Dn mice, which may in part reflect reduced density and/or phenotypic alterations in BFCNs.

In Alzheimer's disease, soluble tau accumulates and deposits as neurofibrillary tangles (NFTs). However, a precise toxic mechanism of tau is not well understood. We hypothesized that overexpression of wild-type tau downregulates brain-derived neurotrophic factor (BDNF), a neurotrophic peptide essential for learning and memory. Two transgenic mouse models of human tau expression and human tau (hTau40)-transfected human neuroblastoma (SH-SY5Y) cells were used to examine the effect of excess or pathologically modified wild-type human tau on BDNF expression. Both transgenic mouse models, with or without NFTs, as well as hTau40-SH-SY5Y cells significantly downregulated BDNF messenger RNA compared with controls. Similarly, transgenic mice overexpressing amyloid-beta (Abeta) significantly downregulated BDNF expression. However, when crossed with tau knockout mice, the resulting animals exhibited BDNF levels that were not statistically different from wild-type mice. These results demonstrate that excess or pathologically modified wild-type human tau downregulates BDNF and that neither a mutation in tau nor the presence of NFTs is required for toxicity. Moreover, our findings suggest that tau at least partially mediates Abeta-induced BDNF downregulation. Therefore, Alzheimer's disease treatments targeting Abeta alone may not be effective without considering the impact of tau pathology on neurotrophic pathways.

Defective autophagy contributes to Alzheimer disease (AD) pathogenesis although evidence is conflicting on whether multiple stages are impaired. Here, for the first time, we have comprehensively evaluated the entire autophagic process specifically in CA1 pyramidal neurons of hippocampus from early and late-stage AD subjects and nondemented controls. CA1 neurons aspirated by laser capture microdissection were analyzed using a custom-designed microarray comprising 578 neuropathology- and neuroscience-associated genes. Striking upregulation of autophagy-related genes, exceeding that of other gene ontology groups, reflected increases in autophagosome formation and lysosomal biogenesis beginning at early AD stages. Upregulated autophagosome formation was further indicated by elevated gene and protein expression levels for autophagosome components and increased LC3-positive puncta. Increased lysosomal biogenesis was evidenced by activation of MiTF/TFE family transcriptional regulators, particularly TFE3 (transcription factor binding to IGHM enhancer 3) and by elevated expression of their target genes and encoded proteins. Notably, TFEB (transcription factor EB) activation was associated more strongly with glia than neurons. These findings establish that autophagic sequestration is both competent and upregulated in AD. Autophagosome-lysosome fusion is not evidently altered. Despite this early disease response, however, autophagy flux is progressively impeded due to deficient substrate clearance, as reflected by autolysosomal accumulation of LC3-II and SQSTM1/p62 and expansion of autolysosomal size and total area. We propose that sustained induction of autophagy in the face of progressively declining lysosomal clearance of substrates explains the uncommonly robust autophagic pathology and neuritic dystrophy implicated in AD pathogenesis.

Glucocorticoid resistance is a risk factor for Alzheimer's disease (AD). Molecular and cellular mechanisms of glucocorticoid resistance in the brain have remained unknown and are potential therapeutic targets. Phosphorylation of glucocorticoid receptors (GR) by brain-derived neurotrophic factor (BDNF) signaling integrates both pathways for remodeling synaptic structure and plasticity. The goal of this study is to test the role of the BDNF-dependent pathway on glucocorticoid signaling in a mouse model of glucocorticoid resistance. We report that deletion of GR phosphorylation at BDNF-responding sites and downstream signaling via the MAPK-phosphatase DUSP1 triggers tau phosphorylation and dendritic spine atrophy in mouse cortex. In human cortex, DUSP1 protein expression correlates with tau phosphorylation, synaptic defects and cognitive decline in subjects diagnosed with AD. These findings provide evidence for a causal role of BDNF-dependent GR signaling in tau neuropathology and indicate that DUSP1 is a potential target for therapeutic interventions.

Although the two pathological hallmarks of Alzheimer's disease (AD), senile plaques composed of amyloid-beta (Abeta) peptides and neurofibrillary tangles (NFTs) consisting of hyperphosphorylated tau, have been studied extensively in postmortem AD and relevant animal and cellular models, the pathogenesis of AD remains unknown, particularly in the early stages of the disease where therapies presumably would be most effective. We and others have demonstrated that Abeta plaques and NFTs are present in varying degrees before the onset and throughout the progression of dementia. In this regard, aged people with no cognitive impairment (NCI), mild cognitive impairment (MCI, a presumed prodromal AD transitional state), and AD all present at autopsy with varying levels of pathological hallmarks. Cognitive decline, a requisite for the clinical diagnosis of dementia associated with AD, generally correlates better with NFTs than Abeta plaques. However, correlations are even higher between cognitive decline and synaptic loss. In this review, we illustrate relevant clinical pathological research in preclinical AD and throughout the progression of dementia in several areas including Abeta and tau pathobiology, single population expression profiling of vulnerable hippocampal and basal forebrain neurons, neuron plasticity, neuroimaging, cerebrospinal fluid (CSF) biomarker studies and their correlation with antemortem cognitive endpoints. In each of these areas, we provide evidence for the importance of studying the pathological hallmarks of AD not in isolation, but rather in conjunction with other molecular, cellular, and imaging markers to provide a more systematic and comprehensive assessment of the multiple changes that occur during the transition from NCI to MCI to frank AD.

Conformational phosphorylation and cleavage events drive the tau protein from a soluble, monomeric state to a relatively insoluble, polymeric state that precipitates the formation of neurofibrillary tangles (NFTs) in projection neurons in Alzheimer's disease (AD), including the magnocellular perikarya located in the nucleus basalis of Meynert (NBM) complex of the basal forebrain. Whether these structural changes in the tau protein are associated with pathogenic changes at the molecular and cellular level remains undetermined during the onset of AD. Here, we examined alterations in gene expression within individual NBM neurons immunostained for pS422, an early tau phosphorylation event, or dual labeled for pS422 and TauC3, a later stage tau neoepitope, from tissue obtained postmortem from subjects who died with an antemortem clinical diagnosis of no cognitive impairment, mild cognitive impairment, or mild/moderate AD. Specifically, pS422-positive pretangles displayed an upregulation of select gene transcripts subserving protein quality control. On the other hand, late-stage TauC3-positive NFTs exhibited upregulation of messenger RNAs involved in protein degradation but also cell survival. Taken together, these results suggest that molecular pathways regulating protein homeostasis are altered during the evolution of NFT pathology in the NBM. These changes likely contribute to the disruption of protein turnover and neuronal survival of these vulnerable NBM neurons during the progression of AD.

Alzheimer's disease (AD) is characterized by a decrease in the enzymatic activity of the enzyme acetylcholinesterase (AChE). AChE is expressed as multiple splice variants, which may serve both cholinergic degradative functions and non-cholinergic functions unrelated with their capacity to hydrolyze acetylcholine. We have recently demonstrated that a prominent pool of enzymatically inactive AChE protein exists in the AD brain. In this study, we analyzed protein and transcript levels of individual AChE variants in human frontal cortex from AD patients by western blot analysis using specific anti-AChE antibodies and by quantitative real-time PCR (qRT-PCR). We found similar protein and mRNA levels of the major cholinergic "tailed"-variant (AChE-T) and the anchoring subunit, proline-rich membrane anchor (PRiMA-1) in frontal cortex obtained from AD patients and non-demented controls. Interestingly, we found an increase in the protein and transcript levels of the non-cholinergic "readthrough" AChE (AChE-R) variants in AD patients compared to controls. Similar increases were detected by western blot using an antibody raised against the specific N-terminal domain, exclusive of alternative N-extended variants of AChE (N-AChE). In accordance with a subset of AChE-R monomers that display amphiphilic properties that are upregulated in the AD brain, we demonstrate that the increase of N-AChE species is due, at least in part, to N-AChE-R variants. In conclusion, we demonstrate selective alterations in specific AChE variants in AD cortex, with no correlation in enzymatic activity. Therefore, differential expression of AChE variants in AD may reflect changes in the pathophysiological role of AChE, independent of cholinergic impairment or its role in degrading acetylcholine.

Epidemiological findings suggest that diabetic individuals are at a greater risk for developing Alzheimer's disease (AD). To examine the mechanisms by which diabetes mellitus (DM) may contribute to AD pathology in humans, we examined brain tissue from streptozotocin-treated type 1 diabetic adult male vervet monkeys receiving twice-daily exogenous insulin injections for 8-20 weeks. We found greater inhibitory phosphorylation of insulin receptor substrate 1 in each brain region examined of the diabetic monkeys when compared with controls, consistent with a pattern of brain insulin resistance that is similar to that reported in the human AD brain. Additionally, a widespread increase in phosphorylated tau was seen, including brain areas vulnerable in AD, as well as relatively spared structures, such as the cerebellum. An increase in active ERK1/2 was also detected, consistent with DM leading to changes in tau-kinase activity broadly within the brain. In contrast to these widespread changes, we found an increase in soluble amyloid-beta (Abeta) levels that was restricted to the temporal lobe, with the greatest increase seen in the hippocampus. Consistent with this localized Abeta increase, a hippocampus-restricted decrease in the protein and mRNA for the Abeta-degrading enzyme neprilysin (NEP) was found, whereas various Abeta-clearing and -degrading proteins were unchanged. Thus, we document multiple biochemical changes in the insulin-controlled DM monkey brain that can link DM with the risk of developing AD, including dysregulation of the insulin-signaling pathway, changes in tau phosphorylation, and a decrease in NEP expression in the hippocampus that is coupled with a localized increase in Abeta. SIGNIFICANCE STATEMENT: Given that diabetes mellitus (DM) appears to increase the risk of developing Alzheimer's disease (AD), understanding the mechanisms by which DM promotes AD is important. We report that DM in a nonhuman primate brain leads to changes in the levels or posttranslational processing of proteins central to AD pathobiology, including tau, amyloid-beta (Abeta), and the Abeta-degrading protease neprilysin. Additional evidence from this model suggests that alterations in brain insulin signaling occurred that are reminiscent of insulin signaling pathway changes seen in human AD. Thus, in anin vivomodel highly relevant to humans, we show multiple alterations in the brain resulting from DM that are mechanistically linked to AD risk.