R. Scott Turner

The X11α Protein Slows Cellular Amyloid Precursor Protein Processing and Reduces Aβ40 and Aβ42 Secretion

Constitutive amyloid precursor protein (APP) metabolism results in the generation of soluble APP (APPs) and Aβ peptides, including Aβ40 and Aβ42–the major component of amyloid plaques in Alzheimer’s disease brain. The phosphotyrosine binding (PTB) domain of X11 binds to a peptide containing a YENPTY motif found in the carboxyl terminus of APP. We have cloned the full-lengthX11 gene now referred to as X11α.Coexpression of X11α with APP results in comparatively greater levels of cellular APP and less APPs, Aβ40, and Aβ42 recovered in conditioned medium of transiently transfected HEK 293 cells. These effects are impaired by a single missense mutation of either APP (Y682G within the YENPTY motif) or X11α (F608V within the PTB domain), which diminishes their interaction, thus demonstrating specificity. The inhibitory effect of X11α on Aβ40 and Aβ42 secretion is amplified by coexpression with the Swedish mutation of APP (K595N/M596L), which promotes its amyloidogenic processing. Pulse-chase analysis demonstrates that X11α prolongs the half-life of APP from ∼2 h to ∼4 h. The effects of X11α on cellular APP and APPs recovery were confirmed in a 293 cell line stably transfected with APP. The specific binding of the PTB domain of X11α to the YENPTY motif-containing peptide of APP appears to slow cellular APP processing and thus reduces recovery of its soluble fragments APPs, Aβ40, and Aβ42 in conditioned medium of transfected HEK 293 cells. X11α may be involved in APP trafficking and metabolism in neurons and thus may be implicated in amyloidogenesis in normal aging and Alzheimer’s disease brain.

Amyloids and Are Generated Intracellularly in Cultured Human Neurons and Their Secretion Increases with Maturation

Previous studies have demonstrated the presence of amyloid β (Aβ) in neurons (NT2N) derived from a human embryonal carcinoma cell line (NT2) by steady state metabolic radiolabeling and immunoprecipitation. We show here that Aβ is present intracellularly since trypsin digestion of intact NT2N cells at 4°C did not eliminate the Aβ recovered in cell lysates. To determine whether both Aβ and Aβ are produced intracellularly, quantitative sandwich enzyme-linked immunosorbent assay (ELISA) was performed using COOH-terminal end-specific anti-Aβ monoclonal antibodies. Sandwich ELISA detected intracellular Aβ and Aβ in NT2N cell lysates at a ratio of 3:1, whereas secreted Aβ and Aβ were recovered in medium conditioned by NT2N cells at a ratio of approximately 20:1. Metabolic steady state and pulse-chase labeling studies demonstrated a 2-h delay in the detection of cell-associated Aβ/Aβ in the medium, suggesting that Aβ is generated at a slow rate intracellularly prior to its secretion. Finally, as NT2N cells mature over time in culture, the secretion of Aβ and Aβ increases more than 5-fold over 7 weeks. This increase in the secretion of Aβ/Aβ in NT2N cells as a function of time may recapitulate a similar phenomenon in the aging brain.

The Chaperone BiP/GRP78 Binds to Amyloid Precursor Protein and Decreases Aβ40 and Aβ42 Secretion

Recent studies of cellular amyloid precursor protein (APP) metabolism demonstrate a β-/γ-secretase pathway resident to the endoplasmic reticulum (ER)/Golgi resulting in intracellular generation of soluble APP (APPsβ) and Aβ42 peptide. Thus, these intracellular compartments may be key sites of amyloidogenic APP metabolism and Alzheimer’s disease pathogenesis. We hypothesized that the ER chaperone immunoglobulin binding protein (BiP/GRP78) binds to and facilitates correct folding of nascent APP. Metabolic labeling and immunoprecipitation of transiently transfected human embryonic kidney 293 cells demonstrated co-precipitation of APP with GRP78, revealing their transient interaction in the ER. Maturation of cellular APP was impaired by this interaction. Furthermore, the levels of APPs, Aβ40, and Aβ42 recovered in conditioned medium were lower compared with cells transfected with APP alone. Co-expression with APP of GRP78 T37G, an ATPase mutant, almost completely blocked cellular APP maturation as well as recovery of APPs, Aβ40, and Aβ42 in conditioned medium. The inhibitory effects of GRP78 and GRP78 T37G on Aβ40 and Aβ42 secretion were magnified by co-expression with the Swedish mutation of APP (K670N/M671L). Collectively, these data suggest a transient and direct interaction of GRP78 with APP in the ER that modulates intracellular APP maturation and processing and may facilitate its correct folding.

X11 interaction with beta-amyloid precursor protein modulates its cellular stabilization and reduces amyloid beta-protein secretion.

The protein interaction domain of the neuronal protein X11 binds to the YENPTY motif within the cytoplasmic domain of β-amyloid precursor protein (βAPP). Amyloid-β protein (Aβ), the major constituent of the amyloid deposited in brain of Alzheimer’s disease patients, is generated by proteolytic processing of βAPP, which occurs in part following βAPP internalization. Because the YENPTY motif has a role in the internalization of βAPP, the effect of X11 binding on βAPP processing was studied in mouse neuroblastoma N2a, human embryonic kidney 293, monkey kidney COS-1, and human glial U251 cell lines transfected with wild type or mutated βAPP cDNAs. Secretion of soluble βAPP via α-secretase activity increased significantly in cells transfected with βAPP variants containing mutations that impair interaction with X11 when compared with cells transfected with wild type cDNA. Cotransfection of βAPP and X11 caused retention of cellular βAPP, decreased secretion of sβAPPα, and decreased Aβ secretion. Thus, βAPP interaction with the protein interaction domain of X11 stabilizes cellular βAPP and thereby participates in the regulation of βAPP processing pathways.

Adaptor protein interactions: modulators of amyloid precursor protein metabolism and Alzheimer's disease risk?

The cytoplasmic C-terminus of APP plays critical roles in its cellular trafficking and delivery to proteases. Adaptor proteins with phosphotyrosine-binding (PTB) domains, including those in the X11, Fe65, and c-Jun N-terminal kinase (JNK)-interacting protein (JIP) families, bind specifically to the absolutely conserved -YENPTY- motif in the APP C-terminus to regulate its trafficking and processing. Compounds that modulate APP-adaptor protein interactions may inhibit Abeta generation by specifically targeting the substrate (APP) instead of the enzyme (beta- or gamma-secretase). Genetic polymorphisms in (or near) adaptor proteins may influence risk of sporadic AD by interacting with APP in vivo to modulate its trafficking and processing to Abeta.

Alzheimer Disease Pathology in Cognitively Healthy Elderly:A Genome-wide Study

Many elderly individuals remain dementia-free throughout their life. However, some of these individuals exhibit Alzheimer disease neuropathology on autopsy, evidenced by neurofibrillary tangles (NFTs) in AD-specific brain regions. We conducted a genome-wide association study to identify genetic mechanisms that distinguish non-demented elderly with a heavy NFT burden from those with a low NFT burden. The study included 299 non-demented subjects with autopsy (185 subjects with low and 114 with high NFT levels). Both a genotype test, using logistic regression, and an allele test provided consistent evidence that variants in the RELN gene are associated with neuropathology in the context of cognitive health. Immunohistochemical data for reelin expression in AD-related brain regions added support for these findings. Reelin signaling pathways modulate phosphorylation of tau, the major component of NFTs, either directly or through β-amyloid pathways that influence tau phosphorylation. Our findings suggest that up-regulation of reelin may be a compensatory response to tau-related or beta-amyloid stress associated with AD even prior to the onset of dementia.

Modulation of Amyloid Precursor Protein Metabolism by X11α/Mint-1 A DELETION ANALYSIS OF PROTEIN-PROTEIN INTERACTION DOMAINS

Modulation of amyloid precursor protein (APP) metabolism plays a pivotal role in the pathogenesis of Alzheimers disease. The phosphotyrosine-binding/protein interaction (PTB/PI) domain of X11α, a neuronal cytosolic adaptor protein, binds to the YENPTY sequence in the cytoplasmic carboxyl terminus of APP. This interaction prolongs the half-life of APP and inhibits Aβ40 and Aβ42 secretion. X11α/Mint-1 has multiple protein-protein interaction domains, a Munc-18 interaction domain (MID), a Cask/Lin-2 interaction domain (CID), a PTB/PI domain, and two PDZ domains. These X11α protein interaction domains may modulate its effect on APP processing. To test this hypothesis, we performed a deletion analysis of X11α effects on metabolism of APP695 Swedish (K595N/M596L) (APPsw) by transient cotransfection of HEK 293 cells with: 1) X11α (X11α-wt, N-MID-CID-PTB-PDZ-PDZ-C), 2) amino-terminal deletion (X11α-ΔN, PTB-PDZ-PDZ), 3) carboxyl-terminal deletion (X11α-ΔPDZ, MID-CID-PTB), or 4) deletion of both termini (PTB domain only, PTB). The carboxyl terminus of X11α was required for stabilization of APPsw in cells. In contrast, the amino terminus of X11α was required to stimulate APPs secretion. X11α, X11α-ΔN, and X11α-PTB, but not X11α-ΔPDZ, were effective inhibitors of Aβ40 and Aβ42 secretion. These results suggest that additional protein interaction domains of X11α modulate various aspects of APP metabolism.

Stable β-Secretase Activity and Presynaptic Cholinergic Markers During Progressive Central Nervous System Amyloidogenesis in Tg2576 Mice

We examined presynaptic cholinergic markers and beta-secretase activity during progressive central nervous system amyloidogenesis in Tg2576 Alzheimer mice (transgenic for human amyloid precursor protein Swedish mutation; hAPPswe). At 14, 18, and 23 months of age there were no significant differences between wild-type and transgenic mice in four distinct central nervous system cholinergic indices--choline acetyltransferase and acetylcholinesterase activities, and binding to vesicular acetylcholine transporter and Na(+)-dependent high-affinity choline uptake sites. A novel enzyme-linked immunosorbent assay measuring only the secreted human beta-secretase cleavage product (APPsbetaswe) of APPswe also revealed no change with aging in Tg2576 mouse brain. In contrast, transgenic but not wild-type mice exhibited an age-dependent increase in soluble Abeta40 and Abeta42 levels and progressive amyloid deposition in brain. Thus, aging Tg2576 mice exhibited presynaptic cholinergic integrity despite progressively increased soluble Abeta40 and Abeta42 levels and amyloid plaque density in brain. Older Tg2576 mice may best resemble preclinical or early stages of human Alzheimers disease with preserved presynaptic cholinergic innervation. Homeostatic APPsbetaswe levels with aging suggest that progressive amyloid deposition in brain results not from increased beta-secretase cleavage of APP but from impaired Abeta/amyloid clearance mechanisms.

Temporoparietal Hypometabolism in Frontotemporal Lobar Degeneration and Associated Imaging Diagnostic Errors

OBJECTIVE To evaluate the cause of diagnostic errors in the visual interpretation of positron emission tomographic scans with fludeoxyglucose F 18 (FDG-PET) in patients with frontotemporal lobar degeneration (FTLD) and patients with Alzheimer disease (AD). DESIGN Twelve trained raters unaware of clinical and autopsy information independently reviewed FDG-PET scans and provided their diagnostic impression and confidence of either FTLD or AD. Six of these raters also recorded whether metabolism appeared normal or abnormal in 5 predefined brain regions in each hemisphere-frontal cortex, anterior cingulate cortex, anterior temporal cortex, temporoparietal cortex, and posterior cingulate cortex. Results were compared with neuropathological diagnoses. SETTING Academic medical centers. PATIENTS Forty-five patients with pathologically confirmed FTLD (n=14) or AD (n=31). RESULTS Raters had a high degree of diagnostic accuracy in the interpretation of FDG-PET scans; however, raters consistently found some scans more difficult to interpret than others. Unanimity of diagnosis among the raters was more frequent in patients with AD (27 of 31 patients [87%]) than in patients with FTLD (7 of 14 patients [50%]) (P=.02). Disagreements in interpretation of scans in patients with FTLD largely occurred when there was temporoparietal hypometabolism, which was present in 7 of the 14 FTLD scans and 6 of the 7 scans lacking unanimity. Hypometabolism of anterior cingulate and anterior temporal regions had higher specificities and positive likelihood ratios for FTLD than temporoparietal hypometabolism had for AD. CONCLUSIONS Temporoparietal hypometabolism in FTLD is common and may cause inaccurate interpretation of FDG-PET scans. An interpretation paradigm that focuses on the absence of hypometabolism in regions typically affected in AD before considering FTLD is likely to misclassify a significant portion of FTLD scans. Anterior cingulate and/or anterior temporal hypometabolism indicates a high likelihood of FTLD, even when temporoparietal hypometabolism is present. Ultimately, the accurate interpretation of FDG-PET scans in patients with dementia cannot rest on the presence or absence of a single region of hypometabolism but rather must take into account the relative hypometabolism of all brain regions.

GABAA Receptor Pharmacology and Subtype mRNA Expression in Human Neuronal NT2-N Cells

Human NT2 teratocarcinoma cells differentiate into neuron-like NT2-N cells when treated with retinoic acid. GABA evoked concentration-dependent whole-cell currents in NT2-N cells with an EC50 of 21.8 μm and a Hill slope of 1.2. GABAA receptor (GABAR) currents reversed atECl− and did not display voltage-dependent rectification. GABAR single channels opened in bursts to a 23 pS main conductance level and a 19 pS subconductance level, with infrequent openings to a 27 pS conductance level. Kinetic properties of the main conductance level were similar to other native and recombinant GABAR channels. Diazepam and zolpidem enhanced GABAR currents with moderate affinity, whereas methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate inhibited GABAR currents. Loreclezole enhanced GABAR currents with high affinity, but furosemide antagonized GABAR currents with low affinity. The neurosteroids alphaxalone and pregnenolone sulfate appropriately modulated GABAR currents. Zinc blocked GABAR currents with low affinity, but lanthanum did not significantly alter NT2-N GABAR currents. Reverse transcription PCR (RT-PCR) performed on RNA from NT2-N cells clearly detected transcripts encoding human α2, α3, α5, β3, γ3, and π subtypes. The combined pharmacological and RT-PCR results are most consistent with a single or predominant GABAR isoform composed of an α2 and/or α3 subtype combined with the β3 and γ3 subtypes. The data do not rule out receptors containing combinations of α2 and/or α3 subtypes with the α5 subtype or receptors with both β1 and β3 subtypes. The presence or absence or the π subunit in functionally expressed receptors could not be determined.