Apolipoprotein E4 genotype compromises brain exosome production.

In addition to being the greatest genetic risk factor for Alzheimer's disease, expression of the ɛ4 allele of apolipoprotein E can lead to cognitive decline during ageing that is independent of Alzheimer's amyloid-ß and tau pathology. In human post-mortem tissue and mouse models humanized for apolipoprotein E, we examined the impact of apolipoprotein E4 expression on brain exosomes, vesicles that are produced within and secreted from late-endocytic multivesicular bodies. Compared to humans or mice homozygous for the risk-neutral ɛ3 allele we show that the ɛ4 allele, whether homozygous or heterozygous with an ɛ3 allele, drives lower exosome levels in the brain extracellular space. In mice, we show that the apolipoprotein E4-driven change in brain exosome levels is age-dependent: while not present at age 6 months, it is detectable at 12 months of age. Expression levels of the exosome pathway regulators tumor susceptibility gene 101 (TSG101) and Ras-related protein Rab35 (RAB35) were found to be reduced in the brain at the protein and mRNA levels, arguing that apolipoprotein E4 genotype leads to a downregulation of exosome biosynthesis and release. Compromised exosome production is likely to have adverse effects, including diminishing a cell's ability to eliminate materials from the endosomal-lysosomal system. This reduction in brain exosome levels in 12-month-old apolipoprotein E4 mice occurs earlier than our previously reported brain endosomal pathway changes, arguing that an apolipoprotein E4-driven failure in exosome production plays a primary role in endosomal and lysosomal deficits that occur in apolipoprotein E4 mouse and human brains. Disruption of these interdependent endosomal-exosomal-lysosomal systems in apolipoprotein E4-expressing individuals may contribute to amyloidogenic amyloid-ß precursor protein processing, compromise trophic signalling and synaptic function, and interfere with a neuron's ability to degrade material, all of which are events that lead to neuronal vulnerability and higher risk of Alzheimer's disease development. Together, these data suggest that exosome pathway dysfunction is a previously unappreciated component of the brain pathologies that occur as a result of apolipoprotein E4 expression.

Brain-Wide Insulin Resistance, Tau Phosphorylation Changes, and Hippocampal Neprilysin and Amyloid-ß Alterations in a Monkey Model of Type 1 Diabetes.

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-ß (Aß) levels that was restricted to the temporal lobe, with the greatest increase seen in the hippocampus. Consistent with this localized Aß increase, a hippocampus-restricted decrease in the protein and mRNA for the Aß-degrading enzyme neprilysin (NEP) was found, whereas various Aß-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 Aß. 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-ß (Aß), and the Aß-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 an in vivo model highly relevant to humans, we show multiple alterations in the brain resulting from DM that are mechanistically linked to AD risk.

Brain apolipoprotein E levels in mice challenged by a Western diet increase in an allele-dependent manner.

Human apolipoprotein E (APOE) is the greatest determinant of genetic risk for memory deficits and Alzheimer's disease (AD). While APOE4 drives memory loss and high AD risk, APOE2 leads to healthy brain aging and reduced AD risk compared to the common APOE3 variant. We examined brain APOE protein levels in humanized mice homozygous for these alleles and found baseline levels to be age- and isoform-dependent: APOE2 levels were greater than APOE3, which were greater than APOE4. Despite the understanding that APOE lipoparticles do not traverse the blood-brain barrier, we show that brain APOE levels are responsive to dietary fat intake. Challenging mice for 6 months on a Western diet high in fat and cholesterol increased APOE protein levels in an allele-dependent fashion with a much greater increase within blood plasma than within the brain. In the brain, APOE2 levels responded most to the Western diet challenge, increasing by 20 % to 30 %. While increased lipoparticles are generally deleterious in the periphery, we propose that higher brain APOE2 levels may represent a readily available pool of beneficial lipid particles for neurons.

Expression and proteolytic processing of the amyloid precursor protein is unaffected by the expression of the three human apolipoprotein E alleles in the brains of mice.

The 3 human apolipoprotein E (APOE) gene alleles modify an individual's risk of developing Alzheimer's disease (AD): compared to the risk-neutral APOE ε3 allele, the ε4 allele (APOE4) is strongly associated with increased AD risk while the ε2 allele is protective. Multiple mechanisms have been shown to link APOE4 expression and AD risk, including the possibility that APOE4 increases the expression of the amyloid precursor protein (APP) (Y-W.A. Huang, B. Zhou, A.M. Nabet, M. Wernig, T.C. Südhof, 2019). In this study, we investigated the impact of APOE genotype on the expression, and proteolytic processing of endogenously expressed APP in the brains of mice humanized for the 3 APOE alleles. In contrast to prior studies using neuronal cultures, we found in the brain that both App gene expression, and the levels of APP holoprotein were not affected by APOE genotype. Additionally, our analysis of APP fragments showed that APOE genotype does not impact APP processing in the brain: the levels of both α- and ß-cleaved soluble APP fragments (sAPPs) were similar across genotypes, as were the levels of the membrane-associated α- and ß-cleaved C-terminal fragments (CTFs) of APP. Lastly, APOE genotype did not impact the level of soluble amyloid beta (Aß). These findings argue that the APOE-allele-dependent AD risk is independent of the brain expression and processing of APP.

Familial partial epilepsy with variable foci: a new family with suggestion of linkage to chromosome 22q12.

Familial partial epilepsy with variable foci (FPEVF) is an autosomal dominant form of partial epilepsy characterized by the presence of epileptic seizures originating from different cerebral lobes in different members of the same family. Linkage to chromosomes 22q12 and 2q36 has been reported, although only six families have been published. We studied a new FPEVF family including nine affected individuals. The phenotype in this family was similar to that previously described and consisted of nocturnal and daytime seizures with semiology suggesting a frontal lobe origin. A video-EEG (electroencephalography) recording of the proband's seizures is presented and revealed hyperkinetic seizures of frontal lobe origin preceded by left frontal spikes. We excluded linkage to chromosome 2q36 and found a suggestion of linkage to chromosome 22q12 with a lod score of 2.64 (h = 0) for marker D22S689.

Age-dependent dysregulation of brain amyloid precursor protein in the Ts65Dn Down syndrome mouse model.

Individuals with Down syndrome develop beta-amyloid deposition characteristic of early-onset Alzheimer's disease (AD) in mid-life, presumably because of an extra copy of the chromosome 21-located amyloid precursor protein (App) gene. App mRNA and APP metabolite levels were assessed in the brains of Ts65Dn mice, a mouse model of Down syndrome, using quantitative PCR, western blot analysis, immunoprecipitation, and ELISAs. In spite of the additional App gene copy, App mRNA, APP holoprotein, and all APP metabolite levels in the brains of 4-month-old trisomic mice were not increased compared with the levels seen in diploid littermate controls. However starting at 10 months of age, brain APP levels were increased proportional to the App gene dosage imbalance reflecting increased App message levels in Ts65Dn mice. Similar to APP levels, soluble amino-terminal fragments of APP (sAPPalpha and sAPPbeta) were increased in Ts65Dn mice compared with diploid mice at 12 months but not at 4 months of age. Brain levels of both Abeta40 and Abeta42 were not increased in Ts65Dn mice compared with diploid mice at all ages examined. Therefore, multiple mechanisms contribute to the regulation towards diploid levels of APP metabolites in the Ts65Dn mouse brain.

Entorhinal cortical defects in Tg2576 mice are present as early as 2-4 months of age.

The entorhinal cortex (EC) is one of the first brain areas to display neuropathology in Alzheimer's disease. A mouse model which simulates amyloid-ß (Aß) neuropathology, the Tg2576 mouse, was used to address these early changes. Here, we show EC abnormalities occur in 2- to 4-month-old Tg2576 mice, an age before Aß deposition and where previous studies suggest that there are few behavioral impairments. First we show, using a sandwich enzyme-linked immunosorbent assay, that soluble human Aß40 and Aß42 are detectable in the EC of 2-month-old Tg2576 mice before Aß deposition. We then demonstrate that 2- to 4-month-old Tg2576 mice are impaired at object placement, an EC-dependent cognitive task. Next, we show that defects in neuronal nuclear antigen expression and myelin uptake occur in the superficial layers of the EC in 2- to 4-month-old Tg2576 mice. In slices from Tg2576 mice that contained the EC, there were repetitive field potentials evoked by a single stimulus to the underlying white matter, and a greater response to reduced extracellular magnesium ([Mg(2+)]o), suggesting increased excitability. However, deep layer neurons in Tg2576 mice had longer latencies to antidromic activation than wild type mice. The results show changes in the EC at early ages and suggest that altered excitability occurs before extensive plaque pathology.

Endogenous murine Aß increases amyloid deposition in APP23 but not in APPPS1 transgenic mice.

Endogenous murine amyloid-ß peptide (Aß) is expressed in most Aß precursor protein (APP) transgenic mouse models of Alzheimer's disease but its contribution to ß-amyloidosis remains unclear. We demonstrate ∼ 35% increased cerebral Aß load in APP23 transgenic mice compared with age-matched APP23 mice on an App-null background. No such difference was found for the much faster Aß-depositing APPPS1 transgenic mouse model between animals with or without the murine App gene. Nevertheless, both APP23 and APPPS1 mice codeposited murine Aß, and immunoelectron microscopy revealed a tight association of murine Aß with human Aß fibrils. Deposition of murine Aß was considerably less efficient compared with the deposition of human Aß indicating a lower amyloidogenic potential of murine Aß in vivo. The amyloid dyes Pittsburgh Compound-B and pentamer formyl thiophene acetic acid did not differentiate between amyloid deposits consisting of human Aß and deposits of mixed human-murine Aß. Our data demonstrate a differential effect of murine Aß on human Aß deposition in different APP transgenic mice. The mechanistically complex interaction of human and mouse Aß may affect pathogenesis of the models and should be considered when models are used for translational preclinical studies.

Chronic anti-murine Aß immunization preserves odor guided behaviors in an Alzheimer's ß-amyloidosis model.

Olfaction is often impaired in Alzheimer's disease (AD) and is also dysfunctional in mouse models of the disease. We recently demonstrated that short-term passive anti-murine-Aß immunization can rescue olfactory behavior in the Tg2576 mouse model overexpressing a human mutation of the amyloid precursor protein (APP) after ß-amyloid deposition. Here we tested the ability to preserve normal olfactory behaviors by means of long-term passive anti-murine-Aß immunization. Seven-month-old Tg2576 and non-transgenic littermate (NTg) mice were IP-injected biweekly with the m3.2 murine-Aß-specific antibody until 16 mo of age when mice were tested in the odor habituation test. While Tg2576 mice treated with a control antibody showed elevations in odor investigation times and impaired odor habituation compared to NTg, olfactory behavior was preserved to NTg levels in m3.2-immunized Tg2576 mice. Immunized Tg2576 mice had significantly less ß-amyloid immunolabeling in the olfactory bulb and entorhinal cortex, yet showed elevations in Thioflavin-S labeled plaques in the piriform cortex. No detectable changes in APP metabolite levels other than Aß were found following m3.2 immunization. These results demonstrate efficacy of chronic, long-term anti-murine-Aß m3.2 immunization in preserving normal odor-guided behaviors in a human APP Tg model. Further, these results provide mechanistic insights into olfactory dysfunction as a biomarker for AD by yielding evidence that focal reductions of Aß may be sufficient to preserve olfaction.

Early endosomal abnormalities and cholinergic neuron degeneration in amyloid-ß protein precursor transgenic mice.

Early endosomal changes, a prominent pathology in neurons early in Alzheimer's disease, also occur in neurons and peripheral tissues in Down syndrome. While in Down syndrome models increased amyloid-ß protein precursor (AßPP) expression is known to be a necessary contributor on the trisomic background to this early endosomal pathology, increased AßPP alone has yet to be shown to be sufficient to drive early endosomal alterations in neurons. Comparing two AßPP transgenic mouse models, one that contains the AßPP Swedish K670N/M671L double mutation at the ß-cleavage site (APP23) and one that has the AßPP London V717I mutation near the γ-cleavage site (APPLd2), we show significantly altered early endosome morphology in fronto-parietal neurons as well as enlargement of early endosomes in basal forebrain cholinergic neurons of the medial septal nucleus in the APP23 model, which has the higher levels of AßPP ß-C-terminal fragment (ßCTF) accumulation. Early endosomal changes correlate with a marked loss of the cholinergic population, which is consistent with the known dependence of the large projection cholinergic cells on endosome-mediated retrograde neurotrophic transport. Our findings support the idea that increased expression of AßPP and AßPP metabolites in neurons is sufficient to drive early endosomal abnormalities in vivo, and that disruption of the endocytic system is likely to contribute to basal forebrain cholinergic vulnerability.

Immunization targeting a minor plaque constituent clears ß-amyloid and rescues behavioral deficits in an Alzheimer's disease mouse model.

Although anti-human ß-amyloid (Aß) immunotherapy clears brain ß-amyloid plaques in Alzheimer's disease (AD), targeting additional brain plaque constituents to promote clearance has not been attempted. Endogenous murine Aß is a minor Aß plaque component in amyloid precursor protein (APP) transgenic AD models, which we show is ∼3%-8% of the total accumulated Aß in various human APP transgenic mice. Murine Aß codeposits and colocalizes with human Aß in amyloid plaques, and the two Aß species coimmunoprecipitate together from brain extracts. In the human APP transgenic mouse model Tg2576, passive immunization for 8 weeks with a murine-Aß-specific antibody reduced ß-amyloid plaque pathology, robustly decreasing both murine and human Aß levels. The immunized mice additionally showed improvements in two behavioral assays, odor habituation and nesting behavior. We conclude that passive anti-murine Aß immunization clears Aß plaque pathology--including the major human Aß component--and decreases behavioral deficits, arguing that targeting minor endogenous brain plaque constituents can be beneficial, broadening the range of plaque-associated targets for AD therapeutics.

Calpastatin modulates APP processing in the brains of ß-amyloid depositing but not wild-type mice.

We report that neuronal overexpression of the endogenous inhibitor of calpains, calpastatin (CAST), in a mouse model of human Alzheimer's disease (AD) ß-amyloidosis, the APP23 mouse, reduces ß-amyloid (Aß) pathology and Aß levels when comparing aged, double transgenic (tg) APP23/CAST with APP23 mice. Concurrent with Aß plaque deposition, aged APP23/CAST mice show a decrease in the steady-state brain levels of the amyloid precursor protein (APP) and APP C-terminal fragments (CTFs) when compared with APP23 mice. This CAST-dependent decrease in APP metabolite levels was not observed in single tg CAST mice expressing endogenous APP or in younger, Aß plaque predepositing APP23/CAST mice. We also determined that the CAST-mediated inhibition of calpain activity in the brain is greater in the CAST mice with Aß pathology than in non-APP tg mice, as demonstrated by a decrease in calpain-mediated cytoskeleton protein cleavage. Moreover, aged APP23/CAST mice have reduced extracellular signal-regulated kinase 1/2 (ERK1/2) activity and tau phosphorylation when compared with APP23 mice. In summary, in vivo calpain inhibition mediated by CAST transgene expression reduces Aß pathology in APP23 mice, with our findings further suggesting that APP metabolism is modified by CAST overexpression as the mice develop Aß pathology. Our results indicate that the calpain system in neurons is more responsive to CAST inhibition under conditions of Aß pathology, suggesting that in the disease state neurons may be more sensitive to the therapeutic use of calpain inhibitors.

In vivo turnover of tau and APP metabolites in the brains of wild-type and Tg2576 mice: greater stability of sAPP in the beta-amyloid depositing mice.

The metabolism of the amyloid precursor protein (APP) and tau are central to the pathobiology of Alzheimer's disease (AD). We have examined the in vivo turnover of APP, secreted APP (sAPP), Abeta and tau in the wild-type and Tg2576 mouse brain using cycloheximide to block protein synthesis. In spite of overexpression of APP in the Tg2576 mouse, APP is rapidly degraded, similar to the rapid turnover of the endogenous protein in the wild-type mouse. sAPP is cleared from the brain more slowly, particularly in the Tg2576 model where the half-life of both the endogenous murine and transgene-derived human sAPP is nearly doubled compared to wild-type mice. The important Abeta degrading enzymes neprilysin and IDE were found to be highly stable in the brain, and soluble Abeta40 and Abeta42 levels in both wild-type and Tg2576 mice rapidly declined following the depletion of APP. The cytoskeletal-associated protein tau was found to be highly stable in both wild-type and Tg2576 mice. Our findings unexpectedly show that of these various AD-relevant protein metabolites, sAPP turnover in the brain is the most different when comparing a wild-type mouse and a beta-amyloid depositing, APP overexpressing transgenic model. Given the neurotrophic roles attributed to sAPP, the enhanced stability of sAPP in the beta-amyloid depositing Tg2576 mice may represent a neuroprotective response.

Autosomal dominant nocturnal frontal lobe epilepsy with a mutation in the CHRNB2 gene.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE; MIM 600513) has been associated with mutations in the genes coding for the alfa-4 (CHRNA4), beta-2 (CHRNB2), and alpha-2 (CHRNA2) subunits of the neuronal nicotinic acetylcholine receptor (nAChR) and for the corticotropin-releasing hormone (CRH). A four-generation ADNFLE family with six affected members was identified. All affected members presented the clinical characteristics of ADNFLE. Interictal awake and sleep EEG recordings showed no epileptiform abnormalities. Ictal video-EEG recordings showed focal seizures with frontal lobe semiology. Mutation analysis of the CHRNB2 gene revealed a c.859G>A transition (Val287Met) within the second transmembrane domain, identical to that previously described in a Scottish ADNFLE family. To our knowledge, this is the third family reported presenting a mutation in CHRNB2. The clinical phenotype appears similar to that described with mutations in CHRNA4, suggesting that mutations in these two subunits lead to similar functional alterations of the nAChR.