BACE1 gene deletion prevents neuron loss and memory deficits in 5XFAD APP/PS1 transgenic mice

Evidence suggests that beta-amyloid (Abeta) peptide triggers a pathogenic cascade leading to neuronal loss in Alzheimer's disease (AD). However, the causal link between Abeta and neuron death in vivo remains unclear since most animal models fail to recapitulate the dramatic cell loss observed in AD. We have recently developed transgenic mice that overexpress human APP and PS1 with five familial AD mutations (5XFAD mice) and exhibit robust neuron death. Here, we demonstrate that genetic deletion of the beta-secretase (BACE1) not only abrogates Abeta generation and blocks amyloid deposition but also prevents neuron loss found in the cerebral cortex and subiculum, brain regions manifesting the most severe amyloidosis in 5XFAD mice. Importantly, BACE1 gene deletion also rescues memory deficits in 5XFAD mice. Our findings provide strong evidence that Abeta ultimately is responsible for neuron death in AD and validate the therapeutic potential of BACE1-inhibiting approaches for the treatment of AD.     

Genetic reductions of β‐site amyloid precursor protein‐cleaving enzyme 1 and amyloid‐β ameliorate impairment of conditioned taste aversion memory in 5XFAD Alzheimer’s disease model mice

Although transgenic mouse models of Alzheimer’s disease (AD) recapitulate amyloid‐β (Aβ)‐related pathologies and cognitive impairments, previous studies have mainly evaluated their hippocampus‐dependent memory dysfunctions using behavioral tasks such as the water maze and fear conditioning. However, multiple memory systems become impaired in AD as the disease progresses and it is important to test whether other forms of memory are affected in AD models. This study was designed to use conditioned taste aversion (CTA) and contextual fear conditioning paradigms to compare the phenotypes of hippocampus‐independent and ‐dependent memory functions, respectively, in 5XFAD amyloid precursor protein/presenilin‐1 transgenic mice that harbor five familial AD mutations. Although both types of memory were significantly impaired in 5XFAD mice, the onset of CTA memory deficits (～9 months of age) was delayed compared with that of contextual memory deficits (～6 months of age). Furthermore, 5XFAD mice that were genetically engineered to have reduced levels of β‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1) (BACE1^+/?·5XFAD) exhibited improved CTA memory, which was equivalent to the performance of wild‐type controls. Importantly, elevated levels of cerebral β‐secretase‐cleaved C‐terminal fragment (C99) and Aβ peptides in 5XFAD mice were significantly reduced in BACE1^+/?·5XFAD mice. Furthermore, Aβ deposition in the insular cortex and basolateral amygdala, two brain regions that are critically involved in CTA performance, was also reduced in BACE1^+/?·5XFAD compared with 5XFAD mice. Our findings indicate that the CTA paradigm is useful for evaluating a hippocampus‐independent form of memory defect in AD model mice, which is sensitive to rescue by partial reductions of the β‐secretase BACE1 and consequently of cerebral Aβ.     

Sominone improves memory impairments and increases axonal density in Alzheimer's disease model mice, 5XFAD

Previously we showed that steroidal sapogenin, sominone improved memory after a single i.p. injection into normal mice. However, it had not been reported that sominone could recover memory deficits in a severe Alzheimer's disease (AD) model animal. Therefore, we aimed to investigate that sominone improved memory impairments in the 5XFAD mouse, model for AD. In the current study, we used sominone that we had synthesized. 5XFAD mice were given 10 μmol/kg sominone intraperitoneally for 9 days. In addition to object recognition memory, axonal density, amyloid plaque number, and activated microglia in the brain were evaluated. Sominone treatment significantly improved object recognition memory compared with vehicle control treatment. Sominone treatment significantly enhanced axonal densities in the frontal cortex and parietal cortex but had no effects on amyloid plaque number and activated microglia. In cultured cortical neurons, the axonal length was significantly reduced by Aβ(1-42) treatment. However, that was markedly recovered 5 days after the treatment with 1 μM sominone. Neuronal loss was not observed in the cortex and hippocampus of 5XFAD mice at 6-8 months of age. These results suggest that memory deficits in AD may be improved by sominone independently of reducing amyloid plaques and neuroinflammation.     

Postsynaptic degeneration as revealed by PSD-95 reduction occurs after advanced Aβ and tau pathology in transgenic mouse models of Alzheimer’s disease

Impairment of synaptic plasticity underlies memory dysfunction in Alzheimer’s disease (AD). Molecules involved in this plasticity such as PSD-95, a major postsynaptic scaffold protein at excitatory synapses, may play an important role in AD pathogenesis. We examined the distribution of PSD-95 in transgenic mice of amyloidopathy (5XFAD) and tauopathy (JNPL3) as well as in AD brains using double-labeling immunofluorescence and confocal microscopy. In wild type control mice, PSD-95 primarily labeled neuropil with distinct distribution in hippocampal apical dendrites. In 3-month-old 5XFAD mice, PSD-95 distribution was similar to that of wild type mice despite significant Aβ deposition. However, in 6-month-old 5XFAD mice, PSD-95 immunoreactivity in apical dendrites markedly decreased and prominent immunoreactivity was noted in neuronal soma in CA1 neurons. Similarly, PSD-95 immunoreactivity disappeared from apical dendrites and accumulated in neuronal soma in 14-month-old, but not in 3-month-old, JNPL3 mice. In AD brains, PSD-95 accumulated in Hirano bodies in hippocampal neurons. Our findings support the notion that either Aβ or tau can induce reduction of PSD-95 in excitatory synapses in hippocampus. Furthermore, this PSD-95 reduction is not an early event but occurs as the pathologies advance. Thus, the time-dependent PSD-95 reduction from synapses and accumulation in neuronal soma in transgenic mice and Hirano bodies in AD may mark postsynaptic degeneration that underlies long-term functional deficits.     

Mitochondrial dysfunction and accumulation of the β-secretase-cleaved C-terminal fragment of APP in Alzheimer's disease transgenic mice

Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD) and may play an important role in the pathogenesis of disease. Emerging evidence indicates that amyloid-β (Aβ) peptides enter mitochondria and may thereby disrupt mitochondrial function in brains of AD patients and transgenic model mice. However, it remains to be determined whether the β-cleaved C-terminal fragment (C99), another neurotoxic fragment of amyloid precursor protein (APP), may accumulate in mitochondria of neurons affected by AD. Using immunoblotting, digitonin fractionation and immunofluorescence labeling techniques, we found that C99 is targeted to mitochondria, in particular, to the mitoplast (i.e., inner membrane and matrix compartments) in brains of AD transgenic mice (5XFAD model). Furthermore, full-length APP (fl-APP) was also identified in mitochondrial fractions of 5XFAD mice. Remarkably, partial deletion of the β-site APP-cleaving enzyme 1 (BACE1^+/−) almost completely abolished mitochondrial targeting of C99 and fl-APP in 5XFAD mice at 6 months of age. However, substantial amounts of C99 and fl-APP accumulation remained in mitochondria of 12-month-old BACE1^+/−·5XFAD mouse brains. Consistent with these changes in mitochondrial C99/fl-APP levels, BACE1^+/− deletion age-dependently rescued mitochondrial dysfunction in 5XFAD mice, as assessed by cytochrome c release from mitochondria, reduced redox or complex activities and oxidative DNA damage. Moreover, BACE1^+/− deletion also improved memory deficits as tested by the spontaneous alternation Y-maze task in 5XFAD mice at 6 months but not at 12 months of age. Taken together, our findings suggest that mitochondrial accumulation of C99 and fl-APP may occur through BACE1-dependent mechanisms and contribute to inducing mitochondrial dysfunction and cognitive impairments associated with AD.     

TrkB reduction exacerbates Alzheimer's disease-like signaling aberrations and memory deficits without affecting β-amyloidosis in 5XFAD mice

Accumulating evidence shows that brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) significantly decrease early in Alzheimer''s disease (AD). However, it remains unclear whether BDNF/TrkB reductions may be mechanistically involved in the pathogenesis of AD. To address this question, we generated 5XFAD transgenic mice with heterozygous TrkB knockout (TrkB^+/–·5XFAD), and tested the effects of TrkB reduction on AD-like features in this mouse model during an incipient stage that shows only modest amyloid-β (Aβ) pathology and retains normal mnemonic function. TrkB^+/– reduction exacerbated memory declines in 5XFAD mice at 4–5 months of age as assessed by the hippocampus-dependent spontaneous alternation Y-maze task, while the memory performance was not affected in TrkB^+/– mice. Meanwhile, TrkB^+/–·5XFAD mice were normal in nest building, a widely used measure for social behavior, suggesting the memory-specific aggravation of AD-associated behavioral impairments. We found no difference between TrkB^+/–·5XFAD and 5XFAD control mice in cerebral plaque loads, Aβ concentrations including total Aβ42 and soluble oligomers and β-amyloidogenic processing of amyloid precursor protein. Interestingly, reductions in hippocampal expression of AMPA/NMDA glutamate receptor subunits as well as impaired signaling pathways downstream to TrkB such as CREB (cAMP response element-binding protein) and Akt/GSK-3β (glycogen synthase kinase-3β) were observed in TrkB^+/–·5XFAD mice but not in 5XFAD mice. Among these signaling aberrations, only Akt/GSK-3β dysfunction occurred in TrkB^+/– mice, while others were synergistic consequences between TrkB reduction and subthreshold levels of Aβ in TrkB^+/–·5XFAD mice. Collectively, our results indicate that reduced TrkB does not affect β-amyloidosis but exacerbates the manifestation of hippocampal mnemonic and signaling dysfunctions in early AD.     

Impairments in remote memory stabilization precede hippocampal synaptic and cognitive failures in 5XFAD Alzheimer mouse model

Although animal models of Alzheimer's disease (AD) recapitulate β-amyloid-dependent hippocampal synaptic and cognitive dysfunctions, it is poorly understood how cortex-dependent remote memory stabilization following initial hippocampal coding is affected. Here, we systematically analyzed biophysical and behavioral phenotypes, including remote memory functions, of 5XFAD APP/PS1 transgenic mice containing five familial AD mutations. We found that 5XFAD mice show hippocampal dysfunctions as observed by reduced levels of baseline transmission and long-term potentiation at Schaffer collateral-CA1 synapses. Hippocampus-dependent memory tested 1 day after contextual fear conditioning was also impaired age-dependently in 5XFAD mice, as correlated with the onset of hippocampal synaptic failures. Importantly, remote memory stabilization during 30 days after training significantly declined in 5XFAD mice at time well before the onset of hippocampal dysfunctions. Our results indicate that 5XFAD mice provide a useful model system to investigate the mechanisms and therapeutic interventions for multiple synaptic and memory dysfunctions associated with AD.     

Reelin depletion is an early phenomenon of Alzheimer's pathology

Alterations in the expression of Reelin (RELN) have been implicated in the pathology of Alzheimer's disease (AD). However, whether these changes are cause or consequence of AD remains to be resolved. To better understand the role of RELN pathway in the development of AD, we examined the expression profile of RELN and its downstream signaling members APOER2, VLDLR, and DAB1 in AD-vulnerable regions of transgenic and wildtype mice as well as in AD patients and controls across disease stages and/or aging. We show that both AD pathology and aging are associated with perturbation of the RELN pathway in a species-, region-, and molecule-specific manner. Further, we show that depletion of RELN, but not its downstream signaling molecules, is detectable long before the onset of amyloid-β pathology in the murine hippocampus and in a pre-clinical AD stage in the human frontal cortex. This early event hints at a possible causative role of RELN decline in the precipitation of AD pathology and supports RELN's potential as a pre-clinical marker for AD.     

Onset of hippocampus‐dependent memory impairments in 5XFAD transgenic mouse model of Alzheimer's disease

The 5XFAD mice are an early‐onset transgenic model of Alzheimer's disease (AD) in which amyloid plaques are first observed between two and four months of age in the cortical layer five and in the subiculum of the hippocampal formation. Although cognitive alterations have been described in these mice, there are no studies that focused on the onset of hippocampus‐dependent memory deficits, which are a hallmark of the prodromal stage of AD. To identify when the first learning and memory impairments appear, 5XFAD mice of two, four, and six months of age were compared with their respective wild‐type littermates using the olfactory tubing maze, which is a very sensitive hippocampal‐dependent task. Deficits in learning and memory started at four months with a substantial increase at six months of age while no olfactory impairments were observed. The volumetric study using magnetic resonance imaging of the whole brain and specific areas (olfactory bulb, striatum, and hippocampus) did not reveal neuro‐anatomical difference. Slight memory deficits appeared at 4 months of age in correlation with an increased astrogliosis and amyloid plaque formation. This early impairment in learning and memory related to the hippocampal dysfunction is particularly suited to assess preclinical therapeutic strategies aiming to delay or suppress the onset of AD. © 2014 Wiley Periodicals, Inc.     

Mechanisms that lessen benefits of β-secretase reduction in a mouse model of Alzheimer's disease

The β-secretase enzyme BACE1 (β-site amyloid precursor protein-cleaving enzyme 1), which initiates amyloid-β (Aβ) production, is an excellent therapeutic target for Alzheimer''s disease (AD). However, recent evidence raises concern that BACE1-inhibiting approaches may encounter dramatic declines in their abilities to ameliorate AD-like pathology and memory deficits during disease progression. Here, we used BACE1 haploinsufficiency as a therapeutic relevant model to evaluate the efficacy of partial inhibition of this enzyme. Specifically, we crossed BACE1^+/− mice with 5XFAD transgenic mice and investigated the mechanisms by which Aβ accumulation and related memory impairments become less sensitive to rescue by BACE1^+/− reduction. Haploinsufficiency lowered BACE1 expression by ∼50% in 5XFAD mice regardless of age in concordance with reduction in gene copy number. However, profound Aβ plaque pathology and memory deficits concomitant with BACE1 equivalent to wild-type control levels remained in BACE1^+/−·5XFAD mice with advanced age (15–18 months old). Therefore, BACE1 haploinsufficiency is not sufficient to block the elevation of BACE1 expression (approximately twofold), which is also reported to occur during human AD progression, in 5XFAD mice. Our investigation revealed that PERK (PKR-endoplasmic reticulum-related kinase)-dependent activation of eIF2α (eukaryotic translation initiation factor-2α) accounts for the persistent BACE1 upregulation in BACE1^+/−·5XFAD mouse brains at 15–18 months of age. Moreover, BACE1 haploinsufficiency was also no longer able to prevent reduction in the expression of neprilysin, a crucial Aβ-degrading enzyme, in 5XFAD mice with advanced age. These findings demonstrate that partial BACE1 suppression cannot attenuate deleterious BACE1-elevating or neprilysin-reducing mechanisms, limiting its capabilities to reduce cerebral Aβ accumulation and rescue memory defects during the course of AD development.     

β‐Secretase‐1 elevation in transgenic mouse models of Alzheimer’s disease is associated with synaptic/axonal pathology and amyloidogenesis: implications for neuritic plaque development

The presence of neuritic plaques is a pathological hallmark of Alzheimer’s disease (AD). However, the origin of extracellular β‐amyloid peptide (Aβ) deposits and the process of plaque development remain poorly understood. The present study attempted to explore plaque pathogenesis by localizing β‐secretase‐1 (BACE1) elevation relative to Aβ accumulation and synaptic/neuritic alterations in the forebrain, using transgenic mice harboring familial AD (FAD) mutations (5XFAD and 2XFAD) as models. In animals with fully developed plaque pathology, locally elevated BACE1 immunoreactivity (IR) coexisted with compact‐like Aβ deposition, with BACE1 IR occurring selectively in dystrophic axons of various neuronal phenotypes or origins (GABAergic, glutamatergic, cholinergic or catecholaminergic). Prior to plaque onset, localized BACE1/Aβ IR occurred at swollen presynaptic terminals and fine axonal processes. These BACE1/Aβ‐containing axonal elements appeared to undergo a continuing process of sprouting/swelling and dystrophy, during which extracellular Aβ IR emerged and accumulated in surrounding extracellular space. These data suggest that BACE1 elevation and associated Aβ overproduction inside the sprouting/dystrophic axonal terminals coincide with the onset and accumulation of extracellular amyloid deposition during the development of neuritic plaques in transgenic models of AD. Our findings appear to be in harmony with an early hypothesis that axonal pathogenesis plays a key or leading role in plaque formation.     

Lipid abnormalities in the brain in adult Down’s syndrome and Alzheimer’s disease

Quantitative analysis by HPTLC of the major lipid classes and dilichol, and of fatty acyl groups of separated phosphoglycerides by capillary GLC, has been carried out on the gray matter of frontal cerebral cortex of brains from six Down’s syndrome (DS) and six Alzheimer’s disease (AD) adults, and six each of two corresponding sets of age-matched controls; specimens of DS and control cerebellum and corpus callosum were also analyzed. In DS frontal cortex, but not in AD frontal cortex, compared to their respective controls there was a decrease in the fraction of phosphatidylethanolamine (PE) and an increase in the fractions of sphingomyelin (SPM) and phosphatidylserine (PS). Abnormalities were not found in the proportions of major lipid classes in DS cerebellum or corpus callosum. The concentration of dolichol was elevated for age in the frontal cortex of DS and of AD. In the phosphoglycerides of DS frontal cortex, the fatty acyl composition showed small, but statistically significant, differences from those of age-matched controls, and some slight abnormalities were also detected in DS corpus callosum. The alterations in DS frontal cortex included decreases in (n-6) and increases in (n-3) groups in choline and ethanolamine phosphoglycerides (CPG and EPG), as had previously been found in EPG and serine phosphoglyceride (SPG) of the DS fetal brain. In DS frontal cortex, the proportion of 22∶4(n-6) groups was decreased in SPG, and in inositol phosphoglyceride (IPG) 18∶1(n-9) was increased. There were also small but significant alterations in DS frontal cortex in the fractions of shorter chain groups in CPG. In marked contrast, most of the fatty acyl abnormalities seen in DS were absent in the AD frontal cortex. It is therefore suggested that some abnormalities in the composition of cerebral membranes present prenatally in DS may persist into adulthood, and are not directly related to AD-type pathology.     

In vivo imaging of microglial activation by positron emission tomography with [11C]PBR28 in the 5XFAD model of Alzheimer's disease

Microglial activation has been linked with deficits in neuronal function and synaptic plasticity in Alzheimer's disease (AD). The mitochondrial translocator protein (TSPO) is known to be upregulated in reactive microglia. Accurate visualization and quantification of microglial density by PET imaging using the TSPO tracer [¹¹C]‐R‐PK11195 has been challenging due to the limitations of the ligand. In this study, it was aimed to evaluate the new TSPO tracer [¹¹C]PBR28 as a marker for microglial activation in the 5XFAD transgenic mouse model of AD. Dynamic PET scans were acquired following intravenous administration of [¹¹C]PBR28 in 6‐month‐old 5XFAD mice and in wild‐type controls. Autoradiography with [³H]PBR28 was carried out in the same brains to further confirm the distribution of the radioligand. In addition, immunohistochemistry was performed on adjacent brain sections of the same mice to evaluate the co‐localization of TSPO with microglia. PET imaging revealed that brain uptake of [¹¹C]PBR28 in 5XFAD mice was increased compared with control mice. Moreover, binding of [³H]PBR28, measured by autoradiography, was enriched in cortical and hippocampal brain regions, coinciding with the positive staining of the microglial marker Iba‐1 and amyloid deposits in the same areas. Furthermore, double‐staining using antibodies against TSPO demonstrated co‐localization of TSPO with microglia and not with astrocytes in 5XFAD mice and human post‐mortem AD brains. The data provided support of the suitability of [¹¹C]PBR28 as a tool for in vivo monitoring of microglial activation and assessment of treatment response in future studies using animal models of AD. GLIA 2016;64:993–1006     

Clinical and pathological evidence for a frontal variant of Alzheimer disease.

OBJECTIVE: To evaluate the clinical and pathological features of a subgroup of patients with Alzheimer disease (AD) who exhibited early and disproportionately severe impairments on tests of frontal lobe functioning. We hypothesized that these patients would exhibit a greater degree of either neurofibrillary tangle (NFT) or senile plaque pathology in the frontal lobes than would patients with typical AD. DESIGN AND OUTCOME MEASURES: We examined the neuropsychological profiles and senile plaque and NFT accumulation in the frontal, entorhinal, temporal, and parietal cortices in 3 patients with AD who exhibited disproportionate frontal impairments during early stages of dementia (frontal AD) and 3 matched patients with typical AD (typical AD). RESULTS: Compared with the typical AD group, the frontal AD group performed significantly worse on 2 tests of frontal lobe functioning and on the Wechsler Adult Intelligence Scale-Revised Block Design test. No significant group differences were found on other tests. Analysis of brain tissue samples demonstrated that, despite comparable entorhinal, temporal, and parietal NFT loads, the frontal AD group showed a significantly higher NFT load in the frontal cortex than the typical AD group. Senile plaque pathology in the frontal and entorhinal cortices did not differentiate the 2 groups. CONCLUSIONS: We identified a subgroup of patients with pathologically confirmed AD who presented in the early stages of dementia with disproportionate impairments on tests of frontal lobe functioning and had a greater-than-expected degree of NFT pathology in the frontal lobes, suggesting the existence of a frontal variant of AD that has distinctive clinical and pathological features.     

Evidence for altered Numb isoform levels in Alzheimer's disease patients and a triple transgenic mouse model

The cell fate determinant Numb exists in four alternatively spliced variants that differ in the length of their PTB (phosphotyrosine-binding domain, either lacking or containing an 11 amino acid insertion) and PRR (proline-rich region, either lacking or containing a 48 amino acid insertion). We previously reported that Numb switches from isoforms containing the PTB insertion to isoforms lacking this insertion in neural cultures subjected to stress induced by trophic factor withdrawal. The switch in Numb isoforms enhances the generation of amyloid-β peptide (Aβ), the principle component of senile plaques in Alzheimer's disease (AD). Here we examine the expression of the Numb isoforms in brains from AD patients and triple transgenic (3xTg) AD mice. We found that levels of the Numb isoforms lacking the PTB insertion are significantly elevated in the parietal cortex but not in the cerebellum of AD patients when compared to control subjects. Levels of Numb isoforms lacking the PTB insertion were also elevated in the cortex but not cerebellum of 12 month-old 3xTg AD mice with Aβ deposits compared to younger 3xTg-AD mice and to non-transgenic mice. Exposure of cultured neurons to Aβ resulted in an increase in the levels of Numb isoforms lacking the PTB domain, consistent with a role for Aβ in the aberrant expression of Numb in vulnerable brain regions of AD patients and mice. Collectively, the data show that altered expression of Numb isoforms in vulnerable neurons occurs during AD pathogenesis and suggest a role for Numb in the disease process.