Knock-in of human BACE1 cleaves murine APP and reiterates Alzheimer-like phenotypes.
ABSTRACT: Key neuropathological hallmarks of Alzheimer's disease (AD) are elevated levels of amyloid ?-peptide (A?) species generated via amyloid precursor protein (APP) endoproteolysis and cleavage by the rate-limiting ?-site enzyme 1 (BACE1). Because rodents do not develop amyloid pathologies, we here investigated whether AD-like endophenotypes can be created in mice by expression of human bace1. To avoid pitfalls of existing models, we introduced hbace1 via knock-in under the control of the CaMKII ? promoter into the safe HPRT locus. We report amyloidogenic processing of murine APP in the hBACE1 mice (termed PLB4), resulting in the formation of toxic APP metabolites that accumulate intra- and extraneuronally in hippocampus and cortex. Pronounced accumulation of A?*56 and A? hexamers in the absence of plaque deposition was detected in brain tissue from symptomatic PLB4 mice. Heightened levels of inflammation (gliosis) also appeared in several AD-related brain regions (dentate gyrus, hippocampal area CA1, piriform and parietal cortices) at 6 and 12 months of age. Behaviorally, deficits in habituation to a novel environment and semantic-like memory (social transmission of food preference) were detected from 3 to 4 months of age. Impairments in spatial learning strategies in long-term reference (water maze) and working memory (Y-maze) tasks presented at 6 months, and were distinct from reductions in locomotor activity and anxiety. Overall, our data indicate for the first time that targeted, subtle forebrain-specific expression through single gene knock-in of hBACE1 is sufficient to generate AD-relevant cognitive impairments amid corresponding histopathologies, confirming human BACE as the key parameter in amyloid pathogenesis.
Project description:Increased amyloid-? (A?) plaque deposition is thought to be the main cause of Alzheimer's disease (AD). ?-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is the key protein involved in A? peptide generation. Excessive expression of BACE1 might cause overproduction of neurotoxins in the central nervous system. Previous studies indicated that BACE1 initially cleaves the amyloid precursor protein (APP) and may subsequently interfere with physiological functions of proteins such as PKA, which is recognized to be closely associated with long-term potentiation (LTP) level and can effectively ameliorate cognitive impairments. Therefore, revealing the underlying mechanism of BACE1 in the pathogenesis of AD might have a significant impact on the future development of therapeutic agents targeting dementia. This study examined the effects of electroacupuncture (EA) stimulation on BACE1, APP, and p-PKA protein levels in hippocampal tissue samples. Memory and learning abilities were assessed using the Morris water maze test after EA intervention. Immunofluorescence, immunohistochemistry, and western blot were employed to assess the distribution patterns and expression levels of BACE1, APP, and p-PKA, respectively. The results showed the downregulation of BACE1 and APP and the activation of PKA by EA. In summary, EA treatment might reduce BACE1 deposition in APP/PS1 transgenic mice and regulate PKA and its associated substrates, such as LTP to change memory and learning abilities.
Project description:Amyloid precursor protein (APP) modulates glutamate release via cytoplasmic and intravesicular interactions with the synaptic vesicle release machinery. The intravesicular domain, called ISVAID, contains the BACE1 cleavage site of APP. We have tested the functional significance of BACE1 processing of APP using App-Swedish (Apps ) knock-in rats, which carry an App mutation that causes familial Alzheimer's disease (FAD) in humans. We show that in Apps rats, ?-cleavage of APP is favored over ?-cleavage. Apps rats show facilitated glutamate, but not GABA, release. Our data support the notion that APP tunes glutamate release, and that BACE1 cleavage of the ISVAID segment of APP facilitates this function. We define this phenomenon as BACE1 on APP-dependent glutamate release (BAD-Glu). Unsurprisingly, Apps rats show no evidence of AD-related pathology at 15 days and 3 months of age, indicating that alterations in BAD-Glu are not caused by pathological lesions. The evidence that a pathogenic APP mutation causes an early enhancement of BAD-Glu suggests that alterations of BACE1 processing of APP in glutamatergic synaptic vesicles could contribute to dementia.
Project description:Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by pathological brain lesions and a decline in cognitive function. ?-Amyloid peptides (A?), derived from proteolytic processing of amyloid precursor protein (APP), play a central role in AD pathogenesis. ?-Site APP cleaving enzyme 1 (BACE1), the transmembrane aspartyl protease which initiates A? production, is axonally transported in neurons and accumulates in dystrophic neurites near cerebral amyloid deposits in AD. BACE1 is modified by S-palmitoylation at four juxtamembrane cysteine residues. S-palmitoylation is a dynamic posttranslational modification that is important for trafficking and function of several synaptic proteins. Here, we investigated the in vivo significance of BACE1 S-palmitoylation through the analysis of knock-in mice with cysteine-to-alanine substitution at the palmitoylated residues (4CA mice). BACE1 expression, as well as processing of APP and other neuronal substrates, was unaltered in 4CA mice despite the lack of BACE1 S-palmitoylation and reduced lipid raft association. Whereas steady-state A? levels were similar, synaptic activity-induced endogenous A? production was not observed in 4CA mice. Furthermore, we report a significant reduction of cerebral amyloid burden and BACE1 accumulation in dystrophic neurites in the absence of BACE1 S-palmitoylation in mouse models of AD amyloidosis. Studies in cultured neurons suggest that S-palmitoylation is required for dendritic spine localization and axonal targeting of BACE1. Finally, the lack of BACE1 S-palmitoylation mitigates cognitive deficits in 5XFAD mice. Using transgenic mouse models, these results demonstrate that intrinsic posttranslational S-palmitoylation of BACE1 has a significant impact on amyloid pathogenesis and the consequent cognitive decline.
Project description:BACKGROUND:Alzheimer's disease (AD) is the most frequent and costly neurodegenerative disorder. Although diverse lines of evidence suggest that the amyloid precursor protein (APP) is involved in its causation, the precise mechanisms remain unknown and no treatments are available to prevent or halt the disease. A favorite hypothesis has been that APP contributes to AD pathogenesis through the cerebral accumulation of the amyloid-? peptide (A?), which is derived from APP through sequential proteolytic cleavage by BACE1 and ?-secretase. However, inhibitors of these enzymes have failed in clinical trials despite clear evidence for target engagement. METHODS:To further elucidate the roles of APP and its metabolites in AD pathogenesis, we analyzed transgenic mice overexpressing wildtype human APP (hAPP) or hAPP carrying mutations that cause autosomal dominant familial AD (FAD), as well as App knock-in mice that do not overexpress hAPP but have two mouse App alleles with FAD mutations and a humanized A? sequence. RESULTS:Although these lines of mice had marked differences in cortical and hippocampal levels of APP, APP C-terminal fragments, soluble A?, A? oligomers and age-dependent amyloid deposition, they all developed cognitive deficits as well as non-convulsive epileptiform activity, a type of network dysfunction that also occurs in a substantive proportion of humans with AD. Pharmacological inhibition of BACE1 effectively reduced levels of amyloidogenic APP C-terminal fragments (C99), soluble A?, A? oligomers, and amyloid deposits in transgenic mice expressing FAD-mutant hAPP, but did not improve their network dysfunction and behavioral abnormalities, even when initiated at early stages before amyloid deposits were detectable. CONCLUSIONS:hAPP transgenic and App knock-in mice develop similar pathophysiological alterations. APP and its metabolites contribute to AD-related functional alterations through complex combinatorial mechanisms that may be difficult to block with BACE inhibitors and, possibly, also with other anti-A? treatments.
Project description:Amyloid-β (Aβ) peptides play a key role in synaptic damage and memory deficits in the early pathogenesis of Alzheimer's disease (AD). Abnormal accumulation of Aβ at nerve terminals leads to synaptic pathology and ultimately to neurodegeneration. β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the major neuronal β-secretase for Aβ generation. However, the mechanisms regulating BACE1 distribution in axons and β cleavage of APP at synapses remain largely unknown. Here, we reveal that dynein-Snapin-mediated retrograde transport regulates BACE1 trafficking in axons and APP processing at presynaptic terminals. BACE1 is predominantly accumulated within late endosomes at the synapses of AD-related mutant human APP (hAPP) transgenic (Tg) mice and patient brains. Defective retrograde transport by genetic ablation of snapin in mice recapitulates late endocytic retention of BACE1 and increased APP processing at presynaptic sites. Conversely, overexpressing Snapin facilitates BACE1 trafficking and reduces synaptic BACE1 accumulation by enhancing the removal of BACE1 from distal AD axons and presynaptic terminals. Moreover, elevated Snapin expression via stereotactic hippocampal injections of adeno-associated virus particles in mutant hAPP Tg mouse brains decreases synaptic Aβ levels and ameliorates synapse loss, thus rescuing cognitive impairments associated with hAPP mice. Altogether, our study provides new mechanistic insights into the complex regulation of BACE1 trafficking and presynaptic localization through Snapin-mediated dynein-driven retrograde axonal transport, thereby suggesting a potential approach of modulating Aβ levels and attenuating synaptic deficits in AD.SIGNIFICANCE STATEMENT β-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) trafficking and synaptic localization significantly influence its β secretase activity and amyloid-β (Aβ) production. In AD brains, BACE1 is accumulated within dystrophic neurites, which is thought to augment Aβ-induced synaptotoxicity by Aβ overproduction. However, it remains largely unknown whether axonal transport regulates synaptic APP processing. Here, we demonstrate that Snapin-mediated retrograde transport plays a critical role in removing BACE1 from presynaptic terminals toward the soma, thus reducing synaptic Aβ production. Adeno-associated virus-mediated Snapin overexpression in the hippocampus of mutant hAPP mice significantly decreases synaptic Aβ levels, attenuates synapse loss, and thus rescues cognitive deficits. Our study uncovers a new pathway that controls synaptic APP processing by enhancing axonal BACE1 trafficking, thereby advancing our fundamental knowledge critical for ameliorating Aβ-linked synaptic pathology.
Project description:Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline with accumulation of amyloid beta (A?) and neurofibrillary tangles that usually begins 15–30 years before clinical diagnosis. Rodent models that recapitulate aggressive A? and/or the pathology of neurofibrillary tangles are essential for AD research. Accordingly, non-invasive early detection systems in these animal models are required to evaluate the phenotypic changes, elucidate the mechanism of disease progression, and facilitate development of novel therapeutic approaches. Although many behavioral tests efficiently reveal cognitive impairments at the later stage of the disease in AD models, it has been challenging to detect such impairments at the early stage. To address this issue, we subjected 4–6-month-old male AppNL?G?F/NL?G?F knock-in (App-KI) mice to touchscreen-based location discrimination (LD), different object–location paired-associate learning (dPAL), and reversal learning tests, and compared the results with those of the classical Morris water maze test. These tests are mainly dependent on the brain regions prone to A? accumulation at the earliest stages of the disease. At 4–6 months, considered to represent the early stage of disease when mice exhibit initial deposition of A? and slight gliosis, the classical Morris water maze test revealed no difference between groups, whereas touchscreen-based LD and dPAL tasks revealed significant impairments in task performance. Our report is the first to confirm that a systematic touchscreen-based behavioral test battery can sensitively detect the early stage of cognitive decline in an AD-linked App-KI mouse model. This system could be applied in future translational research.
Project description:BACKGROUND:?-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the rate-limiting enzyme in the production of amyloid beta (A?), the toxic peptide that accumulates in the brains of Alzheimer's disease (AD) patients. Our previous studies have shown that the clathrin adaptor Golgi-localized ?-ear-containing ARF binding protein 3 (GGA3) plays a key role in the trafficking of BACE1 to lysosomes, where it is normally degraded. GGA3 depletion results in BACE1 stabilization both in vitro and in vivo. Moreover, levels of GGA3 are reduced and inversely related to BACE1 levels in post-mortem brains of AD patients. METHOD:In order to assess the effect of GGA3 deletion on AD-like phenotypes, we crossed GGA3 -/- mice with 5XFAD mice. BACE1-mediated processing of APP and the cell adhesion molecule L1 like protein (CHL1) was measured as well as levels of A?42 and amyloid burden. RESULTS:In 5XFAD mice, we found that hippocampal and cortical levels of GGA3 decreased while BACE1 levels increased with age, similar to what is observed in human AD brains. GGA3 deletion prevented age-dependent elevation of BACE1 in GGA3KO;5XFAD mice. We also found that GGA3 deletion resulted in increased hippocampal levels of A?42 and amyloid burden in 5XFAD mice at 12 months of age. While levels of BACE1 did not change with age and gender in GGAKO;5XFAD mice, amyloid precursor protein (APP) levels increased with age and were higher in female mice. Moreover, elevation of APP was associated with a decreased BACE1-mediated processing of CHL1 not only in 12 months old 5XFAD mice but also in human brains from subjects affected by Down syndrome, most likely due to substrate competition. CONCLUSION:This study demonstrates that GGA3 depletion is a leading candidate mechanism underlying elevation of BACE1 in AD. Furthermore, our findings suggest that BACE1 inhibition could exacerbate mechanism-based side effects in conditions associated with APP elevation (e.g. Down syndrome) owing to impairment of BACE1-mediated processing of CHL1. Therefore, therapeutic approaches aimed to restore GGA3 function and to prevent the down stream effects of its depletion (e.g. BACE1 elevation) represent an attractive alternative to BACE inhibition for the prevention/treatment of AD.
Project description:Rivastigmine (or Exelon) is a cholinesterase inhibitor, currently used as a symptomatic treatment for mild-to-moderate Alzheimer's disease (AD). Amyloid-? peptide (A?) generated from its precursor protein (APP) by ?-secretase (or BACE1) and ?-secretase endoproteolysis. Alternative APP cleavage by ?-secretase (a family of membrane-bound metalloproteases- Adamalysins) precludes the generation of toxic A? and yields a neuroprotective and neurotrophic secreted sAPP? fragment. Several signal transduction pathways, including protein kinase C and MAP kinase, stimulate ?-secretase. We present data to suggest that rivastigmine, in addition to anticholinesterase activity, directs APP processing away from BACE1 and towards ?-secretases. We treated rat neuronal PC12 cells and primary human brain (PHB) cultures with rivastigmine and the ?-secretase inhibitor TAPI and assayed for levels of APP processing products and ?-secretases. We subsequently treated 3×Tg (transgenic) mice with rivastigmine and harvested hippocampi to assay for levels of APP processing products. We also assayed postmortem human control, AD, and AD brains from subjects treated with rivastigmine for levels of APP metabolites. Rivastigmine dose-dependently promoted ?-secretase activity by upregulating levels of ADAM-9, -10, and -17 ?-secretases in PHB cultures. Co-treatment with TAPI eliminated rivastigmine-induced sAPP? elevation. Rivastigmine treatment elevated levels of sAPP? in 3×Tg mice. Consistent with these results, we also found elevated sAPP? in postmortem brain samples from AD patients treated with rivastigmine. Rivastigmine can modify the levels of several shedding proteins and directs APP processing toward the non-amyloidogenic pathway. This novel property of rivastigmine can be therapeutically exploited for disease-modifying intervention that goes beyond symptomatic treatment for AD.
Project description:Voltage-gated sodium channels beta 2 (Nav?2, encoded by SCN2B) is a substrate of ?-site amyloid precursor protein cleaving enzyme 1 (BACE1) and regulates cell surface expression of channels in neurons. Previous studies reported enhanced Nav?2 processing by BACE1 in Alzheimer's disease (AD) model and patients. We investigated whether changes in Nav?2 expression affect neuronal seizure and amyloid precursor protein (APP) processing in an AD mouse model. Our study used eight-month-old APP/presenilin 1 (PS1) mice and transgenic Nav?2 knockdown [by 61% vs. wild type (WT)] APP/PS1 mice (APP/PS1/Nav?2-kd), with age-matched WT and Nav?2 knockdown (Nav?2-kd) mice as controls. We found that Nav?2 knockdown in APP/PS1 mice partially reversed the abnormal Nav?2 cleavage and the changes in intracellular and total Nav1.1? expression. It also restored sodium currents density in hippocampal neurons and neuronal activity, as indicated by EEG tracing; improved Morris water maze performance; and shifted APP amyloidogenic metabolism towards non-amyloidogenic processing. There were no differences in these indicators between WT and Nav?2-kd mice. These results suggest Nav?2 knockdown may be a promising strategy for treating AD.
Project description:Amyloid pathology occurs early in Alzheimer's disease (AD), and has therefore been the focus of numerous studies. Transgenic mouse models have been instrumental to study amyloidosis, but observations might have been confounded by APP-overexpression artifacts. The current study investigated early functional defects in an APP knock-in mouse model, which allows assessing the effects of pathological amyloid-beta (A?) without interference of APP-artifacts. Female APPNL/NL knock-in mice of 3 and 7 months old were compared to age-matched APPNL-F/NL-F mice with increased A?42/40 ratio and initial A?-plaque deposition around 6 months of age. Spatial learning was examined using a Morris water maze protocol consisting of acquisition and reversal trials interleaved with reference memory tests. Functional connectivity (FC) of brain networks was assessed using resting-state functional MRI (rsfMRI). The Morris water maze data revealed that 3 months old APPNL-F/NL-F mice were unable to reach the same reference memory proficiency as APPNL/NL mice after reversal training. This cognitive defect in 3-month-old APPNL-F/NL-F mice coincided with hypersynchronous FC of the hippocampal, cingulate, caudate-putamen, and default-mode-like networks. The occurrence of these defects in APPNL-F/NL-F mice demonstrates that cognitive flexibility and synchronicity of telencephalic activity are specifically altered by early A? pathology without changes in APP neurochemistry.