Tau pathogenicity in Alzheimer's disease and other tauopathies is thought to involve the generation of hyperphosphorylated, truncated, and oligomeric tau species with enhanced neurotoxicity, although the generative mechanisms and the implications for disease therapy are not well understood. Here, we report a striking rescue from mutant tau toxicity in the JNPL3 mouse model of tauopathy. We show that pathological activation of calpains gives rise to a range of potentially toxic forms of tau, directly, and by activating cdk5. Calpain overactivation in brains of these mice is accelerated as a result of the marked depletion of the endogenous calpain inhibitor, calpastatin. When levels of this inhibitor are restored in neurons of JNPL3 mice by overexpressing calpastatin, tauopathy is prevented, including calpain-mediated breakdown of cytoskeletal proteins, cdk5 activation, tau hyperphosphorylation, formation of potentially neurotoxic tau fragments by either calpain or caspase-3, and tau oligomerization. Calpastatin overexpression also prevents loss of motor axons, delays disease onset, and extends survival of JNPL3 mice by 3 months to within the range of normal lifespan. Our findings support the therapeutic promise of highly specific calpain inhibition in the treatment of tauopathies and other neurodegenerative states.
ambulatory movements; calcium; fractionation; insoluble extracts; nesting behavior
Trisomy 21, or Down's syndrome (DS), is the most common genetic cause of intellectual disability. Altered neurotransmission in the brains of DS patients leads to hippocampus-dependent learning and memory deficiency. Although genetic mouse models have provided important insights into the genes and mechanisms responsible for DS-specific changes, the molecular mechanisms leading to memory deficits are not clear. We investigated whether the segmental trisomy model of DS, Ts[Rb(12.1716)]2Cje (Ts2), exhibits hippocampal glutamatergic transmission abnormalities and whether these alterations cause behavioral deficits. Behavioral assays demonstrated that Ts2 mice display a deficit in nest building behavior, a measure of hippocampus-dependent nonlearned behavior, as well as dysfunctional hippocampus-dependent spatial memory tested in the object-placement and the Y-maze spontaneous alternation tasks. Magnetic resonance spectra measured in the hippocampi revealed a significantly lower glutamate concentration in Ts2 as compared with normal disomic (2N) littermates. The glutamate deficit accompanied hippocampal NMDA receptor1 (NMDA-R1) mRNA and protein expression level downregulation in Ts2 compared with 2N mice. In concert with these alterations, paired-pulse analyses suggested enhanced synaptic inhibition and/or lack of facilitation in the dentate gyrus of Ts2 compared with 2N mice. Ts2 mice also exhibited disrupted synaptic plasticity in slice recordings of the hippocampal CA1 region. Collectively, these findings imply that deficits in glutamate and NMDA-R1 may be responsible for impairments in synaptic plasticity in the hippocampus associated with behavioral dysfunctions in Ts2 mice. Thus, these findings suggest that glutamatergic deficits have a significant role in causing intellectual disabilities in DS.
Down's syndrome; glutamate; LTP; NMDA receptors; spatial memory
Cell signaling in response to an array of diverse stress stimuli converges on the phosphorylation of eukaryotic initiation factor-2α (eIF2α). Evidence is accumulating that persistent eIF2α phosphorylation at Ser51 through prolonged overactivation of regulatory kinases occurs in neurodegenerative diseases such as Alzheimer’s disease (AD), leading to shutdown of general translation and translational activation of a subset of mRNAs. Recent advances in the development of gene-based strategies and bioavailable inhibitors, which specifically target one of the eIF2α kinases, have enabled us to investigate pathogenic roles of dysregulated eIF2α phosphorylation pathways. This review provides an overview of animal model studies in this field, focusing particularly on molecular mechanisms by which the dysregulation of eIF2α kinases may account for synaptic and memory deficits associated with AD. A growing body of evidence suggests that correcting aberrant eIF2α kinase activities may serve as disease-modifying therapeutic interventions to treat AD and related cognitive disorders.
Alzheimer’s disease; elF2α; PERK; PKR; BACE1; amyloid-β; ATF4; learning and memory
Emerging evidence suggests that dysregulated translation through phosphorylation of eukaryotic initiation factor-2α (eIF2α) may contribute to Alzheimer’s disease (AD) and related memory impairments. However, the underlying mechanisms remain unclear. Here, we crossed knockout mice for an eIF2α kinase (GCN2: general control nonderepressible-2 kinase) with 5XFAD transgenic mice, and investigated whether GCN2 deletion affects AD-like traits in this model. As observed in AD brains, 5XFAD mice recapitulated significant elevations in the β-secretase enzyme BACE1 and the CREB repressor ATF4 concomitant with a dramatic increase of eIF2α phosphorylation. Contrary to expectation, we found that GCN2−/− and GCN2+/− deficiencies aggravate rather than suppress hippocampal BACE1 and ATF4 elevations in 5XFAD mice, failing to rescue memory deficits as tested by the contextual fear conditioning. The facilitation of these deleterious events resulted in exacerbated β-amyloid accumulation, plaque pathology and CREB dysfunction in 5XFAD mice with GCN2 mutations. Notably, GCN2 deletion caused overactivation of the PKR-endoplasmic reticulum-related kinase (PERK)-dependent eIF2α phosphorylation pathway in 5XFAD mice in the absence of changes in the PKR pathway. Moreover, PERK activation in response to GCN2 deficiency was specific to 5XFAD mice, since phosphorylated PERK levels were equivalent between GCN2−/− and wild-type control mice. Our findings suggest that GCN2 may be an important eIF2α kinase under the physiological condition, whereas blocking the GCN2 pathway under exposure to significant β-amyloidosis rather aggravates eIF2α phosphorylation leading to BACE1 and ATF4 elevations in AD.
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.
Alzheimer’s disease; Mitochondria; C99; Amyloid precursor protein; β-Secretase; BACE1; Mitochondrial dysfunction; Oxidative DNA damage; Learning and memory; 5XFAD
Increasing evidence suggests that reductions in brain-derived neurotrophic factor (BDNF) and its receptor tyrosine receptor kinase B (TrkB) may have a role in the pathogenesis of Alzheimer's disease (AD). However, the efficacy and safety profile of BDNF therapy (eg, gene delivery) remains to be established toward clinical trials. Here, we evaluated the effects of 7,8-dihydroxyflavone (7,8-DHF), a recently identified small-molecule TrkB agonist that can pass the blood–brain barrier, in the 5XFAD transgenic mouse model of AD. 5XFAD mice at 12–15 months of age and non-transgenic littermate controls received systemic administration of 7,8-DHF (5 mg/kg, i.p.) once daily for 10 consecutive days. We found that 7,8-DHF rescued memory deficits of 5XFAD mice in the spontaneous alternation Y-maze task. 5XFAD mice showed impairments in the hippocampal BDNF-TrkB pathway, as evidenced by significant reductions in BDNF, TrkB receptors, and phosphorylated TrkB. 7,8-DHF restored deficient TrkB signaling in 5XFAD mice without affecting endogenous BDNF levels. Meanwhile, 5XFAD mice exhibited elevations in the β-secretase enzyme (BACE1) that initiates amyloid-β (Aβ) generation, as observed in sporadic AD. Interestingly, 7,8-DHF blocked BACE1 elevations and lowered levels of the β-secretase-cleaved C-terminal fragment of amyloid precursor protein (C99), Aβ40, and Aβ42 in 5XFAD mouse brains. Furthermore, BACE1 expression was decreased by 7,8-DHF in wild-type mice, suggesting that BDNF-TrkB signaling is also important for downregulating baseline levels of BACE1. Together, our findings indicate that TrkB activation with systemic 7,8-DHF can ameliorate AD-associated memory deficits, which may be, at least in part, attributable to reductions in BACE1 expression and β-amyloidogenesis.
TrkB agonist; 7,8-dihydroxyflavone; BDNF; Alzheimer's disease; learning and memory; BACE1; Alzheimer's disease; animal models; learning and memory; neurochemistry; TrkB agonist; 7,8-dihydroxyflavone; BDNF; BACE1
The extensive autophagic-lysosomal pathology in Alzheimer disease (AD) brain has revealed a major defect in the proteolytic clearance of autophagy substrates. Autophagy failure contributes on several levels to AD pathogenesis and has become an important therapeutic target for AD and other neurodegenerative diseases. We recently observed broad therapeutic effects of stimulating autophagic-lysosomal proteolysis in the TgCRND8 mouse model of AD that exhibits defective proteolytic clearance of autophagic substrates, robust intralysosomal amyloid-β peptide (Aβ) accumulation, extracellular β-amyloid deposition and cognitive deficits. By genetically deleting the lysosomal cysteine protease inhibitor, cystatin B (CstB), to selectively restore depressed cathepsin activities, we substantially cleared Aβ, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Aβ40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Aβ clearance. Most importantly, lysosomal remediation prevented the marked learning and memory deficits in TgCRND8 mice. Our findings underscore the pathogenic significance of autophagic-lysosomal dysfunction in AD and demonstrate the value of reversing this dysfunction as an innovative therapeutic strategy for AD.
autophagy; lysosome; cathepsin; cystatin B; proteolysis; Alzheimer disease; transgenic
Although evidence is accumulating that diabetes mellitus is an important risk factor for sporadic Alzheimer's disease (AD), the mechanisms by which defects in insulin signaling may lead to the acceleration of AD progression remain unclear. In this study, we applied streptozotocin (STZ) to induce experimental diabetes in AD transgenic mice (5XFAD model) and investigated how insulin deficiency affects the β-amyloidogenic processing of amyloid precursor protein (APP). Two and half months after 5XFAD mice were treated with STZ (90 mg/kg, i.p., once daily for two consecutive days), they showed significant reductions in brain insulin levels without changes in insulin receptor expression. Concentrations of cerebral amyloid-β peptides (Aβ40 and Aβ42) were significantly increased in STZ-treated 5XFAD mice as compared with vehicle-treated 5XFAD controls. Importantly, STZ-induced insulin deficiency upregulated levels of both β-site APP cleaving enzyme 1 (BACE1) and full-length APP in 5XFAD mouse brains, which was accompanied by dramatic elevations in the β-cleaved C-terminal fragment (C99). Interestingly, BACE1 mRNA levels were not affected, whereas phosphorylation of the translation initiation factor eIF2α, a mechanism proposed to mediate the post-transcriptional upregulation of BACE1, was significantly elevated in STZ-treated 5XFAD mice. Meanwhile, levels of GGA3, an adapter protein responsible for sorting BACE1 to lysosomal degradation, are indistinguishable between STZ- and vehicle-treated 5XFAD mice. Moreover, STZ treatments did not affect levels of Aβ-degrading enzymes such as neprilysin and insulin-degrading enzyme (IDE) in 5XFAD brains. Taken together, our findings provide a mechanistic foundation for a link between diabetes and AD by demonstrating that insulin deficiency may change APP processing to favor β-amyloidogenesis via the translational upregulation of BACE1 in combination with elevations in its substrate, APP.
Autophagy, a major degradative pathway for proteins and organelles, is essential for survival of mature neurons. Extensive autophagic-lysosomal pathology in Alzheimer’s disease brain contributes to Alzheimer’s disease pathogenesis, although the underlying mechanisms are not well understood. Here, we identified and characterized marked intraneuronal amyloid-β peptide/amyloid and lysosomal system pathology in the Alzheimer’s disease mouse model TgCRND8 similar to that previously described in Alzheimer’s disease brains. We further establish that the basis for these pathologies involves defective proteolytic clearance of neuronal autophagic substrates including amyloid-β peptide. To establish the pathogenic significance of these abnormalities, we enhanced lysosomal cathepsin activities and rates of autophagic protein turnover in TgCRND8 mice by genetically deleting cystatin B, an endogenous inhibitor of lysosomal cysteine proteases. Cystatin B deletion rescued autophagic-lysosomal pathology, reduced abnormal accumulations of amyloid-β peptide, ubiquitinated proteins and other autophagic substrates within autolysosomes/lysosomes and reduced intraneuronal amyloid-β peptide. The amelioration of lysosomal function in TgCRND8 markedly decreased extracellular amyloid deposition and total brain amyloid-β peptide 40 and 42 levels, and prevented the development of deficits of learning and memory in fear conditioning and olfactory habituation tests. Our findings support the pathogenic significance of autophagic-lysosomal dysfunction in Alzheimer’s disease and indicate the potential value of restoring normal autophagy as an innovative therapeutic strategy for Alzheimer’s disease.
autophagy; lysosome; cystatin B; cathepsin; Alzheimer’s disease
β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) initiates amyloid-β (Aβ) generation that is central to the pathophysiology of Alzheimer’s disease (AD). Therefore, lowering Aβ levels by BACE1 manipulations represents a key therapeutic strategy, but it remains unclear whether partial inhibition of BACE1, as expected for AD treatments, can improve memory deficits. In this study, we used heterozygous BACE1 gene knockout (BACE1+/−) mice to evaluate the effects of partial BACE1 suppression on different types of synaptic and cognitive dysfunctions in Alzheimer’s transgenic mice (5XFAD model). We found that ~50% BACE1 reductions rescued deficits of 5XFAD mice not only in hippocampus-dependent memories as tested by contextual fear conditioning and spontaneous alternation Y-maze paradigms but also in cortex-dependent remote memory stabilization during 30 days after contextual conditioning. Furthermore, 5XFAD-associated impairments in long-term potentiation (a synaptic model of learning and memory) and declines in synaptic plasticity/learning-related BDNF-TrkB signaling pathways were prevented in BACE1+/−·5XFAD mice. Finally, these improvements were related with reduced levels of β-secretase-cleaved C-terminal fragment (C99), Aβ peptides and plaque burden in relevant brain regions of BACE1+/−·5XFAD mice. Therefore, our findings provide compelling evidence for beneficial effects of partially BACE1-inhibiting approaches on multiple forms of functional defects associated with AD.
β-secretase; knockout; amyloid-β; fear conditioning; long-term potentiation; brain-derived neurotrophic factor
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 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 APP/PS1 transgenic mice that harbor five familial AD (FAD) mutations. While both types of memory were significantly impaired in 5XFAD mice, the onset of CTA memory deficits (~9 months of age) was delayed compared to that of contextual memory deficits (~6 months of age). Furthermore, 5XFAD mice genetically engineered to have reduced levels of β-site APP-cleaving enzyme 1 (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 critically involved in CTA performance, was also reduced in BACE1+/−·5XFAD mice compared to 5XFAD mice. Our findings indicate that the CTA paradigm is useful for evaluating a hippocampus-independent form of memory defects in AD model mice, which is sensitive to rescue by partial reductions of the β-secretase BACE1 and consequently of cerebral Aβ.
Alzheimer’s disease; β-secretase; knockout; implicit memory; APP transgenic
It is hypothesized that complex interactions between multiple environmental factors and genetic factors are implicated in sporadic Alzheimer's disease (AD); however, the underlying mechanisms are poorly understood. Importantly, recent evidence reveals that expression and activity levels of the β-site APP cleaving enzyme 1 (BACE1), which initiates amyloid-β (Aβ) production, are elevated in AD brains. In this study, we investigated a molecular mechanism by which sex and stress interactions may accelerate β-amyloidogenesis and contribute to sporadic AD.
We applied 5-day restraint stress (6 h/day) to the male and female 5XFAD transgenic mouse model of AD at the pre-pathological stage of disease, which showed little amyloid deposition under non-stressed control conditions. Exposure to the relatively brief behavioral stress increased levels of neurotoxic Aβ42 peptides, the β-secretase-cleaved C-terminal fragment (C99) and plaque burden in the hippocampus of female 5XFAD mice but not in that of male 5XFAD mice. In contrast, significant changes in the parameters of β-amyloidosis were not observed in the cerebral cortex of stressed male or female 5XFAD mice. We found that this sex- and brain region-specific acceleration of β-amyloidosis was accounted for by elevations in BACE1 and APP levels in response to adverse stress. Furthermore, not only BACE1 mRNA but also phosphorylation of the translation initiation factor eIF2α (a proposed mediator of the post-transcriptional upregulation of BACE1) was elevated in the hippocampus of stressed female 5XFAD mice.
Our results suggest that the higher prevalence of sporadic AD in women may be attributable to the vulnerability of female brains (especially, the hippocampus) to stressful events, which alter APP processing to favor the β-amyloidogenesis through the transcriptional and translational upregulation of BACE1 combined with elevations in its substrate APP.
Previous studies have demonstrated that the formation of spatial, contextual and trace conditioning memories are impaired in animal models of Alzheimer’s disease (AD), consistent with the observations that the first sign of cognitive decline in AD includes difficulties in the acquisition of new information or memory formation. Evidence is accumulating that memory retrieval is a dynamic process in which stored information becomes labile again and needs to be restabilized. However, it is poorly understood how this process referred to as memory reconsolidation is affected in animal models of AD. The present study was designed to use contextual fear conditioning to compare the changes in memory formation and subsequent reconsolidation processes in transgenic mice that overexpress human APP and PS1 harboring five familial AD mutations (5XFAD model). The results clearly demonstrate that cognitive dysfunction starts to occur primarily as reduced levels of contextual learning or memory formation in 5XFAD mice, but it is exacerbated by additional retrieval-dependent retrograde amnesia due to deficient reconsolidation as disease further develops.
Alzheimer’s disease; amyloid; fear conditioning; reconsolidation; amnesia; APP transgenic; 5XFAD mice
β-Site APP-cleaving enzyme 1 (BACE1) initiates amyloid-β (Aβ) generation and thus represents a prime therapeutic target in treating Alzheimer's disease (AD). Notably, increasing evidence indicates that BACE1 levels become elevated in AD brains as disease progresses; however, it remains unclear how the BACE1 upregulation may affect efficacies of therapeutic interventions including BACE1-inhibiting approaches. Here, we crossed heterozygous BACE1 knockout mice with AD transgenic mice (5XFAD model) and compared the abilities of partial BACE1 reduction to rescue AD-like phenotypes at earlier (6-month-old) and advanced (15–18-month-old) stages of disease, which expressed normal (∼100%) and elevated (∼200%) levels of BACE1, respectively. BACE1+/− deletion rescued memory deficits as tested by the spontaneous alternation Y-maze task in 5XFAD mice at the earlier stage and prevented their septohippocampal cholinergic deficits associated with significant neuronal loss. Importantly, BACE1+/− deletion was no longer able to rescue memory deficits or cholinergic neurodegeneration in 5XFAD mice at the advanced stage. Moreover, BACE1+/− deletion significantly reduced levels of Aβ42 and the β-secretase-cleaved C-terminal fragment (C99) in 6-month-old 5XFAD mouse brains, while these neurotoxic β-cleavage products dramatically elevated with age and were not affected by BACE1+/− deletion in 15–18-month-old 5XFAD brains. Interestingly, although BACE1+/− deletion lowered BACE1 expression by ∼50% in 5XFAD mice irrespective of age in concordance with the reduction in gene copy number, BACE1 equivalent to wild-type controls remained in BACE1+/−·5XFAD mice at the advanced age. In accord, phosphorylation of the translation initiation factor eIF2α, an important mediator of BACE1 elevation, was dramatically increased (∼9-fold) in 15–18-month-old 5XFAD mice and remained highly upregulated (∼6-fold) in age-matched BACE1+/−·5XFAD mice. Together, our results indicate that partial reduction of BACE1 is not sufficient to block the phospho-eIF2α-dependent BACE1 elevation during the progression of AD, thus limiting its abilities to reduce cerebral Aβ/C99 levels and rescue memory deficits and cholinergic neurodegeneration.
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.
Alzheimer’s disease; amyloid; synaptic plasticity; LTP; hippocampus; cognition; remote memory; fear conditioning; transgenic; 5XFAD mice
Evidence suggests that β-amyloid (Aβ) peptide triggers a pathogenic cascade leading to neuronal loss in Alzheimer’s disease (AD). However, the causal link between Aβ 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 β-secretase (BACE1) not only abrogates Aβ 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 Aβ ultimately is responsible for neuron death in AD and validate the therapeutic potential of BACE1-inhibiting approaches for the treatment of AD.
Alzheimer’s disease; β-amyloid (Aβ); β-secretase; BACE1 knockout; Tg6799; gliosis; cognitive impairment; spontaneous alternation; Y-maze