Latha Devi

7,8-Dihydroxyflavone, a Small-Molecule TrkB Agonist, Reverses Memory Deficits and BACE1 Elevation in a Mouse Model of Alzheimer's Disease

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 Alzheimers 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.

Mechanisms underlying insulin deficiency-induced acceleration of β-amyloidosis in a mouse model of Alzheimer's disease.

Although evidence is accumulating that diabetes mellitus is an important risk factor for sporadic Alzheimers 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.

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 Alzheimers 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.

PERK mediates eIF2α phosphorylation responsible for BACE1 elevation, CREB dysfunction and neurodegeneration in a mouse model of Alzheimer's disease

Emerging evidence suggests that aberrant phosphorylation of eukaryotic initiation factor-2α (eIF2α) may induce synaptic failure and neurodegeneration through persistent translational inhibition of global protein synthesis. However, elevated phospho-eIF2α also paradoxically causes translational activation of a subset of messenger RNAs such as the β-secretase enzyme, β-site APP-cleaving enzyme 1 (BACE1) and cAMP response element binding protein (CREB) repressor, activating transcription factor 4 (ATF4). Therefore, we tested whether genetic reduction of the eIF2α kinase PERK may prevent these deleterious events and mitigate Alzheimers disease (AD)-like neuropathology and cognitive impairments in the 5XFAD mouse model. PERK haploinsufficiency blocked overactivation of the PERK-eIF2α pathway, as evidenced by significant reductions in phosphorylation of PERK and eIF2α, in 5XFAD mice. PERK haploinsufficiency was sufficient to rescue memory deficits and cholinergic neurodegeneration in this AD model. Notably, PERK haploinsufficiency also prevented BACE1 elevations, resulting in reduced levels of amyloid-β peptides and plaque burden in 5XFAD mice. Moreover, CREB dysfunction was restored in PERK(+/-)·5XFAD mice concomitant with reversal of ATF4 upregulation. Together, these findings suggest that PERK may be a disease-modifying therapeutic target to prevent multiple memory-disrupting mechanisms associated with AD.

Sex- and brain region-specific acceleration of β-amyloidogenesis following behavioral stress in a mouse model of Alzheimer's disease.

BackgroundIt is hypothesized that complex interactions between multiple environmental factors and genetic factors are implicated in sporadic Alzheimers 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.ResultsWe 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.ConclusionsOur 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.

Deletion of the eIF2α Kinase GCN2 fails to rescue the memory decline associated with Alzheimer's disease.

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.

Effects of levetiracetam, an antiepileptic drug, on memory impairments associated with aging and Alzheimer's disease in mice.

Emerging evidence suggests that elevated hippocampal activation may be important for disrupting cognitive functions in aged subjects as well as patients with Alzheimers disease (AD). Therefore, reducing deleterious overactivity of the hippocampus may have therapeutic benefits. This study was designed to compare the effects of levetiracetam, an antiepileptic drug, on memory deficits associated with normal aging and AD in mouse models. Pretraining administration of levetiracetam ameliorated memory impairments of aged C57BL/6 mice (17-20months of age) in the contextual fear conditioning paradigm. Acute levetiracetam immediately after training was also efficacious in rescuing contextual memory decline in aged mice, whereas administration at a later posttraining interval (3h) had no effect. These results suggest that suppressing overexcitation with acute levetiracetam around the time of acquisition or early consolidation may be sufficient to reverse memory decline associated with aging. In contrast, pretraining administration of levetiracetam was not able to rescue memory deficits in 5XFAD transgenic mice harboring amyloid plaque pathologies at moderate (6-8months old) or massive (12-15months old) levels, differentiating between normal aging- and AD-related memory impairments in the responsiveness to acute levetiracetam treatment.

Beneficial Effects of the β-Secretase Inhibitor GRL-8234 in 5XFAD Alzheimer’s Transgenic Mice Lessen During Disease Progression

The β-secretase enzyme BACE1, which initiates the cleavage of amyloid precursor protein (APP) into the amyloid-β (Aβ) peptide, is a prime therapeutic target for Alzheimers disease (AD). However, recent investigations using genetic animal models raise concern that therapeutic BACE1 inhibition may encounter the dramatic reduction of efficacy in ameliorating AD-like pathology and memory deficits during disease progression. Here, we compared the effects of the potent and selective small-molecule BACE1 inhibitor GRL-8234 in different pathological stages of AD mouse model. Specifically, we administered GRL-8234 (33.4 mg/kg, i.p.) once daily for 2 months to 5XFAD transgenic mice, which showed modest (4 months) and massive (10 months of age) Aβ plaque deposition at starting points. Chronic treatments with GRL-8234 reversed memory impairments, as tested by the spontaneous alternation Y-maze task, in the younger 5XFAD group concomitant with significant reductions in cerebral Aβ42 levels. In contrast, only marginal reductions of Aβ42 were observed in 12-month-old 5XFAD mice treated with GRL-8234 and their memory function remained impaired. We found that not only BACE1 but also full-length APP expression was significantly elevated with progressive Aβ accumulation in 5XFAD mice, while GRL-8234 failed to affect these detrimental mechanisms that further accelerate plaque growth in brains of older 5XFAD mice. Therefore, our results provide important insights into the mechanisms by which Aβ accumulation and related memory impairments become less responsive to rescue by BACE1 inhibition during the course of AD development.

Cognitive benefits of memantine in Alzheimer's 5XFAD model mice decline during advanced disease stages.

Memantine, a noncompetitive NMDA receptor antagonist with neuroprotective properties, has been used for the treatment of Alzheimers disease (AD). Administration of memantine to various transgenic AD mice has been reported to improve cognitive deficits, very often completely back to normal wild-type control levels. However, such great benefits of memantine in preclinical studies do not translate into clinical results of this drug, showing only marginal and transient efficacy in moderate to severe AD. To further address in vivo efficacy, we compared the effects of memantine at different disease stages in 5XFAD mice, one of the rapid-onset and most aggressive amyloid models. Specifically, we administered memantine once daily for 30 days to 5XFAD mice, which showed moderate (6-7 months of age) and robust (12-15 months) β-amyloid (Aβ) accumulation. Treatments with memantine (10mg/kg, i.p.) reversed memory impairments in the younger 5XFAD mice, as tested by the contextual fear conditioning and spontaneous alternation Y-maze paradigms. Memantine had no effects on soluble Aβ oligomer or total Aβ42 levels in 5XFAD mouse brains. In contrast, subchronic treatments with memantine showed no behavioral benefits in the older 5XFAD group, which exhibited more profound memory deficits concomitant with highly increased concentrations of Aβ as compared with those of the younger 5XFAD group. Since subchronic memantine at the higher dose (30 mg/kg) impaired memory performances in wild-type controls, we further tested acute administration of 50mg/kg memantine, which was reported to enhance hippocampal adult neurogenesis and memory function. However, this treatment also failed to rescue memory deficits in 12-15-month-old 5XFAD mice. Collectively, our results demonstrate that cognitive benefits of memantine independent of Aβ reductions were no longer observed in the 5XFAD Alzheimer mouse model during advanced stages, which may be reflective of the limited efficacy of memantine in clinical settings.