Faculty Bibliography

Emerging evidence suggests that aberrant phosphorylation of eukaryotic initiation factor-2alpha (eIF2alpha) may induce synaptic failure and neurodegeneration through persistent translational inhibition of global protein synthesis. However, elevated phospho-eIF2alpha also paradoxically causes translational activation of a subset of messenger RNAs such as the beta-secretase enzyme, beta-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 eIF2alpha kinase PERK may prevent these deleterious events and mitigate Alzheimer's disease (AD)-like neuropathology and cognitive impairments in the 5XFAD mouse model. PERK haploinsufficiency blocked overactivation of the PERK-eIF2alpha pathway, as evidenced by significant reductions in phosphorylation of PERK and eIF2alpha, 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-beta 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.

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.

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.

Cell signaling in response to an array of diverse stress stimuli converges on the phosphorylation of eukaryotic initiation factor-2alpha (eIF2alpha). Evidence is accumulating that persistent eIF2alpha 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 eIF2alpha kinases, have enabled us to investigate pathogenic roles of dysregulated eIF2alpha phosphorylation pathways. This review provides an overview of animal model studies in this field, focusing particularly on molecular mechanisms by which the dysregulation of eIF2alpha kinases may account for synaptic and memory deficits associated with AD. A growing body of evidence suggests that correcting aberrant eIF2alpha kinase activities may serve as disease-modifying therapeutic interventions to treat AD and related cognitive disorders.

Emerging evidence suggests that elevated hippocampal activation may be important for disrupting cognitive functions in aged subjects as well as patients with Alzheimer's 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.

Emerging evidence suggests that dysregulated translation through phosphorylation of eukaryotic initiation factor-2alpha (eIF2alpha) may contribute to Alzheimer's disease (AD) and related memory impairments. However, the underlying mechanisms remain unclear. Here, we crossed knockout mice for an eIF2alpha 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 beta-secretase enzyme BACE1 and the CREB repressor ATF4 concomitant with a dramatic increase of eIF2alpha 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 beta-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 eIF2alpha 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 eIF2alpha kinase under the physiological condition, whereas blocking the GCN2 pathway under exposure to significant beta-amyloidosis rather aggravates eIF2alpha 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-beta (Abeta) 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 beta-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 beta-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.

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 beta-secretase enzyme (BACE1) that initiates amyloid-beta (Abeta) generation, as observed in sporadic AD. Interestingly, 7,8-DHF blocked BACE1 elevations and lowered levels of the beta-secretase-cleaved C-terminal fragment of amyloid precursor protein (C99), Abeta40, and Abeta42 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 beta-amyloidogenesis.

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 beta-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-beta peptides (Abeta40 and Abeta42) were significantly increased in STZ-treated 5XFAD mice as compared with vehicle-treated 5XFAD controls. Importantly, STZ-induced insulin deficiency upregulated levels of both beta-site APP cleaving enzyme 1 (BACE1) and full-length APP in 5XFAD mouse brains, which was accompanied by dramatic elevations in the beta-cleaved C-terminal fragment (C99). Interestingly, BACE1 mRNA levels were not affected, whereas phosphorylation of the translation initiation factor eIF2alpha, 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 Abeta-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 beta-amyloidogenesis via the translational upregulation of BACE1 in combination with elevations in its substrate, APP.