Antonella Caccamo

Triple-Transgenic Model of Alzheimer's Disease with Plaques and Tangles: Intracellular Aβ and Synaptic Dysfunction

The neuropathological correlates of Alzheimers disease (AD) include amyloid-beta (Abeta) plaques and neurofibrillary tangles. To study the interaction between Abeta and tau and their effect on synaptic function, we derived a triple-transgenic model (3xTg-AD) harboring PS1(M146V), APP(Swe), and tau(P301L) transgenes. Rather than crossing independent lines, we microinjected two transgenes into single-cell embryos from homozygous PS1(M146V) knockin mice, generating mice with the same genetic background. 3xTg-AD mice progressively develop plaques and tangles. Synaptic dysfunction, including LTP deficits, manifests in an age-related manner, but before plaque and tangle pathology. Deficits in long-term synaptic plasticity correlate with the accumulation of intraneuronal Abeta. These studies suggest a novel pathogenic role for intraneuronal Abeta with regards to synaptic plasticity. The recapitulation of salient features of AD in these mice clarifies the relationships between Abeta, synaptic dysfunction, and tangles and provides a valuable model for evaluating potential AD therapeutics as the impact on both lesions can be assessed.

Amyloid deposition precedes tangle formation in a triple transgenic model of Alzheimer’s disease

Amyloid-beta (Abeta) containing plaques and tau-laden neurofibrillary tangles are the defining neuropathological features of Alzheimers disease (AD). To better mimic this neuropathology, we generated a novel triple transgenic model of AD (3xTg-AD) harboring three mutant genes: beta-amyloid precursor protein (betaAPPSwe), presenilin-1 (PS1M146V), and tauP301L. The 3xTg-AD mice progressively develop Abeta and tau pathology, with a temporal- and regional-specific profile that closely mimics their development in the human AD brain. We find that Abeta deposits initiate in the cortex and progress to the hippocampus with aging, whereas tau pathology is first apparent in the hippocampus and then progresses to the cortex. Despite equivalent overexpression of the human betaAPP and human tau transgenes, Abeta deposition develops prior to the tangle pathology, consistent with the amyloid cascade hypothesis. As these 3xTg-AD mice phenocopy critical aspects of AD neuropathology, this model will be useful in pre-clinical intervention trials, particularly because the efficacy of anti-AD compounds in mitigating the neurodegenerative effects mediated by both signature lesions can be evaluated.

Molecular interplay between mammalian target of rapamycin (mTOR), amyloid-β, and Tau: Effects on cognitive impairments

Accumulation of amyloid-β (Aβ) and Tau is an invariant feature of Alzheimer disease (AD). The upstream role of Aβ accumulation in the disease pathogenesis is widely accepted, and there is strong evidence showing that Aβ accumulation causes cognitive impairments. However, the molecular mechanisms linking Aβ to cognitive decline remain to be elucidated. Here we show that the buildup of Aβ increases the mammalian target of rapamycin (mTOR) signaling, whereas decreasing mTOR signaling reduces Aβ levels, thereby highlighting an interrelation between mTOR signaling and Aβ. The mTOR pathway plays a central role in controlling protein homeostasis and hence, neuronal functions; indeed mTOR signaling regulates different forms of learning and memory. Using an animal model of AD, we show that pharmacologically restoring mTOR signaling with rapamycin rescues cognitive deficits and ameliorates Aβ and Tau pathology by increasing autophagy. Indeed, we further show that autophagy induction is necessary for the rapamycin-mediated reduction in Aβ levels. The results presented here provide a molecular basis for the Aβ-induced cognitive deficits and, moreover, show that rapamycin, an FDA approved drug, improves learning and memory and reduces Aβ and Tau pathology.Accumulation of amyloid-beta (Abeta) and Tau is an invariant feature of Alzheimer disease (AD). The upstream role of Abeta accumulation in the disease pathogenesis is widely accepted, and there is strong evidence showing that Abeta accumulation causes cognitive impairments. However, the molecular mechanisms linking Abeta to cognitive decline remain to be elucidated. Here we show that the buildup of Abeta increases the mammalian target of rapamycin (mTOR) signaling, whereas decreasing mTOR signaling reduces Abeta levels, thereby highlighting an interrelation between mTOR signaling and Abeta. The mTOR pathway plays a central role in controlling protein homeostasis and hence, neuronal functions; indeed mTOR signaling regulates different forms of learning and memory. Using an animal model of AD, we show that pharmacologically restoring mTOR signaling with rapamycin rescues cognitive deficits and ameliorates Abeta and Tau pathology by increasing autophagy. Indeed, we further show that autophagy induction is necessary for the rapamycin-mediated reduction in Abeta levels. The results presented here provide a molecular basis for the Abeta-induced cognitive deficits and, moreover, show that rapamycin, an FDA approved drug, improves learning and memory and reduces Abeta and Tau pathology.

Temporal Profile of Amyloid-β (Aβ) Oligomerization in an in Vivo Model of Alzheimer Disease A LINK BETWEEN Aβ AND TAU PATHOLOGY

Accumulation of amyloid-β (Aβ) is one of the earliest molecular events in Alzheimer disease (AD), whereas tau pathology is thought to be a later downstream event. It is now well established that Aβ exists as monomers, oligomers, and fibrils. To study the temporal profile of Aβ oligomer formation in vivo and to determine their interaction with tau pathology, we used the 3xTg-AD mice, which develop a progressive accumulation of plaques and tangles and cognitive impairments. We show that SDS-resistant Aβ oligomers accumulate in an age-dependent fashion, and we present evidence to show that oligomerization of Aβ appears to first occur intraneuronally. Finally, we show that a single intrahippocampal injection of a specific oligomeric antibody is sufficient to clear Aβ pathology, and more importantly, tau pathology. Therefore, Aβ oligomers may play a role in the induction of tau pathology, making the interference of Aβ oligomerization a valid therapeutic target.

M1 Receptors Play a Central Role in Modulating AD-like Pathology in Transgenic Mice

We investigated the therapeutic efficacy of the selective M1 muscarinic agonist AF267B in the 3xTg-AD model of Alzheimer disease. AF267B administration rescued the cognitive deficits in a spatial task but not contextual fear conditioning. The effect of AF267B on cognition predicted the neuropathological outcome, as both the Abeta and tau pathologies were reduced in the hippocampus and cortex, but not in the amygdala. The mechanism underlying the effect on the Abeta pathology was caused by the selective activation of ADAM17, thereby shifting APP processing toward the nonamyloidogenic pathway, whereas the reduction in tau pathology is mediated by decreased GSK3beta activity. We further demonstrate that administration of dicyclomine, an M1 antagonist, exacerbates the Abeta and tau pathologies. In conclusion, AF267B represents a peripherally administered low molecular weight compound to attenuate the major hallmarks of AD and to reverse deficits in cognition. Therefore, selective M1 agonists may be efficacious for the treatment of AD.

Reduction of Soluble Aβ and Tau, but Not Soluble Aβ Alone, Ameliorates Cognitive Decline in Transgenic Mice with Plaques and Tangles

Increasing evidence points to soluble assemblies of aggregating proteins as a major mediator of neuronal and synaptic dysfunction. In Alzheimer disease (AD), soluble amyloid-β (Aβ) appears to be a key factor in inducing synaptic and cognitive abnormalities. Here we report the novel finding that soluble tau also plays a role in the cognitive decline in the presence of concomitant Aβ pathology. We describe improved cognitive function following a reduction in both soluble Aβ and tau levels after active or passive immunization in advanced aged 3xTg-AD mice that contain both amyloid plaques and neurofibrillary tangles (NFTs). Notably, reducing soluble Aβ alone did not improve the cognitive phenotype in mice with plaques and NFTs. Our results show that Aβ immunotherapy reduces soluble tau and ameliorates behavioral deficit in old transgenic mice.

Age- and region-dependent alterations in Aβ-degrading enzymes: implications for Aβ-induced disorders

Accumulation of amyloid β-protein (Aβ) is a fundamental feature of certain human brain disorders such as Alzheimers disease (AD) and Down syndrome and also of the skeletal muscle disorder inclusion body myositis (IBM). Emerging evidence suggests that the steady-state levels of Aβ are determined by the balance between production and degradation. Although the proteolytic processes leading to Aβ formation have been extensively studied, less is known about the proteases that degrade Aβ, which include insulin-degrading enzyme (IDE) and neprilysin (NEP). Here we measured the steady-state levels of these proteases as a function of age and brain/muscle region in mice and humans. In the hippocampus, which is vulnerable to AD pathology, IDE and NEP steady-state levels diminish as function of age. By contrast, in the cerebellum, a brain region not marked by significant Aβ accumulation, NEP and IDE levels either increase or remain unaltered during aging. Moreover, the steady-state levels of IDE and NEP are significantly higher in the cerebellum compared to the cortex and hippocampus. We further show that IDE is more oxidized in the hippocampus compared to the cerebellum of AD patients. In muscle, we find differential levels of IDE and NEP in fast versus slow twitch muscle fibers that varies with aging. These findings suggest that age- and region-specific changes in the proteolytic clearance of Aβ represent a critical pathogenic mechanism that may account for the susceptibility of particular brain or muscle regions in AD and IBM.

Enhanced Ryanodine Receptor Recruitment Contributes to Ca2+ Disruptions in Young, Adult, and Aged Alzheimer's Disease Mice

Neuronal Ca2+ signaling through inositol triphosphate receptors (IP3R) and ryanodine receptors (RyRs) must be tightly regulated to maintain cell viability, both acutely and over a lifetime. Exaggerated intracellular Ca2+ levels have been associated with expression of Alzheimers disease (AD) mutations in young mice, but little is known of Ca2+ dysregulations during normal and pathological aging processes. Here, we used electrophysiological recordings with two-photon imaging to study Ca2+ signaling in nontransgenic (NonTg) and several AD mouse models (PS1KI, 3xTg-AD, and APPSweTauP301L) at young (6 week), adult (6 months), and old (18 months) ages. At all ages, the PS1KI and 3xTg-AD mice displayed exaggerated endoplasmic reticulum (ER) Ca2+ signals relative to NonTg mice. The PS1 mutation was the predominant “calciopathic” factor, because responses in 3xTg-AD mice were similar to PS1KI mice, and APPSweTauP301L mice were not different from controls. In addition, we uncovered powerful signaling interactions and differences between IP3R- and RyR-mediated Ca2+ components in NonTg and AD mice. In NonTg mice, RyR contributed modestly to IP3-evoked Ca2+, whereas the exaggerated signals in 3xTg-AD and PS1KI mice resulted primarily from enhanced RyR-Ca2+ release and were associated with increased RyR expression across all ages. Moreover, IP3-evoked membrane hyperpolarizations in AD mice were even greater than expected from exaggerated Ca2+ signals, suggesting increased coupling efficiency between cytosolic [Ca2+] and K+ channel regulation. We conclude that lifelong ER Ca2+ disruptions in AD are related to a modulation of RyR signaling associated with PS1 mutations and represent a discrete “calciumopathy,” not merely an acceleration of normal aging.

Dysregulated IP3 Signaling in Cortical Neurons of Knock-In Mice Expressing an Alzheimer's-Linked Mutation in Presenilin1 Results in Exaggerated Ca2+ Signals and Altered Membrane Excitability

Disruptions in intracellular Ca2+ signaling are proposed to underlie the pathophysiology of Alzheimers disease (AD), and it has recently been shown that AD-linked mutations in the presenilin 1 gene (PS1) enhance inositol triphosphate (IP3)-mediated Ca2+ liberation in nonexcitable cells. However, little is known of these actions in neurons, which are the principal locus of AD pathology. We therefore sought to determine how PS1 mutations affect Ca2+ signals and their subsequent downstream effector functions in cortical neurons. Using whole-cell patch-clamp recording, flash photolysis, and two-photon imaging in brain slices from 4-5-week-old mice, we show that IP3-evoked Ca2+ responses are more than threefold greater in PS1M146V knock-in mice relative to age-matched nontransgenic controls. Electrical excitability is thereby reduced via enhanced Ca2+ activation of K+ conductances. Action potential-evoked Ca2+ signals were unchanged, indicating that PS1M146V mutations specifically disrupt intracellular Ca2+ liberation rather than reduce cytosolic Ca2+ buffering or clearance. Moreover, IP3 receptor levels are not different in cortical homogenates, further suggesting that the exaggerated cytosolic Ca2+ signals may result from increased store filling and not from increased flux through additional IP3-gated channels. Even in young animals, PS1 mutations have profound effects on neuronal Ca2+ and electrical signaling: cumulatively, these disruptions may contribute to the long-term pathophysiology of AD.

Age-dependent sexual dimorphism in cognition and stress response in the 3xTg-AD mice

We sought to determine if sex impacts the cognitive and neuropathological phenotype of the 3xTg-AD mice. We find that male and female 3xTg-AD mice show comparable impairments on Morris water maze (MWM) and inhibitory avoidance (IA) at 4 months. Shortly thereafter, however, the cognitive performance varies among the sexes, with females performing worse than males. These behavioral differences are not attributable to differences in Abeta or tau levels. The behavioral effect is transient as from 12 months onward, the disparity is no longer apparent. Because females perform worse than males on stressful tasks, we explored their corticosterone responses and find that young female 3xTg-AD mice show markedly heightened corticosterone response after 5 days of MWM training compared to age-matched male 3xTg-AD mice; this difference is no longer apparent in older mice. Thus, the enhanced corticosterone response of the young female mice likely underlies their poorer performance on stressful tasks.