Lauren M. Billings

Intraneuronal Aβ Causes the Onset of Early Alzheimer’s Disease-Related Cognitive Deficits in Transgenic Mice

Progressive memory loss and cognitive dysfunction are the hallmark clinical features of Alzheimers disease (AD). Identifying the molecular triggers for the onset of AD-related cognitive decline presently requires the use of suitable animal models, such as the 3xTg-AD mice, which develop both amyloid and tangle pathology. Here, we characterize the onset of learning and memory deficits in this model. We report that 2-month-old, prepathologic mice are cognitively unimpaired. The earliest cognitive impairment manifests at 4 months as a deficit in long-term retention and correlates with the accumulation of intraneuronal Abeta in the hippocampus and amygdala. Plaque or tangle pathology is not apparent at this age, suggesting that they contribute to cognitive dysfunction at later time points. Clearance of the intraneuronal Abeta pathology by immunotherapy rescues the early cognitive deficits on a hippocampal-dependent task. Reemergence of the Abeta pathology again leads to cognitive deficits. This study strongly implicates intraneuronal Abeta in the onset of cognitive dysfunction.

Aβ immunotherapy leads to clearance of early, but not late, hyperphosphorylated tau aggregates via the proteasome

Amyloid-beta (Abeta) plaques and neurofibrillary tangles are the hallmark neuropathological lesions of Alzheimers disease (AD). Using a triple transgenic model (3xTg-AD) that develops both lesions in AD-relevant brain regions, we determined the consequence of Abeta clearance on the development of tau pathology. Here we show that Abeta immunotherapy reduces not only extracellular Abeta plaques but also intracellular Abeta accumulation and most notably leads to the clearance of early tau pathology. We find that Abeta deposits are cleared first and subsequently reemerge prior to the tau pathology, indicative of a hierarchical and direct relationship between Abeta and tau. The clearance of the tau pathology is mediated by the proteasome and is dependent on the phosphorylation state of tau, as hyperphosphorylated tau aggregates are unaffected by the Abeta antibody treatment. These findings indicate that Abeta immunization may be useful for clearing both hallmark lesions of AD, provided that intervention occurs early in the disease course.

Glucocorticoids Increase Amyloid-β and Tau Pathology in a Mouse Model of Alzheimer’s Disease

Various environmental and genetic factors influence the onset and progression of Alzheimer’s disease (AD). Dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis, which controls circulating levels of glucocorticoid hormones, occurs early in AD, resulting in increased cortisol levels. Disturbances of the HPA axis have been associated with memory impairments and may contribute to the cognitive decline that occurs in AD, although it is unknown whether such effects involve modulation of the amyloid β-peptide (Aβ) and tau. Using in vitro and in vivo experiments, we report that stress-level glucocorticoid administration increases Aβ formation by increasing steady-state levels of amyloid precursor protein (APP) and β-APP cleaving enzyme. Additionally, glucocorticoids augment tau accumulation, indicating that this hormone also accelerates the development of neurofibrillary tangles. These findings suggest that high levels of glucocorticoids, found in AD, are not merely a consequence of the disease process but rather play a central role in the development and progression of AD.

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.

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.

Memantine Improves Cognition and Reduces Alzheimer’s-Like Neuropathology in Transgenic Mice

Memantine is an N-methyl-d-aspartate receptor antagonist that is approved for the treatment of moderate to severe Alzheimers disease (AD). In this study, three groups of triple-transgenic (3xTg-AD) mice with differing levels of AD-like pathology (6, 9, and 15 months of age) were treated for 3 months with doses of memantine equivalent to those used in humans. After the treatment, memantine-treated mice had restored cognition and significantly reduced the levels of insoluble amyloid-beta (Abeta), Abeta dodecamers (Abeta*56), prefibrillar soluble oligomers, and fibrillar oligomers. The effects on pathology were stronger in older, more impaired animals. Memantine treatment also was associated with a decline in the levels of total tau and hyperphosphorylated tau. Finally, memantine pre-incubation prevented Abeta-induced inhibition of long-term potentiation in hippocampal slices of cognitively normal mice. These results suggest that the effects of memantine treatment on AD brain include disease modification and prevention of synaptic dysfunction.

Learning decreases Aβ*56 and Tau pathology and ameliorates behavioral decline in 3xTg-AD mice

Transgenic mouse models of Alzheimers disease (AD), such as the 3xTg-AD mice, are instrumental for elucidating genetic, pharmacologic, environmental, and behavioral factors that affect the cognitive phenotype. Here we present the novel findings that longitudinal water-maze spatial training produces a significant, albeit transient, improvement in subsequent learning performance and reduces amyloid β (Aβ) and tau neuropathology. The 3xTg-AD mice were trained and tested at 3 month intervals from 2 to 18 months. Separate groups of naive mice were also tested at each age. The improvement in performance seen at 6 and 12 months is dependent on spatial training, because animals that were similarly handled and exposed to swimming without a learning contingency failed to show improved performance. Training before the development of overt neuropathology is required for full expression of the training effect because we found it delays Aβ redistribution to extracellular plaques and reduces Aβ oligomers associated with cognitive decline. In addition, learning leads to decreased glycogen synthase kinase-3β activity, which likely underlies the reduced tau pathology. The previous training effects on both maze performance and neuropathology are attenuated at 15 and 18 months. These findings indicate that, in young and middle-aged 3xTg-AD mice, repeated spatial training can significantly delay the development of neuropathology and decline in spatial memory.

Glutamic Acid Decarboxylase 67 mRNA Regulation in Two Globus Pallidus Neuron Populations by Dopamine and the Subthalamic Nucleus

The globus pallidus (GP) consists of two neuron populations, distinguished according to their immunoreactivity for parvalbumin (PV). The PV-immunoreactive (PV+) neurons project preferentially to “downstream” targets such as the subthalamic and entopeduncular nuclei, whereas neurons lacking PV (PV– neurons) project preferentially to the striatum, suggesting a role for PV– cells in feedback to striatal neurons. Although dopamine D2 antagonist administration induces immediate early gene expression preferentially in PV– GP neurons, little is known about long-term regulation of PV– versus PV+ GP neurons. Nigral 6-hydroxydopamine (6-OHDA) lesions or repeated D2-class antagonist injections have been shown to increase pallidal expression of glutamate decarboxylase (GAD67 isoform) mRNA. This increase in GAD67 is believed to be secondary to activation of excitatory subthalamopallidal projections. The current study examined the effects of subthalamic nucleus (STN) lesion on 6-OHDA- or repeated D2 antagonist-induced changes in GP GAD67 mRNA expression in PV+ and PV– neurons. Five or 21 d after nigral 6-OHDA injections or after 3, 7, or 21 d of D2 antagonist administration, GAD67 mRNA increased in both the PV– and PV+ GP neurons, but the magnitude of the increase was significantly greater in PV– neurons. By contrast, STN lesion resulted in declines in GAD67 mRNA in both cell populations, with the decreases in PV+ neurons exceeding those in PV– neurons. Furthermore, STN lesion completely blocked 6-OHDA- or D2 antagonist-induced GAD67 mRNA increases in PV+ cells but only partly offset the GAD67 mRNA increase in PV– pallidal neurons. Thus, the PV+ and PV– neurons are influenced in qualitatively similar ways by dopamine and the STN, but these cell types exhibit contrasting degrees of regulation by the dopaminergic and STN perturbations. This pattern of results has implications for pallidal control of striatal versus downstream basal ganglia nuclei.

D2 antagonist-induced c-fos in an identified subpopulation of globus pallidus neurons by a direct intrapallidal action

Much research now supports the view that the dopaminergic innervation of the globus pallidus external segment (GP) influences basal ganglia information processing via pallidal dopamine (DA) D2, D3, and possibly D1 receptors. Systemic DA agonists, or systemic or intrapallidal dopamine D2-class antagonist administration, can induce immediate early gene expression (IEG) in the rat GP. In view of the distinct chemical phenotypes and axonal projections of the GP neurons, it is important to characterize the population(s) of pallidal neurons responding to local DA manipulations. Parvalbumin (PV) immunostaining was used to identify one of the two principal GP neuron populations. Awake, behaving rats received intrapallidal infusions of the dopamine D2 antagonist sulpiride (50 or 100 ng), the D1-class antagonist SCH-23390 (100 ng), the D2-class agonist quinpirole (500 ng), the GABA(A) antagonist picrotoxin (0.25, 0.5 or 1 microg) or bicuculline (20 ng), the GABA(A) agonist muscimol (15 ng) or vehicle. Intrapallidal GABA manipulations were used to assess the likelihood that the effects of the DAergic drugs on Fos induction occurred secondarily to altering intrapallidal GABA release. Using Fos and PV double immunolabeling procedures, we found that several treatments induced GP Fos, but that intrapallidal sulpiride induced Fos almost exclusively in PV-lacking pallidal neurons. No other intrapallidal drug-induced Fos showed similar population specificity. These results support evidence suggesting that GP DA can play a unique and critical role in modulating pallidal neuron function, and that the cessation of pallidal dopamine transmission can activate gene expression within the pallidal neuron subpopulation that maintains extensive axonal projections to caudate-putamen.

Dopaminergic modulation of pallidal preproenkephalin mRNA

The present study examines dopaminergic regulation of neuropeptide gene expression within a relatively poorly characterized population of cells, the preproenkephalin (PPE) mRNA containing neurons of the globus pallidus (GP). Rats that received 6-hydroxydopamine (6-OHDA) lesions or repeated D1 or D2 antagonist administration were compared to control animals. One month after 6-OHDA lesions, PPE mRNA was elevated in the GP ipsilateral to the lesion, with a smaller elevation also being observed in the contralateral GP. Repeated administration of eticlopride, but not SCH 23390, also resulted in elevated PPE mRNA expression in the GP. These data reveal a novel effect of decreased dopamine transmission on the GP, and draw attention to this subpopulation of pallidal neurons.