Carlos A. Saura

Loss of Presenilin Function Causes Impairments of Memory and Synaptic Plasticity Followed by Age-Dependent Neurodegeneration

Mutations in presenilins are the major cause of familial Alzheimers disease, but the pathogenic mechanism by which presenilin mutations cause memory loss and neurodegeneration remains unclear. Here we demonstrate that conditional double knockout mice lacking both presenilins in the postnatal forebrain exhibit impairments in hippocampal memory and synaptic plasticity. These deficits are associated with specific reductions in NMDA receptor-mediated responses and synaptic levels of NMDA receptors and alphaCaMKII. Furthermore, loss of presenilins causes reduced expression of CBP and CREB/CBP target genes, such as c-fos and BDNF. With increasing age, mutant mice develop striking neurodegeneration of the cerebral cortex and worsening impairments of memory and synaptic function. Neurodegeneration is accompanied by increased levels of the Cdk5 activator p25 and hyperphosphorylated tau. These results define essential roles and molecular targets of presenilins in synaptic plasticity, learning and memory, and neuronal survival in the adult cerebral cortex.

APP Processing and Synaptic Plasticity in Presenilin-1 Conditional Knockout Mice

We have developed a presenilin-1 (PS1) conditional knockout mouse (cKO), in which PS1 inactivation is restricted to the postnatal forebrain. The PS1 cKO mouse is viable and exhibits no gross abnormalities. The carboxy-terminal fragments of the amyloid precursor protein differentially accumulate in the cerebral cortex of cKO mice, while generation of beta-amyloid peptides is reduced. Expression of Notch downstream effector genes, Hes1, Hes5, and Dll1, is unaffected in the cKO cortex. Although basal synaptic transmission, long-term potentiation, and long-term depression at hippocampal area CA1 synapses are normal, the PS1 cKO mice exhibit subtle but significant deficits in long-term spatial memory. These results demonstrate that inactivation of PS1 function in the adult cerebral cortex leads to reduced Abeta generation and subtle cognitive deficits without affecting expression of Notch downstream genes.

Conditional Inactivation of Presenilin 1 Prevents Amyloid Accumulation and Temporarily Rescues Contextual and Spatial Working Memory Impairments in Amyloid Precursor Protein Transgenic Mice

Accumulation of β-amyloid (Aβ) peptides in the cerebral cortex is considered a key event in the pathogenesis of Alzheimers disease (AD). Presenilin 1 (PS1) plays an essential role in the γ-secretase cleavage of the amyloid precursor protein (APP) and the generation of Aβ peptides. Reduction of Aβ generation via the inhibition of γ-secretase activity, therefore, has been proposed as a therapeutic approach for AD. In this study, we examined whether genetic inactivation of PS1 in postnatal forebrain-restricted conditional knock-out (PS1 cKO) mice can prevent the accumulation of Aβ peptides and ameliorate cognitive deficits exhibited by an amyloid mouse model that overexpresses human mutant APP. We found that conditional inactivation of PS1 in APP transgenic mice (PS1 cKO;APP Tg) effectively prevented the accumulation of Aβ peptides and formation of amyloid plaques and inflammatory responses, although it also caused an age-related accumulation of C-terminal fragments of APP. Short-term PS1 inactivation in young PS1 cKO;APP Tg mice rescued deficits in contextual fear conditioning and serial spatial reversal learning in a water maze, which were associated with APP Tg mice. Longer-term PS1 inactivation in older PS1 cKO;APP Tg mice, however, failed to rescue the contextual memory and hippocampal synaptic deficits and had a decreasing ameliorative effect on the spatial memory impairment. These results reveal that in vivo reduction of Aβ via the inactivation of PS1 effectively prevents amyloid-associated neuropathological changes and can, but only temporarily, improve cognitive impairments in APP transgenic mice.

The role of CREB signaling in Alzheimer's disease and other cognitive disorders.

Abstract Gene expression changes in the brain affect cognition during normal and pathological aging. Progress in understanding the cellular processes regulating gene expression networks in cognition is relevant to develop therapeutic interventions for age-related cognitive disorders. Synaptic efficacy mediating memory storage requires the activation of specific gene expression programs regulated, among others, by the transcription factor cAMP-response element binding protein (CREB). CREB signaling is essential for long-lasting changes in synaptic plasticity that mediates the conversion of short-term memory to long-term memory. CREB signaling has been recently involved in several brain pathological conditions including cognitive and neurodegenerative disorders. The β-amyloid (Aβ) peptide, which plays a crucial role in the pathogenesis of Alzheimer’s disease, alters hippocampal-dependent synaptic plasticity and memory and mediates synapse loss through the CREB signaling pathway. The fact that altered CREB signaling has been implicated in other cognitive disorders including Huntington’s disease and Rubinstein-Taybi and Coffin-Lowry syndromes suggests a crucial role of CREB signaling in cognitive dysfunction. In this review paper, we summarize recent findings indicating a role of CREB and its coactivators CREB binding protein and CREB-regulated transcription coactivator in cognition during normal and pathological aging. We also discuss the development of novel therapeutic strategies based on CREB targeting to ameliorate cognitive decline in aging and cognitive disorders.

Enhanced Akt Signaling Is an Early Pro-survival Response That Reflects N-Methyl-D-aspartate Receptor Activation in Huntington's Disease Knock-in Striatal Cells

Huntingtons disease features the loss of striatal neurons that stems from a disease process that is initiated by mutant huntingtin. Early events in the disease cascade, which predate overt pathology in Hdh CAG knock-in mouse striatum, implicate enhanced N-methyl-d-aspartate (NMDA) receptor activation, with excitotoxity caused by aberrant Ca2+ influx. Here we demonstrate in precise genetic Huntingtons disease mouse and striatal cell models that these early phenotypes are associated with activation of the Akt pro-survival signaling pathway. Elevated levels of activated Ser(P)473-Akt are detected in extracts of HdhQ111/Q111 striatum and cultured mutant STHdhQ111/Q111 striatal cells, compared with their wild type counterparts. Akt activation in mutant striatal cells is associated with increased Akt signaling via phosphorylation of GSK3β at Ser9. Consequent decreased turnover of transcription co-factor β-catenin leads to increased levels of β-catenin target gene cyclin D1. Akt activation is phosphatidylinositol 3-kinase dependent, as demonstrated by increased levels of Ser(P)241-PDK1 kinase and decreased Ser(P)380-PTEN phosphatase. Moreover, Akt activation can be normally stimulated by treatment with insulin growth factor-1 and blocked by treatment with the phosphatidylinositol 3-kinase inhibitor LY294002. However, in contrast to wild type cells, Akt activation in mutant striatal cells can be blocked by the addition of the NMDA receptor antagonist MK-801. Akt activation in mutant striatal cells is Ca2+-dependent, because treatment with EGTA reduces levels of Ser(P)473-Akt. Thus, consistent with excitotoxicity early in the disease process, activation of the Akt pro-survival pathway in mutant knock-in striatal cells predates overt pathology and reflects mitochondrial dysfunction and enhanced NMDA receptor signaling.

Intraneuronal β-Amyloid Accumulation in the Amygdala Enhances Fear and Anxiety in Alzheimer's Disease Transgenic Mice

BACKGROUND Alzheimers disease (AD) is characterized by progressive memory decline and neuropsychiatric symptoms. Despite common emotional symptoms in AD such as anxiety and fear are associated with a more rapid cognitive decline, the pathological mechanisms involved in these behavioral changes remain largely elusive. In this study, we examined the pathological mechanisms of emotional behavior in well-established AD transgenic mice expressing human mutant beta-amyloid (Abeta) precursor protein (APP(Ind) and APP(Sw,Ind)) and tau (3xTg-AD). METHODS We evaluated unconditioned and conditioned fear-induced freezing behavior and spatial memory in APP(Ind), APP(Sw,Ind), and 3xTg-AD transgenic mice. The Abeta and tau pathologies and signaling pathways involved in emotional processing were studied by immunohistochemistry and immunoblotting analyses. RESULTS The APP(Ind)/APP(Sw,Ind) and 3xTg-AD transgenic mice displayed at early ages enhanced innate and conditioned fear symptoms and spatial memory deficits coinciding with enhanced accumulation of Abeta in gamma-aminobutyric acid (GABA)ergic and glutamatergic neurons, respectively, of the basolateral amygdala (BLA). Similarly, the number of neurons with intraneuronal Abeta40 and Abeta42 was significantly increased in the BLA of human AD brains. Fear responses might reflect an influence of anxiety, because the anxiolytic compounds valproate, diazepam, and buspirone reduced efficiently unconditioned and conditioned fear responses in APP transgenic mice. In addition, phosphorylation of extracellular signal-regulated kinase (ERK)1/2, which is critical for acquisition and consolidation of fear conditioning, was increased in the amygdala of APP transgenic mice after cued conditioning. CONCLUSIONS We propose a deleterious role of intraneuronal Abeta on amygdala-dependent emotional responses by affecting the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling pathway.

Long-term memory deficits in Huntington’s disease are associated with reduced CBP histone acetylase activity

Huntingtons disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by an expanded CAG/polyglutamine repeat in the coding region of the huntingtin (htt) gene. Although HD is classically considered a motor disorder, there is now considerable evidence that early cognitive deficits appear in patients before the onset of motor disturbances. Here we demonstrate early impairment of long-term spatial and recognition memory in heterozygous HD knock-in mutant mice (Hdh(Q7/Q111)), a genetically accurate HD mouse model. Cognitive deficits are associated with reduced hippocampal expression of CREB-binding protein (CBP) and diminished levels of histone H3 acetylation. In agreement with reduced CBP, the expression of CREB/CBP target genes related to memory, such c-fos, Arc and Nr4a2, was significantly reduced in the hippocampus of Hdh(Q7/Q111) mice compared with wild-type mice. Finally, and consistent with a role of CBP in cognitive impairment in Hdh(Q7/Q111) mice, administration of the histone deacetylase inhibitor trichostatin A rescues recognition memory deficits and transcription of selective CREB/CBP target genes in Hdh(Q7/Q111) mice. These findings demonstrate an important role for CBP in cognitive dysfunction in HD and suggest the use of histone deacetylase inhibitors as a novel therapeutic strategy for the treatment of memory deficits in this disease.

Evidence That Intramolecular Associations between Presenilin Domains Are Obligatory for Endoproteolytic Processing

Mutations in genes encoding presenilins (PS1 and PS2) cosegregate with the majority of early onset cases of familial Alzheimer’s disease. PS1 and PS2 are polytopic membrane proteins that undergo endoproteolytic cleavage to generate stable NH2- and COOH-terminal derivatives (NTF and CTF, respectively). Several lines of evidence suggest that the endoproteolytic derivatives are likely the functional units of PSin vivo. In the present report, we examine the disposition of PS NTF and CTF assemblies in stable mouse N2a neuroblastoma cell lines expressing human PS polypeptides. We show that exogenous expression of PS1 NTFs neither assemble with endogenous CTF nor exhibit dominant negative inhibitory effects on the endogenous PS1 cleavage and the accumulation of derivatives. In cells co-expressing PS1 and PS2, PS1- and PS2-derived fragments do not form mixed assemblies. In contrast, cells expressing a chimeric PS1/PS2 polypeptide form stable PS1 NTF-PS2 CTF assemblies. Moreover, expression of chimeric PS1/PS2 polypeptides harboring a familial early onset AD-linked mutation (M146L) elevates the production of Aβ42 peptides. Our results provide evidence that assembly of structural domains contained within NH2- and COOH-terminal regions of PS occur prior to endoproteolytic cleavage.

β-Amyloid Disrupts Activity-Dependent Gene Transcription Required for Memory through the CREB Coactivator CRTC1

Activity-dependent gene expression mediating changes of synaptic efficacy is important for memory storage, but the mechanisms underlying gene transcriptional changes in age-related memory disorders are poorly understood. In this study, we report that gene transcription mediated by the cAMP-response element binding protein (CREB)-regulated transcription coactivator CRTC1 is impaired in neurons and brain from an Alzheimers disease (AD) transgenic mouse expressing the human β-amyloid precursor protein (APPSw,Ind). Suppression of CRTC1-dependent gene transcription by β-amyloid (Aβ) in response to cAMP and Ca2+ signals is mediated by reduced calcium influx and disruption of PP2B/calcineurin-dependent CRTC1 dephosphorylation at Ser151. Consistently, expression of CRTC1 or active CRTC1 S151A and calcineurin mutants reverse the deficits on CRTC1 transcriptional activity in APPSw,Ind neurons. Inhibition of calcium influx by pharmacological blockade of L-type voltage-gated calcium channels (VGCCs), but not by blocking NMDA or AMPA receptors, mimics the decrease on CRTC1 transcriptional activity observed in APPSw,Ind neurons, whereas agonists of L-type VGCCs reverse efficiently these deficits. Consistent with a role of CRTC1 on Aβ-induced synaptic and memory dysfunction, we demonstrate a selective reduction of CRTC1-dependent genes related to memory (Bdnf, c-fos, and Nr4a2) coinciding with hippocampal-dependent spatial memory deficits in APPSw,Ind mice. These findings suggest that CRTC1 plays a key role in coupling synaptic activity to gene transcription required for hippocampal-dependent memory, and that Aβ could disrupt cognition by affecting CRTC1 function.

A role for presenilin 1 in regulating the delivery of amyloid precursor protein to the cell surface.

Presenilin 1 (PS1) and presenilin 2 play a critical role in the gamma-secretase processing of amyloid precursor protein (APP) and Notch1. Here, we investigate maturation and intracellular trafficking of APP and other membrane proteins in cells expressing an experimental PS1 deletion mutant (deltaM1,2). Stable expression of deltaM1,2 impairs gamma-secretase processing of Notch1 and delays Abeta secretion. Kinetic studies show enhanced O-glycosylation and sialylation of holo-APP and marked accumulation of APP COOH-terminal fragments (CTFs). Surface biotinylation, live staining, and trafficking studies show increased surface accumulation of holo-APP and CTFs in deltaM1,2 cells resulting from enhanced surface delivery of newly synthesized APP. Expression of a loss-of-function PS1 mutant (D385A) or incubation of cells with gamma-secretase inhibitors also increases surface levels of holo-APP and CTFs. In contrast to APP, glycosylation and surface accumulation of another type I membrane protein, nicastrin, are markedly reduced in deltaM1,2 cells. Finally, expression of deltaM1,2 results in the increased assembly and surface expression of nicotinic acetylcholine receptors, illustrating that PS1s influence on protein trafficking extends beyond APP and other type I membrane protein substrates of gamma-secretase. Collectively, our findings provide evidence that PS1 regulates the glycosylation and intracellular trafficking of APP and select membrane proteins.