Is phosphorylated tau a good biomarker of synapse pathology in Alzheimer's disease?

This scientific commentary refers to 'Distinct brain pathologies associated with Alzheimer's disease biomarker-related phospho-tau 181 and phospho-tau 217 in App knock-in mouse models of amyloid-ß amyloidosis' by Hirota et al. (https://doi.org/10.1093/braincomms/fcac286) and 'Predictive blood biomarkers and brain changes associated with age-related cognitive decline' by Saunders et al. (https://doi.org/10.1093/braincomms/fcad113).

Spatial Memory Training Counteracts Hippocampal GIRK Channel Decrease in the Transgenic APPSw,Ind J9 Alzheimer's Disease Mouse Model.

G-protein-gated inwardly rectifying potassium (GIRK) channels are critical determinants of neuronal excitability. They have been proposed as potential targets to restore excitatory/inhibitory balance in acute amyloidosis models, where hyperexcitability is a hallmark. However, the role of GIRK signaling in transgenic mice models of Alzheimer's disease (AD) is largely unknown. Here, we study whether progressive amyloid-ß (Aß) accumulation in the hippocampus during aging alters GIRK channel expression in mutant ß-amyloid precursor protein (APPSw,Ind J9) transgenic AD mice. Additionally, we examine the impact of spatial memory training in a hippocampal-dependent task, on protein expression of GIRK subunits and Regulator of G-protein signaling 7 (RGS7) in the hippocampus of APPSw,Ind J9 mice. Firstly, we found a reduction in GIRK2 expression (the main neuronal GIRK channels subunit) in the hippocampus of 6-month-old APPSw,Ind J9 mice. Moreover, we found an aging effect on GIRK2 and GIRK3 subunits in both wild type (WT) and APPSw,Ind J9 mice. Finally, when 6-month-old animals were challenged to a spatial memory training, GIRK2 expression in the APPSw,Ind J9 mice were normalized to WT levels. Together, our results support the evidence that GIRK2 could account for the excitatory/inhibitory neurotransmission imbalance found in AD models, and training in a cognitive hippocampal dependent task may have therapeutic benefits of reversing this effect and lessen early AD deficits.

Adenosine A2A Receptor Antagonists Affects NMDA Glutamate Receptor Function. Potential to Address Neurodegeneration in Alzheimer's Disease.

(1) Background. N-methyl d-aspartate (NMDA) ionotropic glutamate receptor (NMDAR), which is one of the main targets to combat Alzheimer's disease (AD), is expressed in both neurons and glial cells. The aim of this paper was to assess whether the adenosine A2A receptor (A2AR), which is a target in neurodegeneration, may affect NMDAR functionality. (2) Methods. Immuno-histo/cytochemical, biophysical, biochemical and signaling assays were performed in a heterologous cell expression system and in primary cultures of neurons and microglia (resting and activated) from control and the APPSw,Ind transgenic mice. (3) Results. On the one hand, NMDA and A2A receptors were able to physically interact forming complexes, mainly in microglia. Furthermore, the amount of complexes was markedly enhanced in activated microglia. On the other hand, the interaction resulted in a novel functional entity that displayed a cross-antagonism, that could be useful to prevent the exacerbation of NMDAR function by using A2AR antagonists. Interestingly, the amount of complexes was markedly higher in the hippocampal cells from the APPSw,Ind than from the control mice. In neurons, the number of complexes was lesser, probably due to NMDAR not interacting with the A2AR. However, the activation of the A2AR receptors resulted in higher NMDAR functionality in neurons, probably by indirect mechanisms. (4) Conclusions. A2AR antagonists such as istradefylline, which is already approved for Parkinson's disease (Nouriast® in Japan and Nourianz® in the US), have potential to afford neuroprotection in AD in a synergistic-like fashion. i.e., via both neurons and microglia.

Potentiation of cannabinoid signaling in microglia by adenosine A2A receptor antagonists.

Neuroprotective M2-skewed microglia appear as promising to alter the course of neurodegenerative diseases and G protein-coupled receptors (GPCRs) are potential targets to achieve such microglial polarization. A common feature of adenosine A2A (A2A R) and cannabinoid CB2 (CB2 R) GPCRs in microglia is that their expression is upregulated in Alzheimer's disease (AD). On the one hand, CB2 R seems a target for neuroprotection, delaying neurodegenerative processes like those associated to AD or Parkinson's diseases. A2A R antagonists reduce amyloid burden and improve cognitive performance and memory in AD animal models. We here show a close interrelationship between these two receptors in microglia; they are able to physically interact and affect the signaling of each other, likely due to conformational changes within the A2A -CB2 receptor heteromer (A2A -CB2 Het). Particularly relevant is the upregulation of A2A -CB2 Het expression in samples from the APPSw ,Ind AD transgenic mice model. The most relevant finding, confirmed in both heterologous cells and in primary cultures of microglia, was that blockade of A2A receptors results in increased CB2 R-mediated signaling. This heteromer-specific feature suggests that A2A R antagonists would potentiate, via microglia, the neuroprotective action of endocannabinoids with implications for AD therapy.

Decreased circulating ErbB4 ectodomain fragments as a read-out of impaired signaling function in amyotrophic lateral sclerosis.

ErbB4 is a transmembrane receptor tyrosine kinase that binds to neuregulins to activate signaling. Proteolytic cleavage of ErbB4 results in release of soluble fragments of ErbB4 into the interstitial fluid. Disruption of the neuregulin-ErbB4 pathway has been suggested to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). This study assesses whether soluble proteolytic fragments of the ErbB4 ectodomain (ecto-ErbB4) can be detected in cerebrospinal fluid (CSF) and plasma, and if the levels are altered in ALS. Immunoprecipitation combined with mass spectrometry or western blotting analyses confirmed the presence of ecto-ErbB4 in human CSF. Several anti-ErbB4-reactive bands, including a 55 kDa fragment, were detected in CSF. The bands were generated in the presence of neuregulin-1 (Nrg1) and were absent in plasma from ErbB4 knockout mice. Ecto-ErbB4 levels were decreased in CSF from ALS patients (n = 20) and ALS with concomitant frontotemporal dementia patients (n = 10), compared to age-matched controls (n = 13). A similar decrease was found for the short ecto-ErbB4 fragments in plasma of the same subjects. Likewise, the 55-kDa ecto-ErbB4 fragments were decreased in the plasma of the two transgenic mouse models of ALS (SOD1G93A and TDP-43A315T). Intracellular ErbB4 fragments were decreased in the frontal cortex from SOD1G93A mice, indicating a reduction in Nrg-dependent induction of ErbB4 proteolytic processing, and suggesting impaired signaling. Accordingly, overexpression of Nrg1 induced by an adeno-associated viral vector increased the levels of the ecto-ErbB4 fragment in the SOD1G93A mice. We conclude that the determination of circulating ecto-ErbB4 fragments could be a tool to evaluate the impairment of the ErbB4 pathway and may be a useful biomarker in ALS.

CRTC1 Function During Memory Encoding Is Disrupted in Neurodegeneration.

BACKGROUND: Associative memory impairment is an early clinical feature of dementia patients, but the molecular and cellular mechanisms underlying these deficits are largely unknown. In this study, we investigated the functional regulation of the cyclic adenosine monophosphate response element binding protein (CREB)-regulated transcription coactivator 1 (CRTC1) by associative learning in physiological and neurodegenerative conditions. METHODS: We evaluated the activation of CRTC1 in the hippocampus of control mice and mice lacking the Alzheimer's disease-linked presenilin genes (presenilin conditional double knockout [PS cDKO]) after one-trial contextual fear conditioning by using biochemical, immunohistochemical, and gene expression analyses. PS cDKO mice display classical features of neurodegeneration occurring in Alzheimer's disease including age-dependent cortical atrophy, neuron loss, dendritic degeneration, and memory deficits. RESULTS: Context-associative learning, but not single context or unconditioned stimuli, induces rapid dephosphorylation (Ser151) and translocation of CRTC1 from the cytosol/dendrites to the nucleus of hippocampal neurons in the mouse brain. Accordingly, context-associative learning induces differential CRTC1-dependent transcription of c-fos and the nuclear receptor subfamily 4 (Nr4a) genes Nr4a1-3 in the hippocampus through a mechanism that involves CRTC1 recruitment to CRE promoters. Deregulation of CRTC1 dephosphorylation, nuclear translocation, and transcriptional function are associated with long-term contextual memory deficits in PS cDKO mice. Importantly, CRTC1 gene therapy in the hippocampus ameliorates context memory and transcriptional deficits and dendritic degeneration despite ongoing cortical degeneration in this neurodegeneration mouse model. CONCLUSIONS: These findings reveal a critical role of CRTC1 in the hippocampus during associative memory, and provide evidence that CRTC1 deregulation underlies memory deficits during neurodegeneration.

Crtc1 activates a transcriptional program deregulated at early Alzheimer's disease-related stages.

Cognitive decline is associated with gene expression changes in the brain, but the transcriptional mechanisms underlying memory impairments in cognitive disorders, such as Alzheimer's disease (AD), are largely unknown. Here, we aimed to elucidate relevant mechanisms responsible for transcriptional changes underlying early memory loss in AD by examining pathological, behavioral, and transcriptomic changes in control and mutant ß-amyloid precursor protein (APPSw,Ind) transgenic mice during aging. Genome-wide transcriptome analysis using mouse microarrays revealed deregulation of a gene network related with neurotransmission, synaptic plasticity, and learning/memory in the hippocampus of APPSw,Ind mice after spatial memory training. Specifically, APPSw,Ind mice show changes on a cAMP-responsive element binding protein (CREB)-regulated transcriptional program dependent on the CREB-regulated transcription coactivator-1 (Crtc1). Interestingly, synaptic activity and spatial memory induces Crtc1 dephosphorylation (Ser151), nuclear translocation, and Crtc1-dependent transcription in the hippocampus, and these events are impaired in APPSw,Ind mice at early pathological and cognitive decline stages. CRTC1-dependent genes and CRTC1 levels are reduced in human hippocampus at intermediate Braak III/IV pathological stages. Importantly, adeno-associated viral-mediated Crtc1 overexpression in the hippocampus efficiently reverses Aß-induced spatial learning and memory deficits by restoring a specific subset of Crtc1 target genes. Our results reveal a critical role of Crtc1-dependent transcription on spatial memory formation and provide the first evidence that targeting brain transcriptome reverses memory loss in AD.

Ras protein activation is a key event in activity-dependent survival of cerebellar granule neurons.

Neuronal activity promotes the survival of cerebellar granule neurons (CGNs) during the postnatal development of cerebellum. CGNs that fail to receive excitatory inputs will die by apoptosis. This process could be mimicked in culture by exposing CGNs to either a physiological concentration of KCl (5 mm or K5) plus N-methyl-d-aspartate (NMDA) or to 25 mm KCl (K25). We have previously described that a 24-h exposure to NMDA (100 µm) or K25 at 2 days in vitro induced long term survival of CGNs in K5 conditions. Here we have studied the molecular mechanisms activated at 2 days in vitro in these conditions. First we showed that NMDA or K25 addition promoted a rapid stimulation of PI3K and a biphasic phosphorylation on Ser-473 of Akt, a PI3K substrate. Interestingly, we demonstrated that only the first wave of Akt phosphorylation is necessary for the NMDA- and K25-mediated survival. Additionally, we detected that both NMDA and K25 increased ERK activity with a similar time-course. Moreover, our results showed that NMDA-mediated activation of the small G-protein Ras is necessary for PI3K/Akt pathway activation, whereas Rap1 was involved in NMDA phosphorylation of ERK. On the other hand, Ras, but not Rap1, mediates K25 activation of PI3K/Akt and MEK/ERK pathways. Because neuroprotection by NMDA or K25 is mediated by Ras (and not by Rap1) activation, we propose that Ras stimulation is a crucial event in NMDA- and K25-mediated survival of CGNs through the activation of PI3K/Akt and MEK/ERK pathways.

ApoER2 processing by presenilin-1 modulates reelin expression.

The reelin signaling protein and its downstream components have been associated with synaptic plasticity and neurotransmission. The reelin signaling pathway begins with the binding of reelin to the transmembrane lipoprotein receptor apolipoprotein E receptor 2 (ApoER2), which in turns induces the sequential cleavage of ApoER2 by the sequential action of α- and γ-secretases. Using conditional-knockout mice of the catalytic component of the γ-secretase complex, presenilin 1 (PS1), we demonstrated increased brain ApoER2 and reelin protein and transcript levels, with no changes in the number of reelin-positive cells. Using the human SH-SY5Y neuroblastoma cell line, we showed that ApoER2 processing occurs in the presence of PS1, producing an intracellular ApoER2 C-terminal fragment. In addition, the pharmacologic inhibition of γ-secretase in SH-SY5Y cells led to increased reelin levels. Overexpression of ApoER2 decreased reelin mRNA levels in these cells. A luciferase reporter gene assay and nuclear fractionation confirmed that increased amounts of intracellular fragment of ApoER2 suppressed reelin expression at a transcriptional level. Chromatin immunoprecipitation experiments corroborated that the intracellular fragment of ApoER2 bound to the RELN promoter region. Our study suggests that PS1/γ-secretase-dependent processing of the reelin receptor ApoER2 inhibits reelin expression and may regulate its signaling.

X-linked inhibitor of apoptosis protein negatively regulates neuronal differentiation through interaction with cRAF and Trk.

X-linked Inhibitor of apoptosis protein (XIAP) has been classically identified as a cell death regulator. Here, we demonstrate a novel function of XIAP as a regulator of neurite outgrowth in neuronal cells. In PC12 cells, XIAP overexpression prevents NGF-induced neuronal differentiation, whereas NGF treatment induces a reduction of endogenous XIAP levels concomitant with the induction of neuronal differentiation. Accordingly, downregulation of endogenous XIAP protein levels strongly increases neurite outgrowth in PC12 cells as well as axonal and dendritic length in primary cortical neurons. The effects of XIAP are mediated by the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinases (ERKs) pathway since blocking this pathway completely prevents the neuritogenesis mediated by XIAP downregulation. In addition, we found that XIAP binds to cRaf and Trk receptors. Our results demonstrate that XIAP plays a new role as a negative regulator of neurotrophin-induced neurite outgrowth and neuronal differentiation in developing neurons.

ATP and noradrenaline activate CREB in astrocytes via noncanonical Ca(2+) and cyclic AMP independent pathways.

In neurons, it is well established that CREB contributes to learning and memory by orchestrating the translation of experience into the activity-dependent (i.e., driven by neurotransmitters) transcription of plasticity-related genes. The activity-dependent CREB-triggered transcription requires the concerted action of cyclic AMP/protein kinase A and Ca(2+) /calcineurin via the CREB-regulated transcription co-activator (CRTC). It is not known, however, whether a comparable molecular sequence occurs in astrocytes, despite the unquestionable contribution of these cells to brain plasticity. Here we sought to determine whether and how ATP and noradrenaline cause CREB-dependent transcription in rat cortical astrocyte cultures. Both transmitters induced CREB phosphorylation (Western Blots), CREB-dependent transcription (CRE-luciferase reporter assays), and the transcription of Bdnf, a canonical regulator of synaptic plasticity (quantitative RT-PCR). We indentified a Ca(2+) and diacylglycerol-independent protein kinase C at the uppermost position of the cascade leading to CREB-dependent transcription. Notably, CREB-dependent transcription was partially dependent on ERK1/2 and CRTC, but independent of cyclic AMP/protein kinase A or Ca(2+) /calcineurin. We conclude that ATP and noradrenaline activate CREB-dependent transcription in cortical astrocytes via an atypical protein kinase C. It is of relevance that the signaling involved be starkly different to the one described in neurons since there is no convergence of Ca(2+) and cyclic AMP-dependent pathways on CRTC, which, moreover, exerts a modulatory rather than a central role. Our data thus point to the existence of an alternative, non-neuronal, glia-based role of CREB in plasticity.

CREB-regulated transcription coactivator 1-dependent transcription in Alzheimer's disease mice.

BACKGROUND/AIMS: Long-term memory requires fine-tuning regulation of gene expression in specific neural circuits of the brain. Transcriptional regulation of gene programs is a key mechanism for memory storage and its deregulation may contribute to synaptic and cognitive dysfunction in memory disorders. The molecular mechanisms underlying changes on activity-dependent gene expression in Alzheimer's disease (AD) are largely unknown. METHODS: We analyzed the expression of activity-dependent genes regulated by the cAMP response element binding protein (CREB) and activation of CREB and its coactivator CREB-regulated transcription coactivator 1 (CRTC1) in control and mutant ß-amyloid precursor protein (APP(Sw,Ind); Swedish and Indiana mutations) transgenic mice. RESULTS: Gene expression analyses revealed specific downregulation of a subset of well-known activity-induced CREB-dependent genes, including c-fos, Bdnf and Nr4a2, in the hippocampus of memory-impaired APP(Sw,Ind) transgenic mice. Activity-dependent CREB transcription induced by calcium/cAMP signals is disrupted through a mechanism involving deregulation of calcium/calcineurin-mediated dephosphorylation and activation of CRTC1. Expression of CRTC1 and pharmacological activation of L-type voltage-gated calcium channels reverse the deficits in CRTC1-mediated transcription in APP(Sw,Ind) neurons. CONCLUSION: Our results suggest that CRTC1 dysfunction caused by Aß accumulation underlies changes in gene expression required for hippocampal-dependent memory in AD transgenic mice.

γ-secretase substrates and their implications for drug development in Alzheimer's disease.

γ-secretase is an aspartyl protease that cleaves a large number of substrates within the membrane environment. This multiprotein complex is responsible for the last cleavage step of the ß-amyloid precursor protein (APP) that generates the amyloid-ß peptide (Aß), one of the primary components of amyloid plaques in Alzheimer's disease (AD). Over the last years, more than 70 type-I membrane proteins have been shown to be cleaved by γ-secretase. The mechanism and function of this cleavage event is yet not well understood. The γ-secretase cleavage of some substrates releases intracellular domains with critical signaling properties. In contrast, the cleavage of other substrates seems to have a mere degradative function. Knowledge about γ-secretase substrates and their function has clear implications for the development of new therapies for AD. Most γ-secretase inhibitors interfere with the cleavage of the Notch receptor, which is known to lead to adverse effects in animal models and in humans. Paradoxically, due to this effect, γ-secretase inhibitors are actively being investigated in cancer. An alternative approach is modulation of γ-secretase, in which small molecules allosterically attenuate the activity to reduce Aß42, the most fibrillogenic species. Although tolerance and efficacy of some γ-secretase inhibitors in AD have shown to be poor in clinical trials, more selective compounds are on the road. As these compounds advance to clinical trials it is critical to understand the mechanism by which γ-secretase recognizes and cleaves this diverse set of substrates to predict possible adverse effects in humans. This knowledge will help to guide drug development in AD and cancer.

Bone morphogenetic protein-6 promotes cerebellar granule neurons survival by activation of the MEK/ERK/CREB pathway.

Bone morphogenetic proteins (BMPs) have been implicated in the generation and postnatal differentiation of cerebellar granule cells (CGCs). Here, we examined the eventual role of BMPs on the survival of these neurons. Lack of depolarization causes CGC death by apoptosis in vivo, a phenomenon that is mimicked in vitro by deprivation of high potassium in cultured CGCs. We have found that BMP-6, but not BMP-7, is able to block low potassium-mediated apoptosis in CGCs. The neuroprotective effect of BMP-6 is not accompanied by an increase of Smad translocation to the nucleus, suggesting that the canonical pathway is not involved. By contrast, activation of the MEK/ERK/CREB pathway by BMP-6 is necessary for its neuroprotective effect, which involves inhibition of caspase activity and an increase in Bcl-2 protein levels. Other pathways involved in the regulation of CGC survival, such as the c-Jun terminal kinase and the phosphatidylinositol 3-kinase (PI3K)-Akt/PKB, were not affected by BMP-6. Moreover, failure of BMP-7 to activate the MEK/ERK/CREB pathway could explain its inability to protect CGCs from low potassium-mediated apoptosis. Thus, this study demonstrates that BMP-6 acting through the noncanonical MEK/ERK/CREB pathway plays a crucial role on CGC survival.

Estradiol facilitates neurite maintenance by a Src/Ras/ERK signalling pathway.

Different reports suggest the estrogens are involved in neuritic outgrowth, maintenance of dendritic morphology and spine formation in the CNS. However, the molecular mechanisms regulated by estrogens on neuronal integrity are not fully understood. We have addressed the relationship between 17beta-estradiol-dependent ERK pathway stimulation and the maintenance of neuritic morphology in cerebellar granule cell cultures (CGC). We report that 17beta-estradiol clearly activates ERK phosphorylation in CGC cultured in low potassium via ERalpha localized in the plasma membrane and without the activation of the insulin-like growth factor-I receptor. 17beta-estradiol activates the ERK pathway through Ras-dependent Src kinase activity. A concomitant activation of the cAMP-response element-binding protein (CREB) is observed. Moreover, we demonstrate that 17beta-estradiol-mediated ERK activation is involved in the maintenance of neuritic arborisation and neuronal morphology in proapoptotic conditions.

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 beta-amyloid (Abeta) peptides in the cerebral cortex is considered a key event in the pathogenesis of Alzheimer's disease (AD). Presenilin 1 (PS1) plays an essential role in the gamma-secretase cleavage of the amyloid precursor protein (APP) and the generation of Abeta peptides. Reduction of Abeta generation via the inhibition of gamma-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 Abeta 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 Abeta 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 Abeta via the inactivation of PS1 effectively prevents amyloid-associated neuropathological changes and can, but only temporarily, improve cognitive impairments in APP transgenic mice.

Reduced beta-amyloid production and increased inflammatory responses in presenilin conditional knock-out mice.

Mutations in presenilins (PS) 1 and 2 are the major cause of familial Alzheimer's disease. Conditional double knock-out mice lacking both presenilins in the postnatal forebrain (PS cDKO mice) exhibit memory and synaptic plasticity impairments followed by progressive neurodegeneration in the cerebral cortex. Here we further investigate the molecular events that may underlie the observed phenotypes and identify additional neuropathological markers in the PS cDKO brain. Enzyme-linked immunosorbent assay analysis showed reduced levels of the toxic beta-amyloid (Abeta) peptides in the cerebral cortex of PS cDKO mice. Interestingly, the reduction in Abeta40 and Abeta42 peptides is similar in PS1 conditional knock-out and PS cDKO mice. We further examined the gene expression profile by oligonucleotide microarrays in the PS cDKO cerebral cortex and found that a high number of genes are differentially expressed, most notably a group of up-regulated inflammatory genes. Quantitative real-time reverse transcription PCR and Western analyses confirmed the elevated levels of glial fibrillary acidic protein, complement component C1q, and cathepsin S, up-regulation of which has been associated with inflammatory responses in various neurodegenerative processes. Immunohistochemical analysis revealed that the increase in complement component C1q is confined to the hippocampal formation, whereas glial fibrillary acidic protein and cathepsin S are up-regulated throughout the entire neocortex and hippocampus. In addition, strong microglial activation occurs in the hippocampus and the deeper cortical layers of PS cDKO mice. These results indicate that the memory impairment and neurodegeneration in PS cDKO mice are not caused by Abeta accumulation and that loss of PS function leads to differential up-regulation of inflammatory markers in the cerebral cortex.

Functional implications of the presenilin dimerization: reconstitution of gamma-secretase activity by assembly of a catalytic site at the dimer interface of two catalytically inactive presenilins.

Presenilins are the catalytic components of gamma-secretase, an intramembrane-cleaving protease whose substrates include beta-amyloid precursor protein (betaAPP) and the Notch receptors. These type I transmembrane proteins undergo two distinct presenilin-dependent cleavages within the transmembrane region, which result in the production of Abeta and APP intracellular domain (from betaAPP) and the Notch intracellular domain signaling peptide. Most cases of familial Alzheimer's disease are caused by presenilin mutations, which are scattered throughout the coding sequence. Although the underlying molecular mechanism is not yet known, the familial Alzheimer's disease mutations produce a shift in the ratio of the long and short forms of the Abeta peptide generated by the gamma-secretase. We and others have previously shown that presenilin homodimerizes and suggested that a presenilin dimer is at the catalytic core of gamma-secretase. Here, we demonstrate that presenilin transmembrane domains contribute to the formation of the dimer. In-frame substitution of the hydrophilic loop 1, located between transmembranes I and II, which modulates the interactions within the N-terminal fragment/N-terminal fragment dimer, abolishes both presenilinase and gamma-secretase activities. In addition, by reconstituting gamma-secretase activity from two catalytically inactive presenilin aspartic mutants, we provide evidence of an active diaspartyl group assembled at the interface between two presenilin monomers. Under our conditions, this catalytic group mediates the generation of APP intracellular domain and Abeta but not Notch intracellular domain, therefore suggesting that specific diaspartyl groups within the presenilin catalytic core of gamma-secretase mediate the cleavage of different substrates.

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 Alzheimer's 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.

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.

Huntington's 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 Huntington's 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 Hdh(Q111/Q111) striatum and cultured mutant STHdh(Q111/Q111) striatal cells, compared with their wild type counterparts. Akt activation in mutant striatal cells is associated with increased Akt signaling via phosphorylation of GSK3beta at Ser9. Consequent decreased turnover of transcription co-factor beta-catenin leads to increased levels of beta-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 Ca(2+)-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.