Increased Foxo3a nuclear translocation and activity is an early neuronal response to βγ-secretase mediated processing of the Amyloid-β-Protein Precursor Protein: Utility of an AβPP-GAL4 reporter assay

Sequential cleavage of the amyloid-β protein precursor (AβPP) by BACE1 (β-secretase) followed by the γ-secretase complex, is strongly implicated in Alzheimer’s Disease (AD) but the initial cellular responses to these cleavage events are not fully defined. β-secretase-mediated AβPP processing yields an extracellular domain (sAβPPβ) and a C-terminal fragment of APP of 99 amino acids (C99). Subsequent cleavage by γ-secretase produces amyloid-β (Aβ) and an APP intracellular domain (AICD). A cellular screen based on the generation of AICD from an AβPP-Gal4 fusion protein was adapted by introducing familial AD (FAD) mutations into the AβPP sequence and linking the assay to Gal4-UAS driven luciferase and GFP expression, to identify responses immediately downstream of AβPP processing in neurons with a focus on the transcription factor Foxo3a which has been implicated in neurodegeneration. The K670N/M671L, E682K, E693G, V717I FAD mutations and the A673T protective mutation, were introduced into the AβPP sequence by site directed mutagenesis. When expressed in mouse cortical neurons AβPP-Gal4-UAS driven luciferase and GFP expression was substantially reduced by γ-secretase inhibitors, lowered by β-secretase inhibitors and enhanced by α-secretase inhibitors suggesting that AICD is a product of the β-secretase pathway. AβPP-Gal4-UAS driven GFP expression was exploited to identify individual neurons undergoing amyloidogenic AβPP processing, revealing increased nuclear localization of Foxo3a and enhanced Foxo3a-mediated transcription downstream of AICD production. Foxo3a translocation was not driven by AICD directly but correlated with reduced Akt phosphorylation. Collectively this suggests that βγ-secretase-mediated AβPP processing couples to Foxo3a which could be an early neuronal signaling response in AD

4-Aminopyridine-induced epileptogenesis depends on activation of mitogen-activated protein kinase ERK

Extracellular signal-regulated kinases such as ERK1 [p44 mitogen-activated protein kinase (MAPK)] and ERK2 (p42 MAPK) are activated in the CNS under physiological and pathological conditions such as ischemia and epilepsy. Here, we studied the activation state of ERK1/2 in rat hippocampal slices during application of the K+ channel blocker 4-aminopyridine (4AP, 50 lM), a procedure that enhances synaptic transmission and leads to the appearance of epileptiform activity. Hippocampal slices superfused with 4AP-containing medium exhibited a marked activation of ERK1/2 phosphorylation that peaked within about 20 min. These effects were not accompanied by changes in the activation state of c-Jun N-terminal kinase (JNK), another member of the MAP kinase superfamily. 4AP-induced ERK1/2 activation was inhibited by the voltage-gated Na+ channel blocker tetrodotoxin (1 lM). We also found that application of the ERK pathway inhibitors U0126 (50 lM) or PD98059 (100 lM) markedly reduced 4AP-induced epileptiform synchronization, thus abolishing ictal discharges in the CA3 area. The effects induced by U0126 or PD98059 were not associated with changes in the amplitude and latency of the field potentials recorded in the CA3 area following electrical stimuli delivered in the dentate hylus. These data demonstrate that activation of ERK1/2 accompanies the appearance of epileptiform activity induced by 4AP and suggest a cause-effect relationship between the ERK pathway and epileptiform synchronization

Computational Modeling of PI3K/AKT and MAPK Signaling Pathways in Melanoma Cancer

Background Malignant melanoma is an aggressive tumor of the skin and seems to be resistant to current therapeutic approaches. Melanocytic transformation is thought to occur by sequential accumulation of genetic and molecular alterations able to activate the Ras/Raf/MEK/ERK (MAPK) and/or the PI3K/AKT (AKT) signalling pathways. Specifically, mutations of B-RAF activate MAPK pathway resulting in cell cycle progression and apoptosis prevention. According to these findings, MAPK and AKT pathways may represent promising therapeutic targets for an otherwise devastating disease. Result Here we show a computational model able to simulate the main biochemical and metabolic interactions in the PI3K/AKT and MAPK pathways potentially involved in melanoma development. Overall, this computational approach may accelerate the drug discovery process and encourages the identification of novel pathway activators with consequent development of novel antioncogenic compounds to overcome tumor cell resistance to conventional therapeutic agents. The source code of the various versions of the model are available as S1 Archive

A Model of Late Long-Term Potentiation Simulates Aspects of Memory Maintenance

Late long-term potentiation (L-LTP) appears essential for the formation of long-term memory, with memories at least partly encoded by patterns of strengthened synapses. How memories are preserved for months or years, despite molecular turnover, is not well understood. Ongoing recurrent neuronal activity, during memory recall or during sleep, has been hypothesized to preferentially potentiate strong synapses, preserving memories. This hypothesis has not been evaluated in the context of a mathematical model representing biochemical pathways important for L-LTP. I incorporated ongoing activity into two such models: a reduced model that represents some of the essential biochemical processes, and a more detailed published model. The reduced model represents synaptic tagging and gene induction intuitively, and the detailed model adds activation of essential kinases by Ca. Ongoing activity was modeled as continual brief elevations of [Ca]. In each model, two stable states of synaptic weight resulted. Positive feedback between synaptic weight and the amplitude of ongoing Ca transients underlies this bistability. A tetanic or theta-burst stimulus switches a model synapse from a low weight to a high weight stabilized by ongoing activity. Bistability was robust to parameter variations. Simulations illustrated that prolonged decreased activity reset synapses to low weights, suggesting a plausible forgetting mechanism. However, episodic activity with shorter inactive intervals maintained strong synapses. Both models support experimental predictions. Tests of these predictions are expected to further understanding of how neuronal activity is coupled to maintenance of synaptic strength.Comment: Accepted to PLoS One. 8 figures at en

Riluzole Attenuates L-DOPA-Induced Abnormal Involuntary Movements Through Decreasing CREB1 Activity

Chronic administration of L-DOPA, the first-line treatment of dystonic symptoms in childhood or in Parkinson's disease, often leads to the development of abnormal involuntary movements (AIMs), which represent an important clinical problem. Although it is known that Riluzole attenuates L-DOPA-induced AIMs, the molecular mechanisms underlying this effect are not understood. Therefore, we studied the behavior and performed RNA sequencing of the striatum in three groups of rats that all received a unilateral lesion with 6-hydroxydopamine in their medial forebrain bundle, followed by the administration of saline, L-DOPA, or L-DOPA combined with Riluzole. First, we provide evidence that Riluzole attenuates AIMs in this rat model. Subsequently, analysis of the transcriptomics data revealed that Riluzole is predicted to reduce the activity of CREB1, a transcription factor that regulates the expression of multiple proteins that interact in a molecular landscape involved in apoptosis. Although this mechanism underlying the beneficial effect of Riluzole on AIMs needs to be confirmed, it provides clues towards novel or existing compounds for the treatment of AIMs that modulate the activity of CREB1 and, hence, its downstream targets

Synaptic NMDA receptor activation stimulates alpha-secretase amyloid precursor protein processing and inhibits amyloid-beta production

Altered amyloid precursor protein (APP) processing leading to increased production and oligomerization of Abeta may contribute to Alzheimer's disease (AD). Understanding how APP processing is regulated under physiological conditions may provide new insights into AD pathogenesis. Recent reports demonstrate that excitatory neural activity regulates APP metabolism and Abeta levels, although understanding of the molecular mechanisms involved is incomplete. We have investigated whether NMDA receptor activity regulates APP metabolism in primary cultured cortical neurons. We report that a pool of APP is localized to the postsynaptic compartment in cortical neurons and observed partial overlap of APP with both NR1 and PSD-95. NMDA receptor stimulation increased nonamyloidogenic alpha-secretase-mediated APP processing, as measured by a 2.5-fold increase in cellular alpha-C-terminal fragment (C83) levels after glutamate or NMDA treatment. This increase was blocked by the NMDA receptor antagonists d-AP5 and MK801 but not by the AMPA receptor antagonist CNQX or the L-type calcium channel blocker nifedipine, was prevented by chelation of extracellular calcium, and was blocked by the alpha-secretase inhibitor TAPI-1. Cotreatment of cortical neurons with bicuculline and 4-AP, which stimulates glutamate release and activates synaptic NMDA receptors, evoked an MK801-sensitive increase in C83 levels. Furthermore, NMDA receptor stimulation caused a twofold increase in the amount of soluble APP detected in the neuronal culture medium. Finally, NMDA receptor activity inhibited both Abeta1-40 release and Gal4-dependent luciferase activity induced by beta-gamma-secretase-mediated cleavage of an APP-Gal4 fusion protein. Altogether, these data suggest that calcium influx through synaptic NMDA receptors promotes nonamyloidogenic alpha-secretase-mediated APP processing

Amyloid precursor protein 695 associates with assembled NR2A- and NR2B-containing NMDA receptors to result in the enhancement of their cell surface delivery

This is a study of the interaction between the two NMDA neurotransmitter receptor subtypes, NR1/NR2A and NR1/NR2B, and amyloid precursor protein (APP) 695, the major APP variant expressed in neurones. APP695 co-immunoprecipitated with assembled NR1-1a/NR2A and NR1-1a/NR2B NMDA receptors following expression in mammalian cells. Single NR1-1a, NR1-2a, NR1-4b(c-Myc), or NR2 subunit transfections revealed that co-association of APP695 with assembled NMDA receptors was mediated via the NR1 subunit; it was independent of the NR1 C1, C2, and C2' cassettes and, the use of an NR1-2a(c-Myc)-trafficking mutant suggested that interaction between the two proteins occurs in the endoplasmic reticulum. The use of antibodies directed against extracellular and intracellular NR2 subunit epitopes for immunoprecipitations suggested that APP/NMDA receptor association was mediated via N-terminal domains. Anti-APP antibodies immunoprecipitated NR1, NR2A, and NR2B immunoreactive bands from detergent extracts of mammalian brain; reciprocally, anti-NR1 or anti-NR2A antibodies co-immunoprecipitated APP immunoreactivity. Immune pellets from brain were sensitive to endoglycosidase H suggesting that, as for heterologous expression, APP and NMDA receptor association occurs in the endoplasmic reticulum. Co-expression of APP695 in mammalian cells resulted in enhanced cell surface expression of both NR1-1a/NR2A and NR1-1a/NR2B NMDA receptors with no increase in total subunit expression. These findings are further evidence for a role of APP in intracellular trafficking mechanisms. Further, they provide a link between two major brain proteins that have both been implicated in Alzheimer's disease