Regulation of glutamate exocytosis from isolated nerve terminals (synaptosomes): Role of presynaptic receptors

Isolated nerve terminals (synaptosomes) can be used as a model to study glutamate exocytosis in the mammalian CNS. ω-CTx GVIA reduced Ca2+ entry into cerebrocortical synaptosomes and inhibited glutamate exocytosis. Furthermore, ω-CTx MVIIC completely inhibited glutamate exocytosis and Ca2+ influx into nerve terminals. From these toxin studies, it is proposed that glutamate transmission in the cerebral cortex may be controlled by class B N-type VSCC and class A P/Q type VSCC. In contrast, dihydropyridines did not alter glutamate release evoked by KCI, supporting the hypothesis that class C/D L-type VSCC are not coupled to release of fast acting transmitters. The role that presynaptic receptors play in the modulation of glutamate release was examined. The non-NMDA receptor agonists AMPA, kainate and domoate potentiated 4AP-evoked glutamate exocytosis and pharmacological analysis indicated the presence of a high-affinity kainate receptor (autoreceptor) on a population of cerebrocortical nerve terminals that is positively linked to glutamate exocytosis. (-)Baclofen, a GABAB receptor agonist, inhibited glutamate release from synaptosomes with a pharmacology that correlates closely with previous data indicating the presence of inhibitory GABAB receptors on glutamatergic nerve terminals (heteroreceptors). A direct reduction of Ca2+ entry into synaptosomes was observed with (-)baclofen and inhibition of glutamate release was insensitive to pertussis toxin (PTX), suggesting that GABAB receptors are negatively linked to VSCC coupled to glutamate exocytosis via a PTX-insensitive G-protein. Finally, the metabotropic glutamate receptor (mGluR) agonists 1S,3R-ACPD and L-AP4 strongly inhibited glutamate exocytosis from adult rat cerebrocortical synaptosomes, indicating that glutamate transmission in the adult mammalian CNS may be subject to modulation by presynaptic inhibitory group II and/or group III mGluRs (autoreceptors)

Protooncogenes Subserve Memory Formation in the Adult CNS

AbstractStudies of the signal transduction mechanisms underlying learning and memory have provided many new insights into the molecular mechanisms underlying associative conditioning in mammals. In this issue of Neuron, Gean and colleagues report the discovery that the PI-3 kinase/AKT(PKB) pathway contributes to LTP and the consolidation of amygdala-dependent cued fear conditioning in rats

Calsyntenin-1 mediates axonal transport of the amyloid precursor protein and regulates Aβ production

Understanding the mechanisms that control processing of the amyloid precursor protein (APP) to produce amyloid-β (Aβ) peptide represents a key area of Alzheimer's disease research. Here, we show that siRNA-mediated loss of calsyntenin-1 in cultured neurons alters APP processing to increase production of Aβ. We also show that calsyntenin-1 is reduced in Alzheimer's disease brains and that the extent of this reduction correlates with increased Aβ levels. Calsyntenin-1 is a ligand for kinesin-1 light chains and APP is transported through axons on kinesin-1 molecular motors. Defects in axonal transport are an early pathological feature in Alzheimer's disease and defective APP transport is known to increase Aβ production. We show that calsyntenin-1 and APP are co-transported through axons and that siRNA-induced loss of calsyntenin-1 markedly disrupts axonal transport of APP. Thus, perturbation to axonal transport of APP on calsyntenin-1 containing carriers induces alterations to APP processing that increase production of Aβ. Together, our findings suggest that disruption of calsyntenin-1-associated axonal transport of APP is a pathogenic mechanism in Alzheimer's disease

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

Extracellular Monomeric and Aggregated Tau Efficiently Enter Human Neurons through Overlapping but Distinct Pathways.

In Alzheimer's disease, neurofibrillary tangle pathology appears to spread along neuronal connections, proposed to be mediated by the release and uptake of abnormal, disease-specific forms of microtubule-binding protein tau MAPT. It is currently unclear whether transfer of tau between neurons is a toxic gain-of-function process in dementia or reflects a constitutive biological process. We report two entry mechanisms for monomeric tau to human neurons: a rapid dynamin-dependent phase typical of endocytosis and a second, slower actin-dependent phase of macropinocytosis. Aggregated tau entry is independent of actin polymerization and largely dynamin dependent, consistent with endocytosis and distinct from macropinocytosis, the major route for aggregated tau entry reported for non-neuronal cells. Anti-tau antibodies abrogate monomeric tau entry into neurons, but less efficiently in the case of aggregated tau, where internalized tau carries antibody with it into neurons. These data suggest that tau entry to human neurons is a physiological process and not a disease-specific phenomenon

AMPA receptor activation promotes non-amyloidogenic amyloid precursor protein processing and suppresses neuronal amyloid-β production

Soluble oligomeric amyloid β peptide (Aβ) generated from processing of the amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer's Disease (AD) and through actions at glutamatergic synapses affects excitability and plasticity. The physiological control of APP processing is not fully understood but stimulation of synaptic NMDA receptors (NMDAR) can suppress Aβ levels through an ERK-dependent increase in α-secretase activity. AMPA-type glutamate receptors (AMPAR) couple to ERK phosphorylation independently of NMDAR activation raising the possibility that stimulation of AMPAR might similarly promote non-amyloidogenic APP processing. We have tested this hypothesis by investigating whether AMPAR directly regulate APP processing in cultured mouse cortical neurons, by analyzing APP C-terminal fragments (CTFs), soluble APP (sAPP), Aβ levels, and cleavage of an APP-GAL4 reporter protein. We report that direct stimulation of AMPAR increases non-amyloidogenic α-secretase-mediated APP processing and inhibits Aβ production. Processing was blocked by the matrix metalloproteinase inhibitor TAPI-1 but was only partially dependent on Ca(2+) influx and ERK activity. AMPAR can therefore, be added to the repertoire of receptors that couple to non-amyloidogenic APP processing at glutamatergic synapses and thus pharmacological targeting of AMPAR could potentially influence the development and progression of Aβ pathology in AD

PrP is a central player in toxicity mediated by soluble aggregates of neurodegeneration-causing proteins

Neurodegenerative diseases are an enormous public health problem, affecting tens of millions of people worldwide. Nearly all of these diseases are characterized by oligomerization and fibrillization of neuronal proteins, and there is great interest in therapeutic targeting of these aggregates. Here, we show that soluble aggregates of α-synuclein and tau bind to plate-immobilized PrP in vitro and on mouse cortical neurons, and that this binding requires at least one of the same N-terminal sites at which soluble Aβ aggregates bind. Moreover, soluble aggregates of tau, α-synuclein and Aβ cause both functional (impairment of LTP) and structural (neuritic dystrophy) compromise and these deficits are absent when PrP is ablated, knocked-down, or when neurons are pre-treated with anti-PrP blocking antibodies. Using an all-human experimental paradigm involving: (1) isogenic iPSC-derived neurons expressing or lacking PRNP, and (2) aqueous extracts from brains of individuals who died with Alzheimer's disease, dementia with Lewy bodies, and Pick's disease, we demonstrate that Aβ, α-synuclein and tau are toxic to neurons in a manner that requires PrPC. These results indicate that PrP is likely to play an important role in a variety of late-life neurodegenerative diseases and that therapeutic targeting of PrP, rather than individual disease proteins, may have more benefit for conditions which involve the aggregation of more than one protein

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

APLP1 and APLP2, members of the APP family of proteins, behave similarly to APP in that they associate with NMDA receptors and enhance NMDA receptor surface expression

The function of amyloid precursor protein (APP) is unknown, although the discovery that it contributes to the regulation of surface expression of N-methyl-D-aspartate (NMDA) receptors has afforded new insights into its functional significance. Since APP is a member of a gene family that contains two other members, amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2), it is important to determine if the related APP proteins possess the same properties as APP with respect to their interactions with NMDA receptors. Following expression in mammalian cells, both APLP1 and APLP2 behaved similarly to APP in that they both co-immunoprecipitated with the two major NMDA receptor subtypes, GluN1/GluN2A and GluN1/GluN2B, via interaction with the obligatory GluN1 subunit. Immunoprecipitations from detergent extracts of adult mammalian brain showed co-immunoprecipitation of APLP1 and APLP2 with GluN2A- and GluN2B-containing NMDA receptors. Furthermore, similarly to APP, APLP1 and APLP2 both enhanced GluN1/GluN2A and GluN1/GluN2B cell surface expression. Thus all three members of the APP gene family behave similarly in that they each contribute to the regulation of cell surface NMDA receptor homeostasis