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19
Section CELLULAR NEUROSCIENCE
Role of Astroglial Calcium/Calcineurin-Mediated Signaling in Alzheimer's Disease: in Search of Potential Mechanisms and Mediators
D. Lim*
Universita del Piemonte Orientale, Novara, Italy. * Presenting e-mail: dmitry.lim@uniupo.it
Alzheimer's disease (AD) is the most common age-related neurological disorder with an enormous social and economical impact. AD is characterized by progressive loss of memory, social deficit and dementia. Currently, there is no cure or preventive therapy for AD and therefore novel approaches for understanding the AD pathogenesis are desperately needed. Deregulation of calcium homeostasis has been proposed to have a crucial role in pathogenic cascade which leads to neurodegeneration Yet, the possibility that the familial AD (FAD)-associated mutations alongside with the soluble beta-amyloid (Ap) oligomers affect calcium-signaling in astroglial cells, leading thus to neuronal dysfunctions at the early stages of disease, has been largely over-looked. We have dissected a cascade of events by which Ap deregulates calcium homeostasis in hippocampal astrocytes. In details: (i) 100 nM Ap42 leads to an increase in cytosolic calcium; (ii) increased Ca2+ leads to activation of calcineurin (CaN), which in turn (iii) directly activates NFAT to up-regulate IP3R1, and (iv) via interaction with Bcl10 and degradation of IkBa activates NF-kB to up-regulate mGluR5 and IP3R2. Furthermore, ATP-induced IP3R1-mediated Ca2+ release and IP3R1 protein were augmented in the hippocampal astrocytes form 3xTg-AD mice, a well characterized AD mouse model in which persenilin 1 (PS1)-M146V FAD-related mutation is expressed in astrocytes. Finally, we show that mGluR5 staining is augmented in hippocampal astrocytes of AD patients in proximity of Ap plaques and co-localized with accumulation of the p65 NF-kB subunit and increased staining of CaNAa. These data indicate that calcium-dependent activation of CaN and NF-kB mediates the remodeling of astroglial calcium signaling toolkit in AD.
Next, using astrocyte-neuronal co-cultures and astrocyte-conditioned medium (ACM) transfer, we found that Ap42-exposed astrocytes, as well as astrocytes form 3xTg-AD mice produce alterations of dendritic spines and reduce MAP2, PSD95 and Syn38 proteins in cultured neurons in a CaN-dependent manner. Searching for a soluble factor(s) we performed multiplex cytokine assay of ACM, but among 12 cytokines assayed only TGF-p was released at a detectable level. Using qPCR we found that TGF-p2 and TGF-p3 are predominantly expressed at mRNA level. Furthermore, Ap42-induced TGF-p3 mRNA up-regulation as well as release of TGF-p3 from 3xTg-AD astrocytes were blocked by a CaN inhibitor. In cultured hippocampal neurons, treatment with TGF-p2 and TGF-p3, but not TGF-p1, produced reduction of neuronal proteins, suggesting that the p2 and p3 isoforms may play a role in the early AD-related neuronal pathology.
Finally, in order to characterize transcriptional alterations that astroglial cells undergo in the early AD we performed a whole-genome microarray study on cultured hippocampal astrocytes from 3xTg-AD mice using Non-Tg astrocytes as a control. A set of 963 genes was differentially expressed in 3xTg-AD with respect to Non-Tg astrocytes. Gene ontology (GO) analysis revealed that among the up-regulated, the genes involved in nucleotide binding, regulation of transcription and mitochondrial function were significantly overrepresented. Instead, among the down-regulated, the overrepresented genes are involved in cell-cell communication and regulation of synaptic transmission.
Taken together, our data indicate that transcriptional Ca2+/CaN-dependent remodeling of astroglial cells, taking place in the early AD stages, may participate to the development of synaptic and neuronal dysfunction.
Role of Synaptic Plasticity in AMPA Receptor Intracellular Trafficking
F. Coussen*, E. Hangen, F. Cordelières, J. Petersen and D. Choquet
Institut Interdisciplinaire de NeuroScience, UMR5297, Université de Bordeaux, 33077 Bordeaux, France. * Presenting e-mail: fcoussen@u-bordeaux.fr
AMPA receptors (AMPARs) mediate fast excitatory synaptic transmission in the central nervous system. Their abundance at the synapse is essential for the establishment and maintenance of synaptic function. Many studies characterized trafficking of AMPARs in spines at basal state or after induced plasticity. Their synaptic localization is dependent on a highly dynamic exocytosis, endocytosis and plasma membrane trafficking events. Our hypothesis is that synaptic localization of AMPARs is also regulated by their earlier intracellular trafficking. However, AMPARs post-ER traffick-
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Section CELLULAR NEUROSCIENCE
ing toward the plasma membrane still remains poorly understood.
Using a new biochemical tool combined with photonic live imaging, we controlled and followed the dynamic secretion of tagged AMPAR subunits in cultured rat hippocampal neurons. We characterize AMPAR trafficking from the ER to the Golgi apparatus and from Golgi to the plasma membrane.
We analyze the characteristics of basal AMPAR transport. We studied the influence of synaptic plasticity in the intracellular transport of GluAl containing AMPAR. Since the scaffold protein SAP97 has been shown to be involved in the intracellular AMPARs traffic via its PDZ interaction with GluAl, we have studied its role in the GluAl vesicular trafficking. We show that an abolishment of the PDZ interaction between GluAl and SAP97 alters the GluAl vesicular trafficking. We are characterizing how interaction of GluAl with SAP97 can regulate the intracellular transport of AMPARs.
Extrasynaptic Protease, MMP-9 in Healthy and Diseased Mind
Leszek Kaczmarek*
Nencki Institute, Warsaw, Poland. * Presenting e-mail: l.kaczmarek@nencki.gov.pl
Matrix metalloproteinase 9, MMP-9 is an extracellularly operating enzyme that has been demonstrated as important regulatory molecule in control of synaptic plasticity, learning and memory. Either genetic or pharmacological inhibition of MMP-9 impairs late phase of long-term potentiation at various pathways, as well as appetitive and spatial memory formation, although aversive learning remains apparently intact in MMP-9 KO mice. MMP-9 is locally translated and released from the excitatory synapses in response to neuronal activity. Extrasynaptic MMP-9 is required for growth and maturation of the dendritic spines to accumulate and immobilize AMPA receptors, making the excitatory synapses more efficacious. Animal studies have implicated MMP-9 in such neuropsychiatric conditions, as e.g., epileptogenesis, autism spectrum disorders, development of addiction, and depression. In humans, MMP-9 appears to contribute to epilepsy, alcohol addiction, Fragile X Syndrome, schizophrenia and bipolar disorder. In aggregate, all those conditions may be considered as relying on alterations of dendritic spines/excitatory synapses and thus understanding the role played by MMP-9 in the synaptic plasticity may allow to elucidate the underpinnings of major neuropsychiatric disorders.
Spontaneous Neurotransmitter Release and Synaptic Plasticity
Elena Nosyreva*, Lisa M. Monteggia, and Ege T. Kavalali
Department of Neuroscience, UT Southwestern Medical Center, Dallas, TX 75390-9111, USA. * Presenting e-mail: elena.nosyreva@utsouthwestern.edu
Spontaneous neurotransmitter release is a salient feature of all presynaptic nerve terminals. Recent studies have shown that these action potential independent release events are essential regulators of synaptic homeostasis; in particular, they are involved in the maintenance of synaptic strength in terms of both presynaptic release rate and postsynaptic sensitivity. Moreover, there is growing evidence that postsynaptic receptors and signaling elements that respond to spontaneous release events diverge from those that respond to evoked release, suggesting a spatial segregation of these two forms of neurotransmission. We have previously shown that application of NMDA receptor antagonists - ketamine (20 |) and MK80l (l0 |M) at rest potentiates synaptic responses in the CA1 regions of rat and mouse hippocampus. This potentiation requires protein synthesis, brain-derived neurotrophic factor expression, eukaryotic elongation factor-2 kinase function, and increased surface expression of AMPA receptors. The same synaptic potentiation could be elicited by deplete neurotransmitter selectively from spontaneously recycling vesicles. In recent experiments, we found that this form of synaptic potentiation does not fully occlude subsequent long-term potentiation elicited by theta burst stimulation (100 Hz theta-burst protocol: 100 Hz, four pulses per burst; 15 bursts at 200 ms intervals). In these experiments, we could detect an additional ~20% increase in synaptic efficacy following ketamine mediated potentiation of responses (~30% above baseline). In this setting, theta burst stimulation alone could elicit up to ~50% potentiation above baseline. Taken together, these findings demonstrate that selective presynaptic impairment of spontaneous release, without alterations in evoked neurotransmission, is sufficient to elicit synaptic potentiation, which shows limited overlap with canonical long term potentiation elicited by repetitive activity.
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