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MMP-9 displays a particular time-response to acute stress: variation in its levels and activity distribution in rat hippocampus Felipe Aguayo, Anibal Pacheco, Gonzalo J. García Rojo, Javier Pizarro-Bauerle, Ana Doberti, Macarena Tejos, Alexandra García-Pérez, Paulina Rojas, and Jenny L Fiedler ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00387 • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 24, 2018
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ACS Chemical Neuroscience
MMP-9 displays a particular time-response to acute stress: variation in its levels and activity distribution in rat hippocampus 1
Felipe I. Aguayo†, 1Aníbal A. Pacheco†, 1Gonzalo J. García-Rojo, 1Javier A. PizarroBauerle, 1Ana V. Doberti, 1Macarena Tejos, 1María A. García-Pérez, 2Paulina S. Rojas and 1 Jenny L. Fiedler*. 1 Laboratorio de Neuroplasticidad y Neurogenética, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile. 2 Facultad de Química y Farmacia. Universidad Andres Bello. † Both authors contributed equally to this study.
Corresponding author: Dr. Jenny L. Fiedler, Department of Biochemistry and Molecular Biology, Faculty of Chemical and Pharmaceutical Sciences, Universidad de Chile, P.O.BOX. 233, Santiago 1, Chile Phone: +562-978-2923, Fax: +562-737-8920, e-mail:
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ABSTRACT
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A single stress exposure facilitates memory formation through neuroplastic processes that
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reshape excitatory synapses in the hippocampus, probably requiring changes in
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extracellular matrix components. We tested the hypothesis that matrix metalloproteinase 9
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(MMP-9), an enzyme that degrades components of extracellular matrix and synaptic
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proteins such as β-dystroglycan (β-DG43), change their activity and distribution in rat
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hippocampus during the acute stress response. After 2.5 h of restraint stress we found that:
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i) MMP-9 levels and potential activity increase in whole hippocampal extracts,
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accompanied by β-DG43 cleavage, and ii) a significant enhancement of MMP-9
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immunoreactivity in dendritic fields such as stratum radiatum and molecular layer of
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hippocampus. After 24 h of stress, we found that i) MMP-9 net activity rises at somatic
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field; i.e., stratum pyramidale and granule cell layers and also at synaptic field, mainly
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stratum radiatum and
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synaptoneurosome fractions are enriched in MMP-9, without variation in its potential
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enzymatic activity, in accordance with the constant level of cleaved β-DG43. These findings
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indicate that stress triggers a peculiar timing response in MMP-9 levels, net activity and
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subcellular distribution in the hippocampus, suggesting its involvement in the processing of
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substrates during the stress response.
molecular layer of
hippocampus
and
ii) hippocampal
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Keywords: acute stress, hippocampus, MMP-9, synaptoneurosomes, β-dystroglycan, zymography
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Introduction
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environment is called neuroplasticity. This response involves synaptic remodeling, which
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includes formation and stabilization of new neural connections that enable important and
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complex processes, such as learning and memory consolidation 1. The mechanisms that
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regulate neuroplasticity can be modulated through several factors. For instance, the stress
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response triggered by environmental challenges (either external or internal) produces a
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myriad of mediators that elicit well-orchestrated physiological modifications that may
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impact neuroplasticity processes. Stress induced by an emotional stressor activates the
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hypothalamic-pituitary-adrenal (HPA) axis, with a consequent release of adrenal
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glucocorticoids (cortisol in human and corticosterone in rat); one of the stress hormones
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that targets several limbic structures, such as the hippocampus 2. This structure is
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implicated in the acquisition of episodic or declarative memory in humans, and spatial
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memory in rodents
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promoting the release of glutamate from cortex and hippocampal areas 5, 6, accompanied by
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the subsequent activation of AMPA and NMDA receptors
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formation negatively regulates HPA axis activity, evidencing their cross regulation 8.
The ability of the brain to change and adapt in response to experience and a fluctuating
3, 4
. Moreover, glucocorticoids trigger fast actions in the brain, 5-7
. Interestingly, hippocampal
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Acute stress seems to facilitate memory formation when the learning takes place in a
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stressful context 9, but usually impairs retrieval
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formation involves neuroplastic processes that allow the reorganization of excitatory
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synapses, changing the balance between the formation and stabilization of dendritic spines
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that may sustain the functionality of neural circuits 11. Several studies support the idea that
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synaptic remodeling requires dynamic changes in the expression of cell-surface adhesion
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molecules (reviewed in
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matrix (ECM)
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proteolytic processing of its components supports a physiological mechanism to shape
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neuronal circuits, such as those described in the hippocampus
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enzymes correspond to matrix metalloproteinases (MMPs), which play a key role in both
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central nervous system (CNS) development and normal brain functioning
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metalloproteinase-9 (MMP-9) and its natural inhibitor, tissue inhibitor of metalloproteinase
13
12
10
. Probably, facilitation of memory
), as well as modifications in components of the extracellular
. The ECM is a meshwork of proteins and proteoglycans in which 14
. One set of degradative 15
. Matrix
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1 (TIMP-1) are expressed at low levels in the mature brain; nonetheless, they are induced in
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response to physiological and pathological conditions 16-18. For instance, increased MMP-9
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expression has been detected in several brain areas, including the hippocampus, following
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fear conditioning
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proteolytic activation of MMP-9 occur during the late-phase of long-term potentiation (L-
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LTP) at CA3–CA1 synapses in the hippocampus
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cultures exposed to glutamate suggest that the signaling of glutamate receptors is required
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for the activation of MMP-9
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growth factors and precursors 24, components of ECM such as laminin and the chondroitin
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sulphate proteoglycan brevican 25, the intercellular adhesion molecules I-CAM 5 26 and the
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neural cell adhesion molecule N-cadherin 27, among others.
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. Furthermore, studies have shown that both the expression and
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20-22
. Experiments with cortical primary
. MMPs cleave a large number of substrates, including
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Some in-vivo exclusive substrates of MMPs have also been described in the brain. For
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instance, Nectin-3 corresponds to a cellular adhesion, Ig-like, transmembrane protein
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predominantly expressed postsynaptically
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synaptic sites are reduced by chronic stress; variations which have been associated to an
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enhancement in gelatinolytic activity of MMP-9
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lower expression of Nectin-3 has been associated with alterations in cognitive function
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and social recognition
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described as a substrate for MMP-2 and MMP-9 in an experimental model of autoimmune
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encephalopathy 30, is also processed in vivo by MMP-9 in response to enhanced excitatory
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neurotransmission in the hippocampus 23. β-DG43 is a dual-function protein which acts as a
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signal transducer between the ECM and the cytoskeleton
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morphogenesis of hippocampal neurons in vitro
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play a key role in neuronal morphology by promoting the elongation of dendritic spines 21,
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33
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excitatory synapse 23. Considering that stress influences glutamate neurotransmission5, 6 and
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promotes remodeling of the brain 34, the aim of this study was to evaluate the consequences
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of acute stress on MMP-9 levels and activity in the hippocampus; variations that may be
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useful to understand the mechanisms that transform an adaptive response into a
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maladaptive one.
29
28
. In the hippocampus, Nectin-3 levels at 29
. From a functional point of view, the
. Additionally, β-dystroglycan 43 (β-DG43), which was first
32
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, and controls dendritic
. Similarly, changes in MMP-9 activity
. Importantly, in the hippocampus, β-DG43 and MMP-9 are colocalized in vivo at the
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Results and discussion
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2.5 hours. This stressor produces the activation of the HPA axis, inducing a fast secretion of
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corticotropin releasing hormone (CRH)
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procedure by measuring fecal output, which is driven by CRH release in the brain
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significant increase in the number of fecal pellets voided by stressed animals was observed
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during the 2.5 h of restraint in comparison to unstressed animals (Fig 1A, P
