Cyclic nucleotide-gated channel subunit glycosylation regulates matrix metalloproteinase-dependent changes in channel gating.

Cyclic-nucleotide gated (CNG) channels are essential for phototransduction within retinal photoreceptors. We have demonstrated previously that the enzymatic activity of matrix metalloproteinase-2 and -9, members of the matrix metalloproteinase (MMP) family of extracellular, Ca(2+)- and Zn(2+)-dependent proteases, enhances the ligand sensitivity of both rod (CNGA1 and CNGB1) and cone (CNGA3 and CNGB3) CNG channels. Additionally, we have observed a decrease in the maximal CNG channel current (Imax) that begins late during MMP-directed gating changes. Here we demonstrate that CNG channels become nonconductive after prolonged MMP exposure. Concurrent with the loss of conductive channels is the increased relative contribution of channels exhibiting nonmodified gating properties, suggesting the presence of a subpopulation of channels that are protected from MMP-induced gating effects. CNGA subunits are known to possess one extracellular core glycosylation site, located at one of two possible positions within the turret loop near the pore-forming region. Our results indicate that CNGA glycosylation can impede MMP-dependent modification of CNG channels. Furthermore, the relative position of the glycosylation site within the pore turret influences the extent of MMP-dependent proteolysis. Glycosylation at the site found in CNGA3 subunits was found to be protective, while glycosylation at the bovine CNGA1 site was not. Relocating the glycosylation site in CNGA1 to the position found in CNGA3 recapitulated CNGA3-like protection from MMP-dependent processing. Taken together, these data indicate that CNGA glycosylation may protect CNG channels from MMP-dependent proteolysis, consistent with MMP modification of channel function having a requirement for physical access to the extracellular face of the channel.

Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels.

Photoreceptor cyclic nucleotide-gated (CNG) channels are the principal ion channels responsible for transduction of the light-induced change in cGMP concentration into an electrical signal. The ligand sensitivity of photoreceptor CNG channels is subject to regulation by intracellular signaling effectors, including calcium-calmodulin, tyrosine kinases and phosphoinositides. Little is known, however, about regulation of channel activity by modification to extracellular regions of CNG channel subunits. Extracellular proteases MMP9 and -2 are present in the interphotoreceptor matrix adjacent to photoreceptor outer segments. Given that MMPs have been implicated in retinal dysfunction and degeneration, we hypothesized that MMP activity may alter the functional properties of photoreceptor CNG channels. For heterologously expressed rod and cone CNG channels, extracellular exposure to MMPs dramatically increased the apparent affinity for cGMP and the efficacy of cAMP. These changes to ligand sensitivity were not prevented by destabilization of the actin cytoskeleton or by disruption of integrin mediated cell adhesion, but could be attenuated by inhibition of MMP catalytic activity. MMP-mediated gating changes exhibited saturable kinetic properties consistent with enzymatic processing of the CNG channels. In addition, exposure to MMPs decreased the abundance of full-length expressed CNGA3 subunits, with a concomitant increase in putative degradation products. Similar gating effects and apparent proteolysis were observed also for native rod photoreceptor CNG channels. Furthermore, constitutive apparent proteolysis of retinal CNGA1 and retinal MMP9 levels were both elevated in aged mice compared with young mice. Together, these results provide evidence that MMP-mediated proteolysis can regulate the ligand sensitivity of CNG channels.

Effects of matrix metalloproteinase inhibition on short- and long-term plasticity of schaffer collateral/CA1 synapses.

It is increasingly evident that matrix metalloproteinases (MMPs), a family of zinc containing extracellular endopeptidases, participate in processes supporting hippocampal synaptic plasticity. The purpose of this study was to further the understanding of MMPs involvement in hippocampal plasticity. Acute hippocampal slices, generated from 20- to 30-day-old male Sprague-Dawley rats, were subjected to various electrophysiologic stimulatory paradigms to produce either short-term or long-term modifications to synaptic efficacy. Slices exposed to broad-spectrum MMP inhibitor, FN-439, exhibited impairments in paired-pulse facilitation, theta-burst facilitation, and long-term depression. Additionally, we observed that MMP inhibition impaired both the induction and stability of long-term potentiation (LTP). Furthermore, evidence indicated that the effect of MMP inhibition on LTP maintenance is dependent upon integrin-directed adhesion, whereas the effects of MMP inhibition on LTP induction are independent of integrin-directed adhesion. Together, these data support a generalized role for MMPs in short-term and long-term hippocampal plasticity and indicate that MMPs are a necessary facet of integrin-mediated cell adhesion supporting LTP stabilization.

Habituation of the head-shake response induces changes in brain matrix metalloproteinases-3 (MMP-3) and -9.

Habituation is defined as a decrease in responsiveness to a repeatedly presented stimulus. The head-shake response (HSR) demonstrates several fundamental properties of habituation including sensitivity to the frequency and intensity of stimulation, and spontaneous recovery. This response shows behavioral plasticity; however the neural plasticity presumed to underlie this behavioral phenomenon has only recently been investigated. The present study initially compared male and female rats and noted equivalent habituation and spontaneous recovery. A second experiment utilized female rats to test the hypothesis that habituation induces changes in neural plasticity. At inter-session intervals (ISIs) of 5 min, 2, 6, and 24 h following HSR habituation independent groups of rats received a second habituation experience, then tissue samples were immediately collected from hippocampal, prefrontal and piriform cortices, and cerebellum. Western blots indicated significant elevations in the expression of matrix metalloproteinase-3 (MMP-3) in hippocampal, prefrontal and piriform cortices at a delay interval of 2 h, and in the prefrontal cortex at 24 h in habituated rats. Increases in active and pro MMP-9 activity were measured by zymography in the hippocampus of habituated rats over yoked controls. Decreases in active MMP-9 activity were seen in the prefrontal cortex, and in pro MMP-9 in the piriform cortex, of habituated as compared with yoked control rats. No changes in MMP-3 or MMP-9 were observed in the cerebellum, and no changes in MMP-2 were seen in any of the four structures examined. These results suggest that habituation of the HSR produced elevations in MMP-3 expression in three of the four structures presently examined, accompanied by increased MMP-9 activity in the hippocampus and decreases in the prefrontal cortex. However, cues present in the test environment appear to have provoked elevations in MMP-3 and -9 independent of those accompanying habituation.

Effects of extracellular matrix-degrading proteases matrix metalloproteinases 3 and 9 on spatial learning and synaptic plasticity.

Rats learning the Morris water maze exhibit hippocampal changes in synaptic morphology and physiology that manifest as altered synaptic efficacy. Learning requires structural changes in the synapse, and multiple cell adhesion molecules appear to participate. The activity of these cell adhesion molecules is, in large part, dependent on their interaction with the extracellular matrix (ECM). Given that matrix metalloproteinases (MMPs) are responsible for transient alterations in the ECM, we predicted that MMP function is critical for hippocampal-dependent learning. In support of this, it was observed that hippocampal MMP-3 and -9 increased transiently during water maze acquisition as assessed by western blotting and mRNA analysis. The ability of the NMDA receptor channel blocker MK801 to attenuate these changes indicated that the transient MMP changes were in large part dependent upon NMDA receptor activation. Furthermore, inhibition of MMP activity with MMP-3 and -9 antisense oligonucleotides and/or MMP inhibitor FN-439 altered long-term potentiation and prevented acquisition in the Morris water maze. The learning-dependent MMP alterations were shown to modify the stability of the actin-binding protein cortactin, which plays an essential role in regulating the dendritic cytoskeleton and synaptic efficiency. Together these results indicate that changes in MMP function are critical to synaptic plasticity and hippocampal-dependent learning.

Ethanol-induced impairment of spatial memory and brain matrix metalloproteinases.

The formation of spatial memory appears to be dependent upon an intact hippocampus capable of the specific biochemical changes associated with synaptic remodeling. Hippocampal damage results in the disruption of synaptic remodeling and the acquisition of spatial memory tasks. Ethanol also disrupts normal hippocampal functioning and spatial memory. The present investigation established a dose-response relationship between ethanol treatment and impairment of spatial memory as measured using the circular water maze task. Intraperitoneal ethanol doses of 1.5 and 2 g/kg significantly increased the latency and distance swam to find the submerged pedestal as compared with a 1 g/kg dose, and 0.15 M NaCl vehicle control treatments. On days 2, 4, and 6 of acquisition animals were sacrificed and brain tissues were retained from the hippocampus, prefrontal neocortex, and cerebellum for measurement of matrix metalloproteinases (MMPs). The results indicated that ethanol treatment interfered with MMP-9, but not MMP-2, activity in the hippocampus, and to a lesser degree in the prefrontal cortex. No changes in the cerebellum were measured. Elevations in MMP activity appear to be a prerequisite to reconfiguration of extracellular matrix cell adhesion molecules thought to be important in the process of synaptic plasticity, which in turn appears to be necessary for memory consolidation. Thus, ethanol-induced impairment in the acquisition of spatial memory tasks may, in part, be due to disruption of brain MMP activity.

Extracellular matrix molecules, long-term potentiation, memory consolidation and the brain angiotensin system.

Considerable evidence now suggests an interrelationship among long-term potentiation (LTP), extracellular matrix (ECM) reconfiguration, synaptogenesis, and memory consolidation within the mammalian central nervous system. Extracellular matrix molecules provide the scaffolding necessary to permit synaptic remodeling and contribute to the regulation of ionic and nutritional homeostasis of surrounding cells. These molecules also facilitate cellular proliferation, movement, differentiation, and apoptosis. The present review initially focuses on characterizing the ECM and the roles of cell adhesion molecules (CAMs), matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), in the maintenance and degradation of the ECM. The induction and maintenance of LTP is described. Debate continues over whether LTP results in some form of synaptic strengthening and in turn promotes memory consolidation. Next, the contribution of CAMs and TIMPs to the facilitation of LTP and memory consolidation is discussed. Finally, possible roles for angiotensins, MMPs, and tissue plasminogen activators in the facilitation of LTP and memory consolidation are described. These enzymatic pathways appear to be very important to an understanding of dysfunctional memory diseases such as Alzheimer's disease, multiple sclerosis, brain tumors, and infections.