Temporal lobe epilepsy (TLE) is a devastating disease in which aberrant synaptic plasticity plays a major role. We identify matrix metalloproteinase (MMP) 9 as a novel synaptic enzyme and a key pathogenic factor in two animal models of TLE: kainate-evoked epilepsy and pentylenetetrazole (PTZ) kindling–induced epilepsy. Notably, we show that the sensitivity to PTZ epileptogenesis is decreased in MMP-9 knockout mice but is increased in a novel line of transgenic rats overexpressing MMP-9. Immunoelectron microscopy reveals that MMP-9 associates with hippocampal dendritic spines bearing asymmetrical (excitatory) synapses, where both the MMP-9 protein levels and enzymatic activity become strongly increased upon seizures. Further, we find that MMP-9 deficiency diminishes seizure-evoked pruning of dendritic spines and decreases aberrant synaptogenesis after mossy fiber sprouting. The latter observation provides a possible mechanistic basis for the effect of MMP-9 on epileptogenesis. Our work suggests that a synaptic pool of MMP-9 is critical for the sequence of events that underlie the development of seizures in animal models of TLE.

Matrix metalloproteinase-9 has recently emerged as an important molecule in control of extracellular proteolysis in the synaptic plasticity. However, no synaptic targets for its enzymatic activity had been identified before. In this report, we show that ␤-dystroglycan comprises such a neuronal activity-driven target for matrix metalloproteinase-9. This notion is based on the following observations. (i) Recombinant, autoactivating matrix metalloproteinase-9 produces limited proteolytic cleavage of ␤-dystroglycan. (ii) In neuronal cultures, ␤-dystroglycan proteolysis occurs in response to stimulation with either glutamate or bicuculline and is blocked by tissue inhibitor of metalloproteinases-1, a metalloproteinase inhibitor. (iii) ␤-Dystroglycan degradation is also observed in the hippocampus in vivo in response to seizures but not in the matrix metalloproteinase-9 knock-out mice. (iv) ␤-Dystroglycan cleavage correlates in time with increased matrix metalloproteinase-9 activity. (v) Finally, ␤-dystroglycan and matrix metalloproteinase-9 colocalize in postsynaptic elements in the hippocampus. In conclusion, our data identify the ␤-dystroglycan as a first matrix metalloproteinase-9 substrate digested in response to enhanced synaptic activity. This demonstration may help to understand the possible role of both proteins in neuronal functions, especially in synaptic plasticity, learning, and memory. Matrix metalloproteinases (MMPs)2 are a family of zinc-dependent endopeptidases acting outside the cells and therefore attributed with digesting extracellular matrix components. These enzymes are produced in a latent form, and after release to extracellular space, they are activated by cleavage off the propeptide (1, 2). MMPs are involved in a number of physiological and pathological conditions, including development, tissue remodeling, inflammation, and tumor metastasis (1-4). Specifically, multiple data show increased expression and activity of MMPs after brain injury and in certain diseases of the central nervous system (5). On the other hand, the physiological roles of MMPs in the adult brain have only recently been appreciated (4). In particular, MMP-9 (also known as gelatinase B) has been implicated in synaptic plasticity, learning, and memory (6, 7). Furthermore, a marked increase in MMP-9 mRNA protein and its enzymatic activity in the hippocampal dentate gyrus after kainate-evoked seizures has been shown (8). Kainate, a glutamate analog, produces excitotoxicity in the CA subfields of the hippocampus, sparing the granule neurons of the dentate gyrus that, however, undergo aberrant plastic changes (9).Despite data implicating MMP-9 in neuronal/synaptic plasticity, no synaptic targets for its enzymatic activity have as yet been identified in neurons. However, recent studies have suggested that this enzyme may digest the 43-kDa ␤-dystroglycan (␤-DG) to release a 30-kDa product from the full-length subunit. First, Yamada et al. (10) have shown that unidentified MMPs digest ␤-DG to reveal the 30-kDa product in the perip...

Neurotransmitters released at synapses mediate Ca2+ signaling in astrocytes in CNS grey matter. Here, we show that ATP and glutamate evoke these Ca2+ signals in white matter astrocytes of the mouse optic nerve, a tract that contains neither neuronal cell bodies nor synapses. We further demonstrate that action potentials along white matter axons trigger the release of ATP and the intercellular propagation of astroglial Ca2+ signals. These mechanisms were studied in astrocytes in intact optic nerves isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP) under control of the human glial fibrillary acidic protein promoter (GFAP) by Fura-2 ratiometric Ca2+ imaging. ATP evoked astroglial Ca2+ signals predominantly via metabotropic P2Y1 and ionotropic P2X7 purinoceptors. Glutamate acted on both AMPA- and NMDA-type receptors, as well as on group I mGlu receptors to induce an increase in astroglial [Ca2+]i. The direct Ca2+ signal evoked by glutamate was small, and the main action of glutamate was to trigger the release of the "gliotransmitter" ATP by a mechanism involving P2X7 receptors; propagation of the glutamate-mediated Ca2+ signal was significantly reduced in P2X7 knock-out mice. Furthermore, axonal action potentials and mechanical stimulation of astrocytes both induced the release of ATP, to propagate Ca2+ signals in astrocytes and neighboring EGFP-negative glia. Our data provide a model of multiphase axon-glial signaling in the optic nerve as follows: action potentials trigger axonal release of ATP, which evokes further release of ATP from astrocytes, and this acts by amplifying the initiating signal and by transmitting an intercellular Ca2+ wave to neighboring glia.

The importance of dystrophin and its associated proteins in normal muscle function is now well established. Many of these proteins are expressed in nonmuscle tissues, particularly the brain. Here we describe the characterization of ␤-dystrobrevin, a dystrophin-related protein that is abundantly expressed in brain and other tissues, but is not found in muscle. ␤-dystrobrevin is encoded by a 2.5-kb alternatively spliced transcript that is found throughout the brain. In common with dystrophin, ␤-dystrobrevin is found in neurons of the cortex and hippocampal formation but is not found in the brain microvasculature. In the brain, ␤-dystrobrevin coimmunoprecipitates with the dystrophin isoforms Dp71 and Dp140. These data provide evidence that the composition of the dystrophinassociated protein complex in the brain differs from that in muscle. This finding may be relevant to the cognitive dysfunction affecting many patients with Duchenne muscular dystrophy.