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Extracellular proteases are critical for normal cell regulation. One such class of ECM proteases, MMPs are a family of extracellular proteases which have been characteristically associated with ECM remodelling including tissue morphogenesis, wound healing, angiogenesis, neuronal growth, cell differentiation, migration, regulation of growth factor and apoptosis. These proteolytic enzymes are thought to be mediators of ECM degradation and collectively have the capacity to breakdown all the ECM protein components [3, 4]. Other than ECM, growth factors, receptors and adhesion molecules are also known substrates for MMPs [3-5]. Because of these activities MMPs can also cause cellular survival, signalling and inflammation .
Other physiological and reparative processes involving MMPs include synaptic remodelling, long term potentiation, Nogo signalling, regulation of neural stem biology and remyelination .
Studies have revealed at least 25 members of MMP family and together these enzymes can break down all constituents of ECM . Due to their inherent proteolytic potential, MMPs production is very tightly regulated and their abnormal activity or over expression along with altered MMPs:TIMPs (Tissue Inhibitors of Metalloproteinases) ratio  has been linked to several diseases and many pathological processes such as aortic aneurysm, atherosclerosis, gastric ulcer, rheumatoid arthritis, periodontal disease, fibrotic lung disease, liver cirrhosis and cancer metastasis.
In central nervous system MMPs have been associated with diseases such as multiple sclerosis (MS), malignant gliomas, stroke, Alzheimer's disease, Parkison's disease and viral infections . In this review we will discuss MMPs and their pathological upregulation in Multiple Sclerosis.
MMPs belong to a structurally similar zinc dependant larger family of metalloproteinases, referred to as metzincins which include ADAMs, astacins and bacterial serralysins. In metzincins family, three histidine residues bind to the zinc ion which is located at their active site. The occurrence of residues is in conserved sequence motif HExxHxxGxxHZ where Z is the family specific residue which in case of MMPs is mostly serine. Also present at active site is an unobscure B-turn, delineated by methionine residue, which is deemed essential for enzymatic activity . There is about 20% homology between metzincin subfamilies which increases markedly at the catalytic domain. Based on protein domain consideration and substrate preference, MMP family members can be further subgrouped into gelatinases, stromelysins, collagenases, membrane-type (MT)-MMPs and 'other MMPs' . On basis of chemical structure, MMPs can be divided into three domains namely: 1) an amino terminal propeptide region, 2) an amino-terminal catalytic domain which contains the zinc-binding motif and 3) a carboxy-terminal domain which is involved in binding of ECM substrate for many MMPs . A hinge region connects the carboxy and amino terminal domains. This hinge region is short in collagenases and longer in 'other MMPs'. Also present is a short signal sequence which is present at the amino terminal of the protein and precedes the propeptide region. This pre-domain is clipped off as the newly formed MMPs are transported to the cell surface. Lastly, the six MT-MMPs are the only members of the MMPs family which are membrane proteins .
Generally all MMPs are secreted from the cells with the exception of six membrane-type MMPs (MT-MMPs). However these MMPs can also stick on cell surface whereby they increase the proteolytic capacity through binding with MT-MMP cell adhesion molecules, integrins and cell surface proteoglycans .
MMPs Gene Expression and Activation
Various cytokines, chemokines and growth factors induce MMP gene expression differentially in different cells at transcriptional level . The regulation of MMP gene expression via transcription is also affected by various transcription factors such as single nucleotide polymorphisms (SNPs) and arrangement of promoter elements in a promoter region. Cis-elements which are known to influence MMP expression include AP1, AP2, Ets, Sp1, Sp3, p53 response elements and retinoic acid.
Most MMPs are activated from their secreted latent proenzyme state by cleavage of propeptide domain. This process involves disruption of cysteine-zinc interaction at the catalytic site. Other already activated MMPs play a pivotal role in final activation of most newly synthesized MMP molecules and this activation occurs extracellularly. For instance, MMP2 activates proMMP1, 9 and 13 and MMP3 is involved in activation of proMMP1, 7, 8, 9 and 13. MMP9 on the other hand is not known to activate other MMPs [1, 7].
Regulation of MMPs Activity
There is tight regulation of MMPs activity as these proteolytic enzymes if let loose can potentially wreck havoc. Their activity is regulated at three levels . Firstly at the level of transcription as most MMPs unlike other proteins, are only transcribed after cell activation rather than being produced constitutively. Factors such as inflammatory cytokines, chemokines, cell-cell, cell-matrix interactions, oncogenes and growth factors stimulate transcription of many of MMPs. Second regulatory step is post-translational modifications. Most MMPs are synthesized as inactive zymogens which are activated by plasminogen-plasmin cascade and other MMPs. Their activation involves disruption of interaction between cysteine and zinc which is followed by removal of propeptide region which culminates in full activation. Other, non proteolytic compounds such as urea and 4-aminophenylmercuric acetate are also known to activate the inactive zymogens . Lastly, the MMP activity is regulated by tissue inhibitors of metalloproteinases (TIMPs). Studies have revealed presence of four TIMPs which bind to catalytic site on MMPs leading to their inactivation .
Role of MMPs in CNS Diseases
Although PCR and RNAse Protection Assays have revealed presence of low levels of MMP 2,3,7,9,11,13,14 and 17 in adult mice central nervous system but by and large MMPs are absent in normal CNS  and their upregulation has been linked to many neurological diseases and they have also been found upregulated post CNS injury .
MS and Upregulation of MMPs in its Pathology
Multiple sclerosis an immune mediated degenerative disease with predisposition for genetically susceptible individuals. It affects around 1 million people worldwide and it is the number one cause of non traumatic disability in young adults in North America .
Although the precise triggering or etiological factors are still unknown but research has revealed that course of this pathological process involves two main processes ; demyelination and neurodegeneration of the central nervous system (CNS) . The hallmark of multiple sclerosis is deposition of demyelinated plaque which causes disruption of blood brain barrier especially during the early phase of its deposition . Although the disease is characterized by plaque deposition, it also involves scarring of axons as well as other broad spectrum signs and symptoms .
MMPs are known to cause tissue damage in MS by two mechanisms; firstly they are secreted by the activated inflammatory cells to degrade endothelial lining of vessels allowing extravasation of inflammatory cells from the blood vessels into parenchyma . This has been confirmed in animal studies where prevention of infiltration or activation of T cells or macrophages lead to amelioration of the disease process .
Second mechanism by which increased expression of MMPs can exacerbate the pathological process is by activating in active forms of certain inflammation mediators for instance tumor necrosis factor alpha which in turn propagate the disease process. Furthermore MMPs also break down the myelin sheath within the CNS parenchyma. The break down products of this proteolytic activity cause further tissue damage initiating a cascade of events resulting in demyleniation and inflammation within the CNS .
Animal Studies involving MMPs in Multiple Sclerosis
Studies revealed increased expression of MMP3,7,9 and 12 in experimentally induced auto-immune encephalomyelitis (EAE), a variant of MS, in rat and mouse models. Induction of EAE in the animals results in initiation of acute inflammatory response in the blood vessels within the CNS. This is followed by fibrin deposition around the vessels with subsequent transmigration of white blood cells within CNS parenchyma .
The relationship between increased MMP expression and disease is most likely a causal one as inhibition of MMP activity ameliorates or prevents EAE [1, 10]. Rats suffering from clinically induced EAE showed elevated expression of mRNA for MMP-3, 7 and 9 . In an animal study by Rosenberg et al.,  it was revealed that MMP-2 when injected in rat brain lead to disruption in the blood brain barrier and when such animals were treated with TIMP-2, the openings in the blood brain barrier were blocked. Further experiments by the same group  also revealed that endogenous production of MMP-9 was induced following intracerebral hemorrhage. They also observed that production of MMP-9 was at peak after 24 hours following TNF-alpha injections which corresponded with opening in blood brain barrier . Others have shown disruption of blood brain barrier and tissue destruction following MMP injection in rat brain as well [13, 15].
EAE animal models have revealed that during peak severity of disease eleven MMPs are simultaneously elevated including MMP-2, 3, 8, 9, 10, 11, 12, 13, 14 and 25 . It has also been shown, that MMP-2 knockout mice have a more severe disease pattern in comparison to the wild type mice. But this exacerbation is due to compensatory increase in MMP-9 in absence of MMP-2 . Furthermore it has also been revealed that EAE could not be induced in MMP-2 and 9 double knockout mice .
Another research group showed that MMP-8 knockout mice show significant reduction in demyelinating lesions, inflammatory cell infiltration and overall severity of EAE disease. Furthermore animals suffering from EAE when treated with MMP-8 inhibitor showed resolution of disease process .
In vitro and in vivo studies show that MMP12 knockout mice macrophages have markedly reduced basement membrane penetration potential [1, 17]. Experiments showed that when T cells which were applied to a monolayer of endothelial cells overlying a collagen matrix were subjected to metalloproteinase inhibitor, they failed to penetrate the collagen matrix where as their capacity to penetrate endothelial cells remained intact .
Human Studies involving MMPs in Multiple Sclerosis
Recent studies have revealed that some of the proteinases which were first discovered in CSF of MS patients 20 years ago, are also MMPs . Research data reveals MMP9 which is not present in CSF of healthy adults is upregulated in neuro inflammatory diseases including MS . In a study by Lee and his colleagues serum analysis also revealed an elevated MMP-9 level in MS patients in comparison to the controls . In another paper published by Leppert and his colleagues it was found that MMP-9 expression was significantly increased in CSF of relapsing MS patients when compared to the control subjects . Similar results were found in another study where it was found that MMP-9 expression was elevated in relapsing patients in comparison to stable patients . Other MMPs have also been implicated in relapsing-remitting MS patients. Experiments have shown increased number of leukocytes expressing MMP-1, 3 and 7 in such patients. There is also an increased expression of MMP7 and 14 mRNA and elevated levels of MMP-2, 14 and TIMP-2 in monocytes of such patients .
Brain tissue of patients dying from MS also revealed that there was increase in the expression of MMP-2, 7 and 9 in inflammatory cells like lymphocytes and macrophages especially around the blood vessels [16, 22, 23].
Studies involving humans have also shown that interferon-B, a drug used for treatment of MS, lowers the serum MMP9 level with reduction in MMP9 expressing leukocytes . This has also been confirmed in vitro as interferon-B has been shown to attenuate MMP9 production by T cells which leads to reduced capacity of T cells to penetrate ECM barriers [1, 25, 26]. Moreover patients who have relapsing disease also show reduced plasma levels of MMP-9 when treated with interferon- B .
There are several challenges linked with relating the role of MMPs in MS. Firstly, any given MMP might behave differently at a given location and under given conditions. For instance, MMP9 produced by a macrophage in close proximity to a myelin sheath may cause demyelindation where as the same molecule may induce remyelination if it is produced by an oligodendrocyte at the tip of its processes . So the challenge lies in identifying all the processes associated with a MMP protease and then evaluating if its overall inhibition is more beneficial or detrimental. Another challenge is to decipher the link of a given MMP with a particular function. For example many of the pharmacological inhibitors available today lack specificity towards members of subfamily which leads to non specific antagonization of other members of the MMP families (10, 11). Ideally the new generation inhibitors should be able to distinguish between subfamilies and members of each subfamily.
Another important aspect is study of MMPs expression at several levels including transcription, protein expression and enzymatic activity. This is crucial as neutrophils have preformed MMPs such as MMP9 (24) which may be the cause of immediate elevation of MMP9 associated with neutrophil infiltration in CNS injuries [28, 29] without detection of significant rise in MMP transcription which is measured immediately after an injury. This highlights that transcription alone my under estimate the MMP9 levels at site of injury.
Further studies of functions of MMPs in MS will shed light on nervous system physiology and pathology and its findings can potentially reveal role of MMPs throughout human body in not just pathological conditions but physiological state as well .
Blood Brain Barrier
MMPs impair BBB
Conversion of pro-forms of inflammatory molecules into mature forms
Produces encephalogens from myelin proteins
MMPs in Multiple Sclerosis: MMPs produced by Leukocytes impair the Blood Brian Barrier initiating a cascade of inflammatory response leading to demyelination and scarring