Dhananjay Duberkar,Department of Neurology, PGIMER

MATRIX METALLOPROTEINASE IN NEUROLOGICAL DISORDERS

WHAT ARE MATRIX METALLOPROTEINASES (MMPs)?

• MMPs have following functional characteristics:

1. Proteinases that degrade at least one component of the extracellular matrix

2. Contain a zinc ion & are inhibited by chelating agents

3. Secreted in latent form, requiring activation for proteolytic activity

4. Inhibited by tissue inhibitors of metalloproteinases (TIMPs)

5. Share common amino acid sequences

WHY ARE WE INTERESTED IN MMPS?

• Increase in research activity pertaining to MMPs & A disintegrin & metalloproteinases (ADAMs), in nervous system

• Implicated in myriad of neurological conditions, & also associated with important neurophysiological functions, including synaptic remodeling & long-term potentiation (LTP)

• More recently, demonstration that MMPs regulate neural stem cell biology & remyelination suggests their importance in regeneration of nervous system

• MMPs & ADAMs are important in neuroinflammation, & recently they are linked to neurodegenerative disorders

ADAMs & TIMPS

INTRODUCTION TO MMPs

METZINCINS

ASTACINS SERRALYSINS MMPs

COLLAGENASE

GELATINASE

STROMEOLYSINS

MT-MMP

ADAMALYSINS

SNAKE VENOM MMPS

ADAMS

GENERAL PROPERTIES OF MMPs

• There are 24 mammalian MMP members (MMP1–28), each product of different gene

• MMP4–6 are no longer used,MMP18 only been cloned from Xenopus, MMP22 is present in chickens & two versions of human MMP23

• These can be subdivided on basis of domain structure

• With exception of the six membrane-type MMPs (MT-MMPs), MMP family members are generally secreted from cells

• Signal peptide & propeptide region form part of cysteine switch, which folds over zinc in catalytic site & maintains latent state

• Cleavage site enables proconvertase furin to activate MMP by cleaving propeptide

• FN binding site is present in MMP2 & 9, connecting them with basal lamina. Catalytic zinc site is present in all MMPs

• Haemopexin domain is joined to catalytic site by hinge region. MMP14 (an MT-MMP) has TMD

• Collagenases– Key feature is ability to cleave interstitial collagens I, II, and III at a

specific site three-fourths from N-terminus– It can also digest number of other ECM & non-ECM molecules

• Gelatinases– Gelatinase A (MMP-2) & gelatinase B (MMP-9) digest denatured

collagens, gelatins & no. of ECM including native type IV, V, XI collagen, laminin

• Stromelysins– Besides digesting ECM components, MMP-3 activates a number of

proMMPs, & its action on partially processed proMMP-1 is critical for generation of fully active MMP-1

• Matrilysins– Matrilysin 1 (MMP-7) & matrilysin 2 (MMP-26), also called

endometase, are in this group– Besides ECM components, MMP-7 processes cell surface

molecules such as pro–-defensin, Fas-ligand, pro–tumor necrosis factor (TNF)-, & E-cadherin to generate soluble forms hence acting as ‘sheddases’

• Membrane-Type MMPs (6)– 4 are type I transmembrane proteins (MMP-14,15, 16,& 24),& 2

are GPI anchored proteins (MMP-17 & 25)

– With exception of MT4-MMP, all capable of activating proMMP-2 & digest number of ECM molecules, & MT1-MMP has collagenolytic activity on type I, II, & III collagens

ADAMs

• Transmembrane proteins that bind to integrins & important in intracellular signaling & cell adhesion

• Chara. function - proteolytic processing of membrane-anchored precursors & subsequent release of mature proteins .This process is referred to as ‘protein ECTODOMAIN SHEDDING’

• Proteins modulated include cell-surface receptor Notch & its ligand Delta, important in CNS development, amyloid precursor protein implicated in AD(ADAM-10 an α- secretase) ,TNF-α (ADAM-17 known as TACE- TNF-α converting enzyme), & ligands of EGF receptor

• ADAMs are implicated in cellular proliferation, migration, differentiation, & survival, in axonal growth & myelination

• TIMPs

• TIMPs are small proteins & these enzymes are codified by highly conserved genes and have overlapping functions

• Four TIMPs have been identified

• TIMPs are inhibitory against most MMPs with some predilections: TIMP1 inhibits MMP9, TIMP2 inhibits MMP2 &, paradoxically, contributes to activation of pro-MMP2

• TIMP3 has role in cellular growth, cellular death, & tissue repair

ACTIONS OF TIMP – 3

Activation of ProMMPs

• MMPs can be activated by proteinases or in vitro by chemical agents, such as thiol-modifying agents

• Activation of proMMPs by plasmin is relevant in vivo

• Activated MMPs can participate in processing other MMPs

• Fine regulatory mechanisms to control destructive enzymes, TIMPs may interfere with activation by interacting with intermediate MMP before it is fully activated

Role of MMPs in CNS

1. MMPs regulate CNS development –

• MMP’s are expressed during nervous system ontogeny & CNS abnormalities occur when their activity is perturbed or impaired

• In particular role in neurogenesis ,angiogenesis, development of oligodendrocytes & myelin

2. MMPs have beneficial role in healthy CNS -

• Within adult CNS MMPs function in remodeling ECM. ECM will in turn regulate cell migration & survival

• Neurons remodel their synaptic connections in response to various stimuli. Synaptic environment including ECM , play imp role in neuronal plasticity & learning & memory

3. MMPs in repair of adult CNS – • It is important that following initial & likely detrimental up

regulation of several MMPs after injury, delayed, subtle & restricted expression of specific MMPs have beneficial role

• MMP-9 expressed 7 days after demyelinating insult to the spinal cord of mice promotes maturation of oligodendrocytes & their formation of myelin

• Delayed expression of MMP-2 after traumatic spinal cord is necessary for ECM remodeling & functional recovery.

MMPS IN THE CNS: WHERE THE GOOD GUYS GO BAD?

NEUROINFLAMMATION

• CNS injury initiates cascade of events defined as neuroinflammation

• Cytokine & chemokine response associated with production of free radicals & proteases

• TNFα, interleukin 1β, & chemokines, are implicated and tissue responses differ with specific Injury

• Factors that affect outcome of inflammatory process include presence of hypoxia–ischemia, duration of injury, presence of infection & types of cells involved

MMPs in hypoxia–ischemia

• Basal lamina around cerebral blood vessels contains ECM proteins, including laminin, fibronectin, heparan sulphate, & type IV collagen

• Proteolysis of BBB by MMPs results in loss of basal lamina proteins, which increases the risk of hemorrhage

• MMP2, MMP3, and MMP9 increase permeability of BBB. Inhibitors of MMPs can reduce damage to BBB

• In ischemia with reperfusion, MMPs are induced & disrupt BBB; for instance, in Mmp9 knockout model, focal ischemic lesions decreased the damage to the blood–brain barrier & infarct size

MMP IN AUTOIMMUNE DISORDER & INFECTION

• Immunological or infectious pathogen, main injury site is blood vessels alone. Whenever BBB is affected, MMP9 is key factor in injury process

• Exacerbation of acute MS increases MMP9 concentrations in CSF & treatment with steroid reduces MMP9 concentrations

• Treatment with MMP inhibitor GM-6001 suppresses development of clinical experimental allergic encephalomyelitis in mice

• In rats with experimental allergic neuritis, a broad spectrum MMP inhibitor, BB-1101, which also inhibits TACE, reduces damage to nerves

Metalloproteinases regulate myelinogenesis. A. MMP9/12 in maturation of oligodendrocytes precursor cells to oligodendrocytes & extension of their processes MMP9 and 12 are known to influence myelin formation during development, this is achieved through effect of these MMPs on bioavailability ofIGF1. IGFBP6,. B. Demyelination in adult mouse spinal cord, MMP9 regulates remyelination. mechanism is clearance of NG2 proteoglycans, which constitutes an inhibitory barrier for maturation of oligodendrocytes & their subsequent reformation of myelin.

Matrix metalloproteinase in neuroinflammation & MS: Neuroscientist 2002; 8; 586

MMPS IN NEURODEGENERATION

MMP in vascular cognitive impairment

• MMPs are induced by hypoxic hypoperfusion in white matter • During hypoxia, HIF1α increases, leading to expression of

many genes implicated in injury & repair

• Furin contributes to activation of MMPs implicated in injury

• HIF1α stimulates expression of substances that mediate repair, including VEGF & TGFβ

• In rat model of VCI , hypoxic hypoperfusion induces MMPs & thus increases permeability of BBB in white matter

Possible mechanism for white matter injury in vascular cognitive impairmentHIF1α concentration, turns on cassettes of genes associated with injury such as FURIN, & increases expression of VEGF and TGFβ, which are important in repair. Furin leads to activation of MMP2 through activation of MT-MMP. MMP2 can disrupt tight junction proteins & open the BBB, leading to edema. Edema might also cause demyelination. Additionally, MMP2 might attack myelin and can activate endothelin 1, which causes vasoconstriction through calcium metabolism in small muscle. Vasoconstriction aggravates hypoxic state. Conversely, on the repair side, VEGF and TGFβ activate angiopoietin 2, which acts through the secretion of MMPs to initiate angiogenesis and neurogenesis.

(E) Immunostaining with antibodies to MMP3 detects an immunoreactiveastrocytes (arrow).

(F) Immunostaining with antibodies to MMP3 detects an MMP3-positive macrophage around a fibrotic blood vessel in demyelinated area (arrow).

MMPs & ADAMs in AD• MMPs participate in formation & clearance of amyloid-β

peptides (Aβ)

• MMPs are induced endogenously by amyloid molecules in blood vessels, astrocytes, & microglia

• Astrocytes exposed to Aβ1-40 secrete MMP2,3 & 9

• When Aβ is deposited in tissues around plaques, there is activation of microglia & astrocytosis, this inflammatory response might contribute to neuronal Death

• Increase in expression of MMPs in brain tissue & blood of patients with AD is probably part of inflammatory response

α-secretase cleaves APP molecule, soluble fragment of APP is produced (APPsα), which can be further metabolized by proteases & cleared from brain across BBB. However, if β-secretase initiates cleavage of APP to form interim product that γ-secretase cleaves, then the Aβ molecule is formed. Most common form of Aβ is Aβ1-40, which is degraded by MMPs cleared through BBB : a smaller amount of Aβ1-42 is formed, but this can clump into fibrils that accumulate in amyloid plaques. MMP2 and MMP9 degrade Aβ & aid in its clearance from brain across BBB. Furthermore, microglia are activated by Aβ, adding to the amount of MMP9 available. Aβ=am

ADAMs are part of α- secretase

MMPs in Parkinson’s disease

• In vitro, apoptotic dopaminergic neurons release MMP3, which activates microglia , suggesting that, MMP3 is signalling molecule in addition.

• TNFα released from microglia leads to neuronal death;

• Primary mouse mesencephalic cells in culture die when treated with BH4 (tetrahydrobiopterin), selective dopaminergic neuronal toxin; treatment with MMP3 inhibitor NNGH (N-isobutyl-N-[4-methoxyphenylsulfonyl]-glycylhydroxamic acid) prolongs cell survival by decreasing TNFα release from activated microglia.

Determinants of beneficial or detrimental roles

• At least 3 factors contribute1. Stage of CNS injury –

• it is likely that in early phase after insult, when many metalloproteinases are induced or released into milieu that normally has low levels of protease MMPs are detrimental

• Indeed, in rodents, early & short-term treatment (for 3 days) of spinal cord injury or intracerebral hemorrhage with metalloproteinase inhibitors attenuates extent of neurotoxicity & subsequent loss of function

• More latterly after injury, when metalloproteinases are expressed discreetly and locally

2. Type of injury inflicted

• Type of injury affects profile of inflammatory cells at injury site & available MMP substrates expressed in that microenvironment

• chronic diseases such as MS & EAE entail significant representation by T lymphocytes, which promote inflammation & neurotoxicity, whereas in acute spinal cord injury, injury site is infiltrated with neutrophils & macrophages rather than T cells

• In different cellular contexts, & with differences in gene expression in these cell types, substrates for,& functions of, MMP differ

3. Pathophysiology of disorder involved

• In EAE, recovery from disease depends on switch of CD4+ T lymphocytes from proinflammatory Th1 subset to Th2 environment in order for inflammation to subside

• MMP12 may be required for this switch,& its absence results in

animals with more inflamed CNS & greater EAE severity

• By contrast, in cord injury, which represents insult less dependent on T cells, MMP12 may not be required to alter this ratio

• Rather, MMP12 expression increases activation of microglia/ macrophages, which exacerbate damage to injured cord

Targeting MMP in Neurological conditions

MMP inhibitors

• Early recognition that excessive MMP expression contributes to diseases such as cancer led to active development of inhibitors

• One of first hydroxamates developed was batimastat (BB94). Marimastat second generation hydroxamate with increased bioavailability after oral intake,& has been tested in gliomas

• Tetracycline antibiotics inhibits MMP enzymatic activity has led to use of these & chemically modified tetracyclines without anti-microbial activity (CMTs) as MMP inhibitors

• Only compound approved for use based on its ability to inhibit MMPs is low-dose doxycycline formulation, Periostat

MMP IN DEMYELINATING & INFLAMMATORY DISORDERS

• One of most potent tetracyclines to inhibit gelatinases, is minocycline. minocycline in EAE reduces disease activity

• Minocycline was found to reduce relapse rate & gad enhancing MRI lesions in pilot trial in RRMS

• CMT as MMP inhibitors acts by binding Zn2+ & Ca2+, latter being required to maintain proper enzyme conformation

• CMTs also down regulate expression of MMPs, & to decrease oxidative activation of pro-MMPs into active enzymes

• Statins, are also described to reduce production of MMPs by cells or to inhibit MMP activity

MMP INHIBITOR IN STROKE

• Antibodies to MMPs & broad-spectrum MMP inhibitors, BB-94, 1101, & GM-6001, reduce BBB damage, infarct size, & cell death

• Protect brain from hemorrhagic complications of alteplase by reducing permeability of BBB & preventing alteplase from entering brain & activating MMPs

• MMP inhibitors in stroke needs to be given early in first few days after injury to prevent blocking recovery phase

• MRI has shown in rats that, although BB-1101 blocked early opening of BBB at 3hr after 90-min MCA occlusion with reperfusion, inhibitor had no effect on lesion size at 48 h, & recovery over 3 weeks was slowed

MMP INHIBITORS IN OTHER DISEASES

• MMP inhibitors were beneficial in animal studies of MS,GBS , meningitis & vascular dementia

• MMPs in bacterial meningitis contribute to damage seen in children with meningitis despite use of appropriate antibiotics

• Water-soluble inhibitor of MMPs & TACE, TNF-484, is effective in animals

• Doxycycline blocks secondary damage in experimental bacterial meningitis in rodents

• Doxycycline has been used in treatment of MMP-mediated vascular diseases & reduced vascular damage in animal model

• Even more speculative is possible use of MMP inhibitors in treatment of Alzheimer’s disease

• Role of MMPs & ADAMs in AD is complex because of their

dual function

• Theoretically, treatment with MMP inhibitors could impede amyloid clearance because MMP9 seems to facilitate removal of the amyloid peptides

• Use of MMP inhibitors in PD might show great promise as death of dopaminergic neurons seems to be associated with release of MMPs by the activated cells around them

WHY WE ARE NOT USING MMPs?

• Major obstacle is low specificity & poor solubility particularly those with hydroxymate base

• Other obstacle is poor understanding of time to initiate & time to stop treatment

• Need to assess beneficial & deleterious effects in detail

• Selective inhibition VS broad spectrum inhibition

• Side effects

CONCLUSIONS

• Metalloproteinases have both beneficial and detrimental functions in the CNS

• Issue of MMPs as causative factors in disease is active area of investigation, but their possible role as facilitators of CNS recovery needs greater consideration.

• Research needed to understand diverse roles of proteases to design specific drugs & devise therapeutic strategies

• The potential use of MMP inhibitors to treat CNS diseases is an exciting prospect, but it is important to balance risk with the benefits of this treatment