Brain Extracellular Matrix in Neurodegeneration

ECM modulators in the CNS

The two major systems that modify the adult brain matrix are the serine protease tissue plasminogen activators (tPAs) and matrix metalloproteinases (MMPs). The MMPs are synthesized as proenzymes with removal of a propeptide as a prerequisite for activation. They are synchronously expressed with tissue inhibitors of metalloproteinases (TIMPs), which form tight complexes with the enzyme. Activation of MMPs can occur through plasmin generation by tPAs, thus functionally linking the two proteolytic systems. The tPAs are also secreted in a precursor form as single‐chain polypeptides and in vitro are converted by plasmin to active double‐chain forms. Regulation of tPA activity in the nervous system can occur at multiple levels: transcription, translation, secretion and inhibition by anti‐proteases. Despite high levels of tPA mRNA in the hippocampal CA1 region, protein and enzymatic assays show no evidence of tPA, suggesting stringent translational control ⁽³⁹⁾. Synaptic signaling can release this control and can lead to tPA secretion. Inhibition of tPA activity is under tight control by a variety of serpin family anti‐proteases (eg, protease‐nexin 1, neuroserpin and plasminogen activator inhibitor 1) ⁽³⁹⁾. Some tPA effects are plasminogen independent (eg, cleavage of NR1 subunit of the NMDA receptor (NMDAR) enhancement of N‐methyl‐D‐aspartic acid (NMDA)‐mediated intracellular calcium levels or binding of the low‐density lipoprotein (LDL) receptor‐related protein) and promote upregulation of MMP‐9, degradation of the ECM resulting in synaptic plasticity or neuronal degeneration ⁽⁵⁵⁾. The mechanisms of activation of tPAs and MMPs in vivo are still poorly understood. The detailed molecular biology and biochemistry of these enzyme systems have been extensively documented elsewhere ²⁷, ³⁴, ⁵², ⁶³, ⁹⁵, ⁹⁶. For purposes of this review, these proteolytic and GAG‐degrading enzymes, such as heparanase and chondroitinase, are key components in modulating cellular interactions with ECM (eg, proteolytic cleavage of core proteins or degradation of GAG side chains). MMPs are generally secreted; however, they can be localized to cell surfaces by binding to cell adhesion molecules or cell surface proteoglycans and integrins or through transmembrane domains of membrane‐type MMPs. Another subgroup of metalloproteases is the A disintegrin and metalloproteinases (ADAMs) and A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS). ADAMs are membrane‐anchored enzymes that possess both proteolytic and adhesive activities. They are involved in shedding of cytokines, receptors and growth factors as well as adhesion to integrins. Tumor necrosis factor alpha (TNFα)‐converting enzyme or ADAM17 is the most characterized sheddase releasing TNFα from the cell surface ⁽¹⁶⁾. ADAMTSs are secreted enzymes that are involved in the cleavage of ECM proteoglycans and in collagen processing ⁽⁷¹⁾. MMP secretion has been shown to contribute to the migration ability of monocytes as well as astrocyte motility ⁵⁶, ⁶², ⁶⁷, ⁶⁹, ⁷⁴, ⁸². MMPs can also be secreted by neurons, oligodendrocytes, microglia and endothelial cells ⁽⁵²⁾. The MMP inhibitors (TIMPs) are expressed in neurons and astrocytes in the adult CNS ⁽²⁵⁾. tPA is expressed both in neurons and microglial cells and is highly expressed in regions involved in learning and memory. The activity of tPA in neural tissue is correlated with neurite outgrowth, regeneration and migration, suggesting that it might be involved in neuronal plasticity ⁽⁵⁸⁾.

Ischemic stroke

In the penumbra of an infarct, gliosis delineates regions of apoptotic cell death. Within the region of glial scar, stroke induces the growth‐inhibitory CSPGs, such as aggrecan, phosphacan and versican, the semaphorin IIIa receptor neuropilin, and myelin‐associated glycoprotein. Stroke also induces growth‐promoting proteins in this region of glial scar, including GAP43, cytoskeleton‐associated protein 23, myristoylated alanine‐rich protein kinase C substrate and small proline repeat rich protein 1. Thus, the glial scar of a stroke is a narrow region that closely borders the infarct and contains increased expression of both growth‐promoting and growth‐inhibitory molecules. The peri‐infarct region that lies just outside of the glial scar has reduced levels of growth‐inhibitory molecules. This growth‐permissive zone corresponds to the region of post‐stroke axonal sprouting ⁽²¹⁾.

Al'Qteishat et al ⁽²⁾ performed a thorough immunohistochemical analysis of post‐mortem tissues from stroke patients and found enhanced deposition of HA around blood vessels and, paradoxically, intracellularly and in the nuclei of peri‐infarct neurons. Western blot and immunohistochemistry showed upregulation of HA synthases 1 and 2 and hyaluronidases 1 and 2 in inflammatory cells in stroke and peri‐infarct regions. Hyaluronidase 1 was upregulated in microvessels and intracellularly in neurons. The expression of HA‐binding protein CD44 and tumor necrosis factor‐stimulated gene 6 was mainly increased in infiltrating mononuclear cells from inflammatory regions. The results demonstrated a significant change in the enzymes responsible for HA synthesis and degradation together with upregulation of HA receptors. This may reflect tissue remodeling after stroke and may be partially responsible for increased angiogenesis and neuronal migration ⁽²⁾.

A commonly used animal model of human stroke is permanent middle cerebral artery occlusion. This model has demonstrated that, in addition to central neuronal loss, the peri‐infarct regions have shown loss of PNN marker Wisteria floribunda agglutinin (WFA) staining and, to a lesser degree, loss of CSPG core protein staining ⁽⁴¹⁾. Beginning at 24 h, osteopontin protein was expressed by endogenous microglia and by infiltrating macrophages at 48 h in the infarct. By day 5, osteopontin mRNA was restricted to the infarct region. The osteopontin receptor integrin α_vβ₃ was expressed in peri‐infarct astrocytes at 5 and 15 days. It has been hypothesized that the osteopontin is deposited in the matrix and serves as a barrier for the glial scar formation by astrocytes ⁽³³⁾.

In summary, beyond the extensive destruction of ischemic tissue, in the penumbra of infarcts, abundant alterations occur in the ECM. Some of these alterations contribute to growth‐inhibiting glial scars, while others are associated with neuronal growth‐promoting regions. In addition to endogenous neuroglial cell modulation, infiltrating inflammatory cells take part in ECM remodeling.

Kainic acid (KA) excitotoxicity

KA‐induced seizures in rodents represent a well‐established animal model for human temporal lobe epilepsy ⁽¹¹⁾. KA produces neuronal excitation resulting in seizures within hours following intraperitoneal injection. At 2–3 days after treatment, neurodegeneration of both necrotic and apoptotic character can be observed in the limbic system, in particular the hippocampus and amygdala, in addition to other brain regions ⁽⁴⁷⁾. ECM changes that have been documented in this model include reduction in laminin and phosphacan, and increases in neurocan and ADAMTS‐cleaved brevican.

Laminin, an important constituent of the ECM, has been shown to play a key role in KA excitotoxicity ⁽²³⁾. In wild‐type mice, 2 h after injection of KA, neurons had normal morphology, but laminin had almost completely disappeared in CA1 and CA3. Two days after injection, neurons in CA1, CA2 and CA3 had degenerated. These results indicate that laminin degradation precedes neuronal loss after KA treatment and occurs in exactly the same hippocampal regions that eventually experience neuronal degeneration. The CA3 region that showed more laminin expression was more resistant to excitotoxin‐induced neuronal cell death. This correlation between high laminin expression and neuronal resistance to excitotoxin further implicates laminin as an important factor in maintaining cellular viability. While loss of laminin attachment was deleterious in combination with kainate injection, it was not by itself significantly toxic. Infusion of anti‐laminin antibodies was toxic only in conjunction with KA injection ⁽²³⁾.

KA‐induced convulsions caused prolonged changes in the CSPGs neurocan and phosphacan. Okamoto et al ⁽⁶⁸⁾ showed increased neurocan staining that co‐localized with glial fibrillary‐associated protein staining. Phosphacan staining was reduced in areas of pyramidal cell loss in the hippocampus and around parvalbumin‐positive neurons. Interestingly, compared to other classes of neurons undergoing degeneration, the parvalbumin‐positive neurons were relatively conserved ⁽⁶⁸⁾.

Mediators of ECM destruction in the KA model have been studied in mice deficient in tPA or plasminogen. Mice with either of these enzymes knocked out were resistant to neuronal destruction induced by excitotoxins. Because infusion of excess tPA into the wild‐type hippocampus does not kill neurons, excitotoxin treatment must cause additional cellular changes beyond ECM alterations to induce neurodegeneration.

Using the KA model, Yuan et al ⁽⁹⁸⁾ assessed co‐localization of ADAMTS or its activity in regions of synaptic loss or neuronal death that could indicate a functional role for the protease in degeneration. They demonstrated that increased ADAMTS cleavage of brevican was found 24 h after KA administration and was sustained in several susceptible brain regions. An increase in the mRNA of ADAMTS‐1/4 in the seized animals was accompanied by elevation of the ADAMTS‐cleaved brevican ⁽⁹⁸⁾.

In summary, many of the neurodegenerative changes associated with KA excitotoxicity are linked to ECM alterations. Degeneration of the ECM does not directly cause neurodegeneration but rather makes neurons susceptible to activation‐associated cell death. Therefore, supporting neuronal‐ECM interactions appears to be a reasonable approach to blocking neurodegeneration in this model.

Multiple sclerosis (MS)

MS is a demyelinating disease associated with inflammation, gliosis and variable axon loss. While most of the disease process is focused in the white matter, recently, gray matter lesions have been demonstrated ⁽¹³⁾. In a recent review, van Horssen summarized the ECM changes in MS ⁽⁴⁵⁾. Classical MS lesions showed accumulation of laminin, HA and MMP‐19. The active MS lesions showed accumulation of laminin in the parenchyma, which was normally associated with the basement membrane of endothelial cells. This laminin accumulation was not seen in inactive MS lesions.

CD68‐positive macrophages in the vicinity of the lesion expressed high levels of transforming growth factor β1 (TGFβ1), which is known to modulate ECM production ⁽⁴³⁾. MMP‐19 was upregulated in MS lesions and was expressed by microglia/macrophages ⁽⁴⁴⁾. As opposed to tenascin‐R and tenascin‐C that accumulate in chronic MS lesions, there was a striking loss of both tenascin‐R and tenascin‐C in the edge of acute lesions where the macrophage density is greatest. This loss extended into adjacent, histologically normal white matter ⁽⁴⁰⁾. In chronic demyelinated lesions, a high‐molecular‐weight HA synthesized by astrocytes accumulated. This was thought to inhibit remyelination as oligodendrocyte progenitor cells do not mature into myelin‐forming cells in demyelinating lesions where high‐molecular‐weight HA is present ⁽⁶⁾. The inflammatory response of MS is associated with modulation of the ECM that is incommensurate with regeneration. Manipulation of this response could be key to anatomical and functional recovery.

Human immunodeficiency virus dementia (HIVD)

Human immunodeficiency virus (HIV)‐1‐associated dementia (AIDS dementia complex) is marked by deficits in motor control, cognition and behavior ⁽⁶⁴⁾. Achim and Wiley ⁽¹⁾ reported that at autopsy, AIDS patients who had clinical dementia during terminal stages of their disease demonstrated human immunodeficiency virus encephalitis (HIVE). The constellation of neuropathological changes associated with HIVE includes activated and infected macrophages, astrocytosis and neurodegeneration ⁽¹⁸⁾. As neurons are not infected by HIV, the etiology of neurodegeneration must be indirect. Current leading theories suggest that neurodegeneration results from neurotoxic viral or inflammatory factors. The ability of activated monocytes and macrophages to penetrate the ECM and to migrate to target tissues is dependent on ECM‐degrading proteins like MMPs and heparanase. This degradation of ECM components could result in disintegration of the structure that physically and physiologically supports neurons, and thus could lead to degeneration.

Belichenko et alstudied the ECM in HIVE cases and found that each of the nine cases with HIVE was completely devoid of labeled ECM components, while only 44% of HIV‐infected non‐encephalitic cases had ECM loss ⁽⁹⁾. Although PNNs were lost in HIVE, there was no loss of parvalbumin‐, calbindin‐ and calretinin‐positive neurons that are frequently surrounded by these nets. ECM destruction was not confined to microglial nodules, suggesting that if macrophages were the source of released ECM‐degrading enzymes, these would need to diffuse throughout the brain parenchyma. Alternatively, macrophages could activate astrocytes to secrete enzymes responsible for the metabolism of the ECM. Staining for Vicia villosa agglutinin, a lectin that binds to N‐acetyl‐galactoseamine residues on CS or DS, was lost before the core protein staining, suggesting that the degradation of the GAG chains occurs before the actual degradation of the core protein ⁽⁹⁾. A more indirect indication of GAG chain degradation in lentiviral encephalitis is the presence of HA in the cerebrospinal fluid (CSF) ⁽⁴⁹⁾.

Support of these findings of ECM degradation in AIDS comes from the simian immunodeficiency virus (SIV) encephalitis model in rhesus and pigtailed macaques. Disruption of brain ECM was present in SIV cases without encephalitis, but was more severe in cases with encephalitis ⁽⁵⁷⁾. This suggests that ECM changes occur prior to parenchymal macrophage infiltration. ECM changes in SIV encephalitis (SIVE) occur concurrently with reduced staining for the pre‐ and postsynaptic marker synaptophysin and microtubule‐associated protein 2 (MAP2). One possible source of ECM degradation activity is the induced microglial/macrophage expression of ADAMTS‐1 and ADAMTS‐4 ⁽⁵⁷⁾ and MMP‐2 and MMP‐7, as observed by their elevation in the CSF of patients with HIVD ⁽²⁴⁾. Other sources of ECM‐degrading enzymes include astrocytes or lymphocytes as shown in the coronavirus‐induced encephalitis in mice, where increased mRNA levels of MMP‐3 and MMP‐12 as well as the inhibitor TIMP‐1 were found. In this model, MMP‐3 expression was restricted to astrocytes and TIMP‐1 expression was localized with CD4‐positive cells. However, when T‐cells were removed from this model using irradiation–immunosuppression, mice that had high viral load in the CNS showed increased mRNA levels of MMP‐3, MMP‐12 and TIMP‐1, suggesting that viral replication directly triggers upregulation of these proteins ⁽¹⁰⁰⁾.

In summary, lentiviral infection is associated with neurodegeneration and disruption of CNS ECM. While SIV systemic infection may elicit brain ECM disruption, a more pronounced disruption is associated with SIVE. As observed in other viral encephalitides, this ECM disruption occurs independently of local lymphocyte response and appears tightly associated with macrophage activation and infiltration.

Production of toxic proteolytic cleavage products

In addition to the above‐mentioned ECM changes mediated by MMPs and tPA, one study showed an additional mechanism that could contribute to neurodegeneration. HIV‐infected macrophages secreted MMP‐2, which metabolizes the chemokine stromal cell‐derived factor (SDF)‐1 to the cleaved form. This cleaved form is more toxic than the whole protein ⁽⁹⁹⁾. Dose‐dependent neuronal death was observed in human cholinergic cell line incubated with increasing concentration of MMP‐2 but not with a mutant MMP‐2. Treatment with neutralizing antibodies against MMP‐2 or inhibitor of MMP‐2 reduced neuronal death. The cleaved SDF‐1 causes apoptosis and dose‐dependent neuronal death. Mice that were stereotactically injected with cleaved SDF‐1 showed more microglial reactivity and astrocytosis than animals injected with the whole SDF‐1 ⁽⁹⁹⁾. Thus, in addition to ECM remodeling, protelolytic activity can induce protein gain of toxicity.

Induction of chemotaxis and neuroinflammtory response

Chemokines and their receptors play a key role in directing the migration of mononuclear cells into the CNS under normal physiological conditions and in pathological states. Plasmin or MMP‐mediated proteolysis of chemokines is one way by which these proteolytic enzymes can influence leukocyte trafficking. MMP proteolysis can activate or inhibit the biological functions of chemokines ⁽⁸⁹⁾. Apoptotic cells trigger the recruitment of phagocytic cells through release of chemoattractants. It has been shown that apoptotic neurons release the active form of MMP‐3 that elicits microglial secretion of TNFα⁽⁴⁸⁾. The chemokine fractalkine is cleaved by MMPs after excitotoxic injury. Although inhibition of MMPs by batimastat inhibits fractalkine cleavage, it does not confer neuroprotection. Release of fractalkine from neurons plays a pivotal role in attracting microglia and other cells derived from the monocytic lineage ⁽²²⁾. Mice genetically deficient for the chemokine monocyte chemotactic protein‐1 (MCP‐1) display decreased microglial recruitment and resist excitotoxic neurodegeneration. MCP‐1 undergoes proteolytic processing mediated by plasmin, enhancing the potency of MCP‐1 in in vitro migration assays. Infusion of the cleaved form of MCP‐1 into the CNS restores microglial recruitment and excitotoxicity in plasminogen‐deficient mice ⁽⁸¹⁾. In summary, plasmin‐ or MMP‐mediated proteolysis of chemokines can increase their potency in inducing microglial activation and trafficking of immune cells into the CNS.

Expression of regeneration‐inhibitory CSPGs

In the trauma model of unilateral knife lesion, increased staining of neurocan was observed. In vitro, neurocan was shown to inhibit neurite outgrowth and being deposited on the substratum around astrocytes but not on the cell surface. These findings support the role of astrocytes within the region of glial scar as a source of growth‐inhibitory CSPGs ⁽⁴⁾. Unsulfated chondroitin, chondroitin 4‐sulfate and chondroitin 6‐sulfate were found associated with SPs, NFTs and dystrophic neurites ⁽²⁹⁾. These results suggest that CSPG could contribute or be responsible for the regression of neurites around SPs in AD. Abnormal CSPG accumulation could disrupt cell adhesion or growth factor utilization ⁽²⁹⁾. Deposition of CSPG has been found at the periphery of SP cores. It has been hypothesized that neurotoxicity is not simply the consequence of β‐amyloid aggregation. Implantation of nitrocellulose impregnated with β‐amyloid in the cortex of neonatal rats caused reactive gliosis in addition to deposition of CSPGs. Secondary glial cell reaction to the insoluble molecule and CSPGs, along with other proteoglycans, may be an additional link in the chain of events leading to neuronal dystrophy and possibly neuronal death ⁽²⁰⁾.

Thus, neurite growth‐inhibitory CSPGs are induced in neuropathological states like SPs or glial scar regions and, therefore, could play a role in inhibiting regeneration of damaged neurons.

Associated HSPG deposition and protein aggregation

The HSPGs agrin, glypican and syndecans 1–3 were found to be incorporated into SPs ⁽⁹⁰⁾. HSPGs normally associated with basement membrane of blood vessels were lost in affected areas in AD but were present in congophilic angiopathy (CA) in AD. HSPGs were also demonstrated in “primitive plaques” (defined as those having little or no amyloid core), suggesting that their accumulation takes place during early stages of plaque development. HSPGs were localized to astrocytes and neurons in close proximity of SPs and CA, suggesting that both may be involved in the deposition of HSPGs at these sites and may, therefore, play a role in plaque development. Ultrastructural studies specifically localized the HSPGs to amyloid fibrils present in SPs and CA. This close association between amyloid fibrils and HSPGs suggests that HSPGs have an important role in amyloidosis ⁽⁸³⁾. Other support for this idea comes from the work of Liu et al, who found that agrin co‐localizes with α‐synuclein in neuronal Lewy bodies in the SN of Parkinson's disease brain. Agrin was found to accelerate formation of protofibrils by α‐synuclein and to decrease the half‐time of fibril formation ⁽⁵¹⁾. In summary, HSPGs have been found to be associated with protein aggregation and deposition, and it has been hypothesized that changes in the metabolism of the ECM could contribute to amyloid neurodegenerative diseases.