Neuroprotective role of MMP-9 overexpression in the brain of Alzheimer's 5xFAD mice.

Accumulation of amyloid-ß (Αß) peptide is believed to play a central role in the pathogenesis of Alzheimer's disease (AD). Lowering Aß levels in the brain may thus improve synaptic and cognitive deficits observed in AD patients. In the non-amyloidogenic pathway, the amyloid-ß precursor protein (APP) is cleaved within the Aß peptide sequence by α-secretases, giving rise to the potent neurotrophic N-terminal fragment sΑPPα. We have previously reported that gelatinase B/matrix metalloproteinase 9 (MMP-9), a matrix metalloproteinase critically involved in neuronal plasticity, acts as α-secretase both in vitro and in vivo and reduces Aß levels in vitro. In the present study, we demonstrate that neuronal overexpression of MMP-9 in a transgenic AD mouse model harboring five familial AD-related mutations (5xFAD) resulted in increased sAPPα levels and decreased Aß oligomers without affecting amyloid plaque load in the brain. Functionally, overexpression of MMP-9 prevented the cognitive deficits displayed by 5xFAD mice, an improvement that was accompanied by increased levels of the pre-synaptic protein synaptophysin and mature brain-derived neurotrophic factor (BDNF) in the brain. These results suggest that in vivo activation of endogenous MMP-9 could be a promising target for interference with development and/or progression of AD.

Oleuropein, an anti-oxidant polyphenol constituent of olive promotes α-secretase cleavage of the amyloid precursor protein (AßPP).

Over the past decade, intense focus has been dedicated on investigating processes involved in the proteolysis of amyloid precursor protein (AßPP) and ß-amyloid (Aß) peptide metabolism, as possible targets for Alzheimer's disease (AD) therapy. To this goal, considerable research has been targeted on potential therapeutic use of compounds promoting non-amyloidogenic processing of AßPP. One of these compounds, oleuropein, a polyphenol constituent of extra virgin olive oil exhibiting a wide range of pharmacological properties, was shown to interact non-covalently with Aß, an interaction that might be related to a potential protective role of oleuropein against Aß aggregation. In the present study, it was demonstrated that oleuropein treatment of HEK293 cells stably transfected with the isoform 695 of human AßPP (APP695) leads to markedly elevated levels of sAPPα and to significant reduction of Aß oligomers. These effects were associated with increased activity of matrix metalloproteinase 9 (MMP-9), whereas no significant alterations in the expression of secretases TACE, ADAM-10 or BACE-1 were observed. Similar results were obtained using the human neuroblastoma cell line SK-N-SH. The experimental data reveal an anti-amyloidogenic effect of oleuropein and suggest a possible protective role for oleuropein against AD, extending the spectrum of beneficial properties of this naturally occurring polyphenol.

Enhanced neuronal plasticity and elevated endogenous sAPPα levels in mice over-expressing MMP9.

Evidence accumulating during the past few years points to a significant role of matrix metalloproteinase 9 (MMP9) enzymatic activity in synaptic plasticity and cognitive processes. We have previously demonstrated that MMP9 is involved in receptor-mediated α-secretase-like cleavage of APP in vitro, resulting in increased secretion of sAPPα, the soluble N-terminal product of the non-amyloidogenic pathway known to be involved in neuronal plasticity and memory formation. To study the in vivo role of MMP9, we have generated transgenic mice over-expressing MMP9 in the brain. Herein, we demonstrate that MMP9 transgenic animals display enhanced performance in the non-spatial novel object recognition and the spatial water-maze task and that their enhanced performance was accompanied by increased dendritic spine density in the hippocampus and cortex following behavioural testing. Consistent with the above observations, the electrophysiological analysis revealed prolonged maintenance of long-term synaptic potentiation in hippocampal slices from MMP9 transgenic mice. Moreover, elevated sAPPα levels in the hippocampus and cortex of MPP9 transgenic animals were also observed. Overall, our results extend previous findings on the physiological role of MMP9 in neuronal plasticity and furthermore reveal that, APP may be one of the physiological proteolytic targets of MMP9 in vivo.

Expression levels of matrix metalloproteinase (MMP)-9 and its specific inhibitor TIMP-1, in septic and aseptic arthritis of the knee.

PURPOSE: In cases of septic knee arthritis, there is excess of matrix metalloproteinases (MMPs) over tissue inhibitors of metalloproteinases (TIMPs), due to enhanced expression and activation that are induced by bacteria in comparison with rheumatic or degenerative arthritis. The aim of this study was to explore the expression levels of synovial gelatinase MMP-9 and its specific inhibitor TIMP-1 in septic and aseptic arthritis and their potential use as additional aids to clinical investigation. METHODS: Gelatin zymography and western blot analysis were applied in effusions from knees of the patients with septic (SA-10 patients), rheumatic (RA-10 patients) and osteoarthritis (OA-10 patients). RESULTS: Zymographic analysis revealed that all samples contained latent MMP-2 activity, albeit activated MMP-2 appeared in more of the septic than aseptic effusions. MMP-9 was not detected in osteoarthritic synovial fluid samples. Only trace amounts of MMP-9 activity were detected in 4 of 10 patients with RA, whereas higher MMP-9 levels were evident in all samples from SA (P = 0.0241). In immunoblotting assays, samples from SA showed significantly higher levels of MMP-9 compared with samples from RA (P = 0.0052), confirming zymographic results. Although no significant difference in TIMP-1 levels was observed, the estimated MMP-9/TIMP-1 ratio of septic effusions was significantly higher compared with aseptic ones (P = 0.0029). CONCLUSIONS: The data presented suggest enhanced expression and activation of MMP-9 in septic native knee arthritis compared with aseptic. The presence of high levels of MMP-9 with concomitantly increased MMP-9/TIMP-1 ratio and activated gelatinases in effusions, independent of neutrophilic counts, may be indicative for infection.

The Prophylactic and Multimodal Activity of Two Isatin Thiosemicarbazones against Alzheimer's Disease In Vitro.

Alzheimer's disease (AD) is a multifactorial disorder strongly involving the formation of amyloid-ß (Aß) oligomers, which subsequently aggregate into the disease characteristic insoluble amyloid plaques, in addition to oxidative stress, inflammation and increased acetylcholinesterase activity. Moreover, Aß oligomers interfere with the expression and activity of Glycogen synthase kinase-3 (GSK3) and Protein kinase B (PKB), also known as AKT. In the present study, the potential multimodal effect of two synthetic isatin thiosemicarbazones (ITSCs), which have been previously shown to prevent Aß aggregation was evaluated. Both compounds resulted in fully reversing the Aß-mediated toxicity in SK-NS-H cells treated with exogenous Aß peptides at various pre-incubation time points and at 1 µM. Cell survival was not recovered when compounds were applied after Aß cell treatment. The ITSCs were non-toxic against wild type and 5xFAD primary hippocampal cells. They reversed the inhibition of Akt and GSK-3ß phosphorylation in 5xFAD cells. Finally, they exhibited good antioxidant potential and moderate lipoxygenase and acetylcholinesterase inhibition activity. Overall, these results suggest that isatin thiosemicarbazone is a suitable scaffold for the development of multimodal anti-AD agents.

Temporal Pattern of Neuroinflammation Associated with a Low Glycemic Index Diet in the 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is associated with brain amyloid-ß (Aß) peptide accumulation and neuroinflammation. Currants, a low glycemic index dried fruit, and their components display pleiotropic neuroprotective effects in AD. We examined how diet containing 5% Corinthian currant paste (CurD) administered in 1-month-old 5xFAD mice for 1, 3, and 6 months affects Aß levels and neuroinflammation in comparison to control diet (ConD) or sugar-matched diet containing 3.5% glucose/fructose (GFD). No change in serum glucose or insulin levels was observed among the three groups. CurD administered for 3 months reduced brain Aß42 levels in male mice as compared to ConD and GFD, but after 6 months, Aß42 levels were increased in mice both on CurD and GFD compared to ConD. CurD for 3 months also reduced TNFα and IL-1ß levels in male and female mouse cortex homogenates compared to ConD and GFD. However, after 6 months, TNFα levels were increased in cortex homogenates of mice both on CurD and GFD as compared to ConD. A similar pattern was observed for TNFα-expressing cells, mostly co-expressing the microglial marker CD11b, in mouse hippocampus. IL-1ß levels were similarly increased in the brain of all groups after 6 months. Furthermore, a time dependent decrease of secreted TNFα levels was found in BV2 microglial cells treated with currant phenolic extract as compared to glucose/fructose solution. Overall, our findings suggest that a short-term currant consumption reduces neuroinflammation in 5xFAD mice as compared to sugar-matched or control diet, but longer-term intake of currant or sugar-matched diet enhances neuroinflammation.

An apolipoprotein E4 fragment can promote intracellular accumulation of amyloid peptide beta 42.

Apolipoprotein E (apoE) plays a crucial role in lipid transport in circulation and the brain. The apoE4 isoform is a major risk factor for Alzheimer's disease (AD). ApoE4 is more susceptible to proteolysis than other apoE isoforms and apoE4 fragments have been found in brains of AD patients. These apoE4 fragments have been hypothesized to be involved in the pathogenesis of AD, although the mechanism is not clear. In this study we examined the effect of lipid-free apoE4 on amyloid precursor protein processing and 40-amino-acid Aß variant and 42-amino-acid Aß variant levels in human neuroblastoma SK-N-SH cells. We discovered that a specific apoE4 fragment, apoE4[Δ(166-299)], can promote the cellular uptake of extracellular 40-amino-acid Aß variant and 42-amino-acid Aß variant either generated after amyloid precursor protein transfection or added exogenously. A longer length fragment, apoE4[Δ(186-299)], or full-length apoE4 failed to elicit this effect. ApoE4[Δ(166-299)] effected a 20% reduction of cellular sphingomyelin levels, as well as changes in cellular membrane micro-fluidity. Following uptake, approximately 50% of 42-amino-acid Aß variant remained within the cell for at least 24 h, and led to increased formation of reactive oxygen species. Overall, our findings suggest a direct link between two early events in the pathogenesis of AD, apoE4 proteolysis and intraneuronal presence of amyloid beta peptide.

TIMP-1 interaction with αvß3 integrin confers resistance to human osteosarcoma cell line MG-63 against TNF-α-induced apoptosis.

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine affecting diverse cellular responses. TNF-α is cytotoxic in many systems, but it can also act as an anti-apoptotic signal to promote cell survival pathways activated through integrins and extracellular matrix components. This is particularly evident in cancer cells. To unravel the basis of resistance to TNF-α-induced apoptosis, human osteosarcoma MG-63 cell line was used. Our data showed that resistance to apoptosis was accompanied by high levels of TIMP-1 expression in part mediated by NF-κB activation, whereas under apoptotic conditions, in the presence of cycloheximide (CHX), TIMP-1 and αvß3 integrin protein levels were significantly reduced. Silencing TIMP-1 using siRNA led to increased apoptosis following treatment with TNF-α, whereas exogenously-added recombinant TIMP-1 reduced the extent of apoptosis. Immunoprecipitation and confocal microscopy experiments demonstrated that TIMP-1 interacted with αvß3 integrins. The biological role of this interaction was revealed by the use of echistatin, an antagonist of αvß3 integrin. In the presence of echistatin, decreased protection against apoptosis by recombinant TIMP-1 was observed.

Selective binding of integrins from different renal cell types to the NC1 domain of alpha3 and alpha1 chains of type IV collagen.

BACKGROUND: Cells interact with type IV collagen (Col IV) via integrins through the triple-helical and NC1 domains. We examined interactions of human glomerular and proximal tubular epithelial cells with recombinant alpha1 and alpha3 NC1 chains of Col IV, to explore the ability of different cell types to interact with Col IV of different trimer composition. METHODS: Interactions of TSV-40-immortalized human glomerular epithelial cells (HGECs), HPV-16-immortalized human proximal tubular epithelial (HK-2) cells and primary human mesangial cells (MES) with recombinant alpha1 and alpha3 NC1 chains of Col IV were examined by affinity chromatography and solid-phase binding assays. The expression of integrin-regulated metalloproteinases was examined by zymography. RESULTS: HGECs bound to both alpha3 and alpha1(IV)NC1, albeit there was preferential binding to alpha3(IV)NC1, through the alpha3beta1 and alpha2beta1 integrin receptors; HK-2 cells and MES bound almost exclusively to alpha1(IV)NC1 via the alpha3beta1/alphavbeta3 and alpha1beta1/alpha2beta1 receptors, respectively. It was demonstrated that the expression of MMP-2 and MMP-9 by HGECs was down-regulated in the presence of alpha3(IV)NC1. CONCLUSIONS: The observed data indicate that the isoform NC1 chains of Col IV serve for selective, integrin-mediated cell binding which probably triggers different signaling mechanisms, resulting in the activation of specific transcription factors and the modulation of gene expression.

A New Perspective in Utilizing MMP-9 as a Therapeutic Target for Alzheimer's Disease and Type 2 Diabetes Mellitus.

Matrix metalloprotease 9 (MMP-9) is a 92 kDa type IV collagenase and a member of the family of endopeptidases. MMP-9 is involved in the degradation of extracellular matrix components, tissue remodeling, cellular receptor stripping, and processing of various signaling molecules. In the CNS, the effects of MMP-9 are quite complex, since it exerts beneficial effects including neurogenesis, angiogenesis, myelogenesis, axonal growth, and inhibition of apoptosis, or destructive effects including apoptosis, blood-brain barrier disorder, and demyelination. Likewise, in the periphery, physiological events, as the involvement of MMP-9 in angiogenesis, for instance in wound healing, can be turned into pathological, such as in tumor metastasis, depending on the state of the organism. Alzheimer's disease is a neurodegenerative disorder, characterized by amyloid accumulation and deposition in the brain. Amyloidogenesis, however, also occurs in diseases of the periphery, such as type II diabetes mellitus, where an analogous type of amyloid, is deposited in the pancreas. Interestingly, both diseases exhibit similar pathology and disease progression, with insulin resistance being a major common denominator. Hence, combinatorial strategies searching new or existing molecules to apply for therapeutic use for both diseases are gaining momentum. MMP-9 is extensively studied due to its association with a variety of physiological and pathological processes. Consequently, meticulous design could render MMP-9 into a potential therapeutic target for Alzheimer's disease and type 2 diabetes mellitus; two seemingly unrelated diseases.

Elevation of MMP-9 Levels Promotes Epileptogenesis After Traumatic Brain Injury.

Posttraumatic epilepsy (PTE) is a recurrent seizure disorder that often develops secondary to traumatic brain injury (TBI) that is caused by an external mechanical force. Recent evidence shows that the brain extracellular matrix plays a major role in the remodeling of neuronal connections after injury. One of the proteases that is presumably responsible for this process is matrix metalloproteinase-9 (MMP-9). The levels of MMP-9 are elevated in rodent brain tissue and human blood samples after TBI. However, no studies have described the influence of MMP-9 on the development of PTE. The present study used controlled cortical impact (CCI) as a mouse model of TBI. We examined the detailed kinetics of MMP-9 levels for 1 month after TBI and observed two peaks after injury (30 min and 6 h after injury). We tested the hypothesis that high levels of MMP-9 predispose individuals to the development of PTE, and MMP-9 inhibition would protect against PTE. We used transgenic animals with either MMP-9 knockout or MMP-9 overexpression. MMP-9 overexpression increased the number of mice that exhibited TBI-induced spontaneous seizures, and MMP-9 knockout decreased the appearance of seizures. We also evaluated changes in responsiveness to a single dose of the chemoconvulsant pentylenetetrazol. MMP-9-overexpressing mice exhibited a significantly shorter latency between pentylenetetrazol administration and the first epileptiform spike. MMP-9 knockout mice exhibited the opposite response profile. Finally, we found that the occurrence of PTE was correlated with the size of the lesion after injury. Overall, our data emphasize the contribution of MMP-9 to TBI-induced structural and physiological alterations in brain circuitry that may lead to the development of PTE.

Overexpression of matrix metalloproteinase-9 (MMP-9) rescues insulin-mediated impairment in the 5XFAD model of Alzheimer's disease.

A hallmark of Alzheimer's disease (AD) is the accumulation of oligomeric amyloid-ß (Aß) peptide, which may be primarily responsible for neuronal dysfunction. Insulin signaling provides a defense mechanism against oligomer-induced neuronal loss. We previously described the neuroprotective role of matrix metalloproteinase 9 (MMP-9) in decreasing the formation of Aß oligomers. In the present study, we examined the role of MMP-9 on the insulin survival pathway in primary hippocampal cultures and hippocampal cell extracts from 3 month-old wild type, AD (5XFAD), MMP-9-overexpressing (TgMMP-9), and double transgenic mice (5XFAD/TgMMP-9). The data demonstrate that the insulin pathway was compromised in samples from 5XFAD mice, when compared to the wild type and TgMMP-9. This was due to enhanced phosphorylation of IRS1 at Serine 636 (pIRS1-Ser636), which renders IRS1 inactive and prevents insulin-mediated signaling. In 5XFAD/TgMMP-9 samples, the insulin survival pathway was rescued through enhanced activation by phosphorylation of IRS1 at Tyrosine 465 (pIRS1-Tyr465), downstream increased phosphorylation of Akt and GSK-3ß, and decreased phosphorylation of JNK kinase. Oligomeric Aß levels decreased and BDNF levels increased in 5XFAD/TgMMP-9 mice, compared to 5XFAD mice. Our findings indicate that overexpression of MMP-9 rescued insulin survival signaling in vitro and in early stages in the 5XFAD model of AD.

The ability of apolipoprotein E fragments to promote intraneuronal accumulation of amyloid beta peptide 42 is both isoform and size-specific.

The apolipoprotein (apo) E4 isoform is the strongest risk factor for late-onset Alzheimer's disease (AD). ApoE4 is more susceptible to proteolysis than apoE2 and apoE3 isoforms and carboxyl-terminal truncated apoE4 forms have been found in AD patients' brain. We have previously shown that a specific apoE4 fragment, apoE4-165, promotes amyloid-peptide beta 42 (Aß42) accumulation in human neuroblastoma SK-N-SH cells and increased intracellular reactive oxygen species formation, two events considered to occur early in AD pathogenesis. Here, we show that these effects are allele-dependent and absolutely require the apoE4 background. Furthermore, the exact length of the fragment is critical since longer or shorter length carboxyl-terminal truncated apoE4 forms do not elicit the same effects. Structural and thermodynamic analyses showed that apoE4-165 has a compact structure, in contrast to other carboxyl-terminal truncated apoE4 forms that are instead destabilized. Compared however to other allelic backgrounds, apoE4-165 is structurally distinct and less thermodynamically stable suggesting that the combination of a well-folded structure with structural plasticity is a unique characteristic of this fragment. Overall, our findings suggest that the ability of apoE fragments to promote Aß42 intraneuronal accumulation is specific for both the apoE4 isoform and the particular structural and thermodynamic properties of the fragment.

ECM in brain aging and dementia.

An essential component of the brain extracellular space is the extracellular matrix contributing to the spatial assembly of cells by binding cell-surface adhesion molecules, supporting cell migration, differentiation, and tissue development. The most interesting and complex functions of the central nervous system are the abilities to encode new information (learning) and to store this information (memory). The creation of perineuronal nets, consisting mostly of chondroitin sulfate proteoglycans, stabilizes the synapses and memory trails and forms protective shields against neurodegenerative processes but terminates plasticity and the potential for recovery of the tissue. Age-related changes in the extracellular matrix composition and the extracellular space volume and permissivity are major determinants of the onset and development of the most common neurodegenerative disorder, Alzheimer's disease. In this regard, heparan sulfate proteoglycans, involved in amyloid clearance from the brain, play an important role in Alzheimer's disease and other types of neurodegeneration. Additional key players in the modification of the extracellular matrix are matrix metalloproteinases. Recent studies show that the extracellular matrix and matrix metalloproteinases are important regulators of plasticity, learning, and memory and might be involved in different neurological disorders like epilepsy, schizophrenia, addiction, and dementia. The identification of molecules and mechanisms that modulate these processes is crucial for the understanding of brain function and dysfunction and for the design of new therapeutic approaches targeting the molecular mechanism underlying these neurological disorders.

Neural ECM proteases in learning and synaptic plasticity.

Recent studies implicate extracellular proteases in synaptic plasticity, learning, and memory. The data are especially strong for such serine proteases as thrombin, tissue plasminogen activator, neurotrypsin, and neuropsin as well as matrix metalloproteinases, MMP-9 in particular. The role of those enzymes in the aforementioned phenomena is supported by the experimental results on the expression patterns (at the gene expression and protein and enzymatic activity levels) and functional studies, including knockout mice, specific inhibitors, etc. Counterintuitively, the studies have shown that the extracellular proteolysis is not responsible mainly for an overall degradation of the extracellular matrix (ECM) and loosening perisynaptic structures, but rather allows for releasing signaling molecules from the ECM, transsynaptic proteins, and latent form of growth factors. Notably, there are also indications implying those enzymes in the major neuropsychiatric disorders, probably by contributing to synaptic aberrations underlying such diseases as schizophrenia, bipolar, autism spectrum disorders, and drug addiction.

Pharmacogenetic inhibition of eIF4E-dependent Mmp9 mRNA translation reverses fragile X syndrome-like phenotypes.

Fragile X syndrome (FXS) is the leading genetic cause of autism. Mutations in Fmr1 (fragile X mental retardation 1 gene) engender exaggerated translation resulting in dendritic spine dysmorphogenesis, synaptic plasticity alterations, and behavioral deficits in mice, which are reminiscent of FXS phenotypes. Using postmortem brains from FXS patients and Fmr1 knockout mice (Fmr1(-/y)), we show that phosphorylation of the mRNA 5' cap binding protein, eukaryotic initiation factor 4E (eIF4E), is elevated concomitant with increased expression of matrix metalloproteinase 9 (MMP-9) protein. Genetic or pharmacological reduction of eIF4E phosphorylation rescued core behavioral deficits, synaptic plasticity alterations, and dendritic spine morphology defects via reducing exaggerated translation of Mmp9 mRNA in Fmr1(-/y) mice, whereas MMP-9 overexpression produced several FXS-like phenotypes. These results uncover a mechanism of regulation of synaptic function by translational control of Mmp-9 in FXS, which opens the possibility of new treatment avenues for the diverse neurological and psychiatric aspects of FXS.

Calcitonin-induced NF-κB activation up-regulates fibronectin expression in MG63 osteosarcoma cells.

Salmon calcitonin has been used extensively as a therapeutic tool in the regulation of bone remodeling. However, there is a growing body of evidence indicating that the calcitonin peptides are involved in regulation of cell growth, differentiation, survival and tissue development. In the present study, we investigated the effect of calcitonin in cell matrix interactions in MG63 cell line. Our results demonstrated that calcitonin increases cell growth of MG63 osteosarcoma cells in parallel with serine/threonine protein kinase B (AKT/PKB) activation. Moreover, calcitonin induced up-regulation of fibronectin expression in a nuclear factor-kappa B (NF-κB)-dependent manner, accompanied by enhanced enzymatic activity of matrix metalloproteinase-9 (MMP-9) and increased expression of tissue inhibitors of MMP-1 and -2. MMP-9 stimulation with calcitonin was accompanied by an increase in protein expression of the α5ß1 integrin receptor. To our knowledge, our results demonstrate, for the first time, that calcitonin is a potent inducer of fibronectin, an extacellular matrix component that is suggested to have a pro-oncogenic and healing effect, in a NF-κB-dependent manner.

Matrix metalloproteinase-9 participates in NGF-induced α-secretase cleavage of amyloid-ß protein precursor in PC12 cells.

Amyloid-ß protein precursor (AßPP) is a ubiquitously expressed glycoprotein, which under physiological conditions can be cleaved following two alternative routes; the non-amyloidogenic and the amyloidogenic pathway. Shift of AßPP processing in favor of the amyloidogenic pathway is a key event in the pathogenesis of Alzheimer's disease (AD). Among the factors that regulate AßPP processing, nerve growth factor (NGF) appears to play an important role; abnormal NGF signaling has been implicated in the onset of AD. In the present study, we used PC12 cells to study the effects of NGF on AßPP processing and provide evidence that NGF, through binding to its high affinity receptor, TrkA moderately down-regulates the expression of the ß-secretase ß-site AßPP cleaving enzyme-1 and, most importantly, upregulates the expression of two enzymes with α-secretase activity, a disintegrin and metalloprotease-17 and to a greater extent matrix metalloproteinase-9 (MMP9) in a phosphoinositide kinase-3 dependent manner. Finally, we demonstrate that MMP9 actively participates in NGF-induced α-secretase cleavage of AßPP, thus it contributes to the shift of AßPP processing towards the non-amyloidogenic pathway precluding the formation of neurotoxic Aß peptides.

Abeta(1-40)-induced secretion of matrix metalloproteinase-9 results in sAPPalpha release by association with cell surface APP.

To understand matrix metalloproteinase-9 (MMP-9) involvement in Alzheimer's disease, we examined mechanisms mediating increased expression of MMP-9 in the presence of Abeta(1-40) and the role of MMP-9 on amyloid precursor protein (APP) processing. Up-regulation of MMP-9 expressed by SK-N-SH cells in the presence of Abeta(1-40) was mediated by alpha(3)beta(1) and alpha(2)beta(1) integrin receptors. Overexpression of MMP-9 or treatment of HEK/APP695 cells with activated recombinant MMP-9 resulted in enhanced secretion of soluble APP (sAPPalpha), a product of alpha-secretase cleavage, and reduction of Abeta release. MMP-9 effect was enhanced by phorbol 12-mysistrate-13-acetate (PMA), an alpha-secretase activator and inhibited by EDTA or SB-3CT, an MMP-9 inhibitor. Additionally, immunoprecipitation and confocal microscopy demonstrated that MMP-9 and APP695 were associated on the cell surface. These results indicate that Abeta peptide increases MMP-9 secretion through integrins; MMP-9 then directly processes cell surface APP695 with an alpha-secretase like activity, substantially reducing the levels of secreted Abeta peptide.

Effects of collagen IV on neuroblastoma cell matrix-related functions.

Integrin-mediated interactions with collagen IV and its domains were examined in a human neuroblastoma cell line (SK-N-SH). By adhesion assays we demonstrated that neuroblastoma cells bound to solid-phase intact collagen IV and synthetic cell-binding peptide HEP-III, derived from the collagenous part of the molecule, but not to the main noncollagenous NC1 domain or to the synthetic cell-binding peptide HEP-I, derived from this domain. Monoclonal antibodies against beta1, alpha3, and alpha(v)beta3 integrins resulted in inhibition of cell binding to collagenous substrates by 95, 30, and 35%, respectively. By flow cytometry and immunoblotting it was shown that culture of SK-N-SH cells on collagen IV resulted in alteration in the expression of major neuroblastoma cell integrins. Binding to collagen IV induced the expression and activation of matrix metalloproteinases A and B (MMP-2, MMP-9), with a concomitant increase at the protein level of tissue-specific inhibitors of metalloproteinases (TIMP-1, TIMP-2). Finally, the expression of MMP-2 was significantly up-regulated by anti-alpha3beta1 antibodies, whereas ligation of anti-alpha(v)beta3 antibodies resulted in a modest down-regulation of MMP-2. Our results indicate that the presence of collagen IV modulates the expression of integrins, which are used for binding to this glycoprotein, and MMP-2 secreted by SK-N-SH cells.