Tumor-reactive antibodies evolve from non-binding and autoreactive precursors.

The tumor microenvironment hosts antibody-secreting cells (ASCs) associated with a favorable prognosis in several types of cancer. Patient-derived antibodies have diagnostic and therapeutic potential; yet, it remains unclear how antibodies gain autoreactivity and target tumors. Here, we found that somatic hypermutations (SHMs) promote antibody antitumor reactivity against surface autoantigens in high-grade serous ovarian carcinoma (HGSOC). Patient-derived tumor cells were frequently coated with IgGs. Intratumoral ASCs in HGSOC were both mutated and clonally expanded and produced tumor-reactive antibodies that targeted MMP14, which is abundantly expressed on the tumor cell surface. The reversion of monoclonal antibodies to their germline configuration revealed two types of classes: one dependent on SHMs for tumor binding and a second with germline-encoded autoreactivity. Thus, tumor-reactive autoantibodies are either naturally occurring or evolve through an antigen-driven selection process. These findings highlight the origin and potential applicability of autoantibodies directed at surface antigens for tumor targeting in cancer patients.

Proteogenomics of Non-smoking Lung Cancer in East Asia Delineates Molecular Signatures of Pathogenesis and Progression.

Lung cancer in East Asia is characterized by a high percentage of never-smokers, early onset and predominant EGFR mutations. To illuminate the molecular phenotype of this demographically distinct disease, we performed a deep comprehensive proteogenomic study on a prospectively collected cohort in Taiwan, representing early stage, predominantly female, non-smoking lung adenocarcinoma. Integrated genomic, proteomic, and phosphoproteomic analysis delineated the demographically distinct molecular attributes and hallmarks of tumor progression. Mutational signature analysis revealed age- and gender-related mutagenesis mechanisms, characterized by high prevalence of APOBEC mutational signature in younger females and over-representation of environmental carcinogen-like mutational signatures in older females. A proteomics-informed classification distinguished the clinical characteristics of early stage patients with EGFR mutations. Furthermore, integrated protein network analysis revealed the cellular remodeling underpinning clinical trajectories and nominated candidate biomarkers for patient stratification and therapeutic intervention. This multi-omic molecular architecture may help develop strategies for management of early stage never-smoker lung adenocarcinoma.

Rapid Mobilization Reveals a Highly Engraftable Hematopoietic Stem Cell.

Hematopoietic stem cell transplantation is a potential curative therapy for malignant and nonmalignant diseases. Improving the efficiency of stem cell collection and the quality of the cells acquired can broaden the donor pool and improve patient outcomes. We developed a rapid stem cell mobilization regimen utilizing a unique CXCR2 agonist, GROß, and the CXCR4 antagonist AMD3100. A single injection of both agents resulted in stem cell mobilization peaking within 15 min that was equivalent in magnitude to a standard multi-day regimen of granulocyte colony-stimulating factor (G-CSF). Mechanistic studies determined that rapid mobilization results from synergistic signaling on neutrophils, resulting in enhanced MMP-9 release, and unexpectedly revealed genetic polymorphisms in MMP-9 that alter activity. This mobilization regimen results in preferential trafficking of stem cells that demonstrate a higher engraftment efficiency than those mobilized by G-CSF. Our studies suggest a potential new strategy for the rapid collection of an improved hematopoietic graft.

Ciliary Hedgehog Signaling Restricts Injury-Induced Adipogenesis.

Injured skeletal muscle regenerates, but with age or in muscular dystrophies, muscle is replaced by fat. Upon injury, muscle-resident fibro/adipogenic progenitors (FAPs) proliferated and gave rise to adipocytes. These FAPs dynamically produced primary cilia, structures that transduce intercellular cues such as Hedgehog (Hh) signals. Genetically removing cilia from FAPs inhibited intramuscular adipogenesis, both after injury and in a mouse model of Duchenne muscular dystrophy. Blocking FAP ciliation also enhanced myofiber regeneration after injury and reduced myofiber size decline in the muscular dystrophy model. Hh signaling through FAP cilia regulated the expression of TIMP3, a secreted metalloproteinase inhibitor, that inhibited MMP14 to block adipogenesis. A pharmacological mimetic of TIMP3 blocked the conversion of FAPs into adipocytes, pointing to a strategy to combat fatty degeneration of skeletal muscle. We conclude that ciliary Hh signaling by FAPs orchestrates the regenerative response to skeletal muscle injury.

Cellular and Molecular Mechanisms of MT1-MMP-Dependent Cancer Cell Invasion.

Metastasis is responsible for most cancer-associated deaths. Accumulating evidence based on 3D migration models has revealed a diversity of invasive migratory schemes reflecting the plasticity of tumor cells to switch between proteolytic and nonproteolytic modes of invasion. Yet, initial stages of localized regional tumor dissemination require proteolytic remodeling of the extracellular matrix to overcome tissue barriers. Recent data indicate that surface-exposed membrane type 1-matrix metalloproteinase (MT1-MMP), belonging to a group of membrane-anchored MMPs, plays a central role in pericellular matrix degradation during basement membrane and interstitial tissue transmigration programs. In addition, a large body of work indicates that MT1-MMP is targeted to specialized actin-rich cell protrusions termed invadopodia, which are responsible for matrix degradation. This review describes the multistep assembly of actin-based invadopodia in molecular details. Mechanisms underlying MT1-MMP traffic to invadopodia through endocytosis/recycling cycles, which are key to the invasive program of carcinoma cells, are discussed.

An mTORC1-GRASP55 signaling axis controls unconventional secretion to reshape the extracellular proteome upon stress.

Cells communicate with their environment via surface proteins and secreted factors. Unconventional protein secretion (UPS) is an evolutionarily conserved process, via which distinct cargo proteins are secreted upon stress. Most UPS types depend upon the Golgi-associated GRASP55 protein. However, its regulation and biological role remain poorly understood. Here, we show that the mechanistic target of rapamycin complex 1 (mTORC1) directly phosphorylates GRASP55 to maintain its Golgi localization, thus revealing a physiological role for mTORC1 at this organelle. Stimuli that inhibit mTORC1 cause GRASP55 dephosphorylation and relocalization to UPS compartments. Through multiple, unbiased, proteomic analyses, we identify numerous cargoes that follow this unconventional secretory route to reshape the cellular secretome and surfactome. Using MMP2 secretion as a proxy for UPS, we provide important insights on its regulation and physiological role. Collectively, our findings reveal the mTORC1-GRASP55 signaling hub as the integration point in stress signaling upstream of UPS and as a key coordinator of the cellular adaptation to stress.

ETS transcription factors regulate precise matrix metalloproteinase expression and follicle rupture in Drosophila.

Drosophila matrix metalloproteinase 2 (MMP2) is specifically expressed in posterior follicle cells of stage-14 egg chambers (mature follicles) and is crucial for the breakdown of the follicular wall during ovulation, a process that is highly conserved from flies to mammals. The factors that regulate spatiotemporal expression of MMP2 in follicle cells remain unknown. Here, we demonstrate crucial roles for the ETS-family transcriptional activator Pointed (Pnt) and its endogenous repressor Yan in the regulation of MMP2 expression. We found that Pnt is expressed in posterior follicle cells and overlaps with MMP2 expression in mature follicles. Genetic analysis demonstrated that pnt is both required and sufficient for MMP2 expression in follicle cells. In addition, Yan was temporally upregulated in stage-13 follicle cells to fine-tune Pnt activity and MMP2 expression. Furthermore, we identified a 1.1 kb core enhancer that is responsible for the spatiotemporal expression of MMP2 and contains multiple pnt/yan binding motifs. Mutation of pnt/yan binding sites significantly impaired the Mmp2 enhancer activity. Our data reveal a mechanism of transcriptional regulation of Mmp2 expression in Drosophila ovulation, which could be conserved in other biological systems.

Eta-secretase-like processing of the amyloid precursor protein (APP) by the rhomboid protease RHBDL4.

The amyloid precursor protein (APP) is a key protein in Alzheimer's disease synthesized in the endoplasmic reticulum (ER) and translocated to the plasma membrane where it undergoes proteolytic cleavages by several proteases. Conversely, to other known proteases, we previously elucidated rhomboid protease RHBDL4 as a novel APP processing enzyme where several cleavages likely occur already in the ER. Interestingly, the pattern of RHBDL4-derived large APP C-terminal fragments resembles those generated by the η-secretase or MT5-MMP, which was described to generate so-called Aη fragments. The similarity in large APP C-terminal fragments between both proteases raised the question of whether RHBDL4 may contribute to η-secretase activity and Aη-like fragments. Here, we identified two cleavage sites of RHBDL4 in APP by mass spectrometry, which, intriguingly, lie in close proximity to the MT5-MMP cleavage sites. Indeed, we observed that RHBDL4 generates Aη-like fragments in vitro without contributions of α-, ß-, or γ-secretases. Such Aη-like fragments are likely generated in the ER since RHBDL4-derived APP-C-terminal fragments do not reach the cell surface. Inherited, familial APP mutations appear to not affect this processing pathway. In RHBDL4 knockout mice, we observed increased cerebral full-length APP in comparison to wild type (WT) in support of RHBDL4 being a physiologically relevant protease for APP. Furthermore, we found secreted Aη fragments in dissociated mixed cortical cultures from WT mice, however significantly fewer Aη fragments in RHBDL4 knockout cultures. Our data underscores that RHBDL4 contributes to the η-secretease-like processing of APP and that RHBDL4 is a physiologically relevant protease for APP.

Downregulation of PIK3IP1/TrIP on T cells is controlled by TCR signal strength, PKC and metalloprotease-mediated cleavage.

The protein known as PI3K-interacting protein (PIK3IP1), or transmembrane inhibitor of PI3K (TrIP), is highly expressed by T cells and can modulate PI3K activity in these cells. Several studies have also revealed that TrIP is rapidly downregulated following T cell activation. However, it is unclear as to how this downregulation is controlled. Using a novel monoclonal antibody that robustly stains cell-surface TrIP, we demonstrate that TrIP is lost from the surface of activated T cells in a manner dependent on the strength of signaling through the T cell receptor (TCR) and specific downstream signaling pathways, in particular classical PKC isoforms. TrIP expression returns by 24 hours after stimulation, suggesting that it may play a role in resetting TCR signaling at later time points. We also provide evidence that ADAM family proteases are required for both constitutive and stimulation-induced downregulation of TrIP in T cells. Finally, by expressing truncated forms of TrIP in cells, we identify the region in the extracellular stalk domain of TrIP that is targeted for proteolytic cleavage.

TRPV4 mediates IL-1-induced Ca2+ signaling, ERK activation and MMP expression.

Ca2+ permeation through TRPV4 in fibroblasts is associated with pathological matrix degradation. In human gingival fibroblasts, IL-1ß binding to its signaling receptor (IL-1R1) induces activation of extracellular regulated kinase (ERK) and MMP1 expression, processes that require Ca2+ flux across the plasma membrane. It is not known how IL-1R1, which does not conduct Ca2+, generates Ca2+ signals in response to IL-1. We examined whether TRPV4 mediates the Ca2+ fluxes required for ERK signaling in IL-1 stimulated gingival fibroblasts. TRPV4 was immunostained in fibroblasts of human gingival connective tissue and in focal adhesions of cultured mouse gingival fibroblasts. Human gingival fibroblasts treated with IL-1ß showed no change of TRPV4 expression but there was increased MMP1 expression. In mouse, gingival fibroblasts expressing TRPV4, IL-1 strongly increased [Ca2+]i. Pre-incubation of cells with IL-1 Receptor Antagonist blocked Ca2+ entry induced by IL-1 or the TRPV4 agonist GSK101. Knockout of TRPV4 or expression of a non-Ca2+-conducting TRPV4 pore-mutant or pre-incubation with the TRPV4 inhibitor RN1734, blocked IL-1-induced Ca2+ transients and expression of the mouse interstitial collagenase, MMP13. Treatment of mouse gingival fibroblasts with GSK101 phenocopied Ca2+ and ERK responses induced by IL-1; these responses were absent in TRPV4-null cells or cells expressing a non-conducting TRPV4 pore-mutant. Immunostained IL-1R1 localized with TRPV4 in adhesions within cell extensions. While TRPV4 immunoprecipitates analyzed by mass spectrometry showed no association with IL-1R1, TRPV4 associated with Src-related proteins and Src co-immunoprecipitated with TRPV4. Src inhibition reduced IL-1-induced Ca2+ responses. The functional linkage of TRPV4 with IL-1R1 expands its repertoire of innate immune signaling processes by mediating IL-1-driven Ca2+ responses that drive matrix remodeling in fibroblasts. Thus, inhibiting TRPV4 activity may provide a new pharmacological approach for blunting matrix degradation in inflammatory diseases.

Disorganisation of basement membrane zone architecture impairs melanocyte residence in vitiligo.

The basement membrane zone is the interface between the epidermis and dermis, and it is disrupted in several skin conditions. Here, we report the results of a comprehensive investigation into the structural and molecular factors of the basement membrane zone in vitiligo, a dermatological disorder characterised by depigmented patches on the skin. Using electron microscopy and immunofluorescence staining, we confirmed abnormal basement membrane zone morphology and disrupted basement membrane zone architecture in human vitiliginous skin. Furthermore, we identified elevated expression of matrix metalloproteinase 2 (MMP2) in human dermal fibroblasts as a key factor responsible for basement membrane zone matrix degradation. In our in vitro and ex vivo models, overexpression of MMP2 in fibroblasts led to basement membrane zone disruption and melanocyte disappearance. Importantly, we reveal that the loss of melanocytes in vitiligo is primarily linked to their weakened adhesion to the basement membrane, mediated by binding between integrin ß1 and laminin and discoidin domain receptor 1 and collagen IV. Finally, inhibition of matrix metalloproteinase 2 expression reversed depigmentation in a mouse model of vitiligo. In conclusion, our research shows the importance of basement membrane zone integrity in melanocyte residence and offers new avenues for therapeutic interventions to address this challenging skin condition. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Degradomic Identification of Membrane Type 1-Matrix Metalloproteinase as an ADAMTS9 and ADAMTS20 Substrate.

The secreted metalloproteases ADAMTS9 and ADAMTS20 are implicated in extracellular matrix proteolysis and primary cilium biogenesis. Here, we show that clonal gene-edited RPE-1 cells in which ADAMTS9 was inactivated, and which constitutively lack ADAMTS20 expression, have morphologic characteristics distinct from parental RPE-1 cells. To investigate underlying proteolytic mechanisms, a quantitative terminomics method, terminal amine isotopic labeling of substrates was used to compare the parental and gene-edited RPE-1 cells and their medium to identify ADAMTS9 substrates. Among differentially abundant neo-amino (N) terminal peptides arising from secreted and transmembrane proteins, a peptide with lower abundance in the medium of gene-edited cells suggested cleavage at the Tyr314-Gly315 bond in the ectodomain of the transmembrane metalloprotease membrane type 1-matrix metalloproteinase (MT1-MMP), whose mRNA was also reduced in gene-edited cells. This cleavage, occurring in the MT1-MMP hinge, that is, between the catalytic and hemopexin domains, was orthogonally validated both by lack of an MT1-MMP catalytic domain fragment in the medium of gene-edited cells and restoration of its release from the cell surface by reexpression of ADAMTS9 and ADAMTS20 and was dependent on hinge O-glycosylation. A C-terminally semitryptic MT1-MMP peptide with greater abundance in WT RPE-1 medium identified a second ADAMTS9 cleavage site in the MT1-MMP hemopexin domain. Consistent with greater retention of MT1-MMP on the surface of gene-edited cells, pro-MMP2 activation, which requires cell surface MT1-MMP, was increased. MT1-MMP knockdown in gene-edited ADAMTS9/20-deficient cells restored focal adhesions but not ciliogenesis. The findings expand the web of interacting proteases at the cell surface, suggest a role for ADAMTS9 and ADAMTS20 in regulating cell surface activity of MT1-MMP, and indicate that MT1-MMP shedding does not underlie their observed requirement in ciliogenesis.

Metformin represses the carcinogenesis potentially induced by 50 Hz magnetic fields in aged mouse fibroblasts via inhibition of NF-kB.

Aging is a risk factor for various human disorders, including cancer. Current literature advocates that the primary principles of aging depend on the endogenous stress-induced DNA damage caused by reactive oxygen species 50 Hz low-frequency magnetic field was suggested to induce DNA damage and chromosomal instability. NF-kB, activated by DNA damage, is upregulated in age-related cancers and inhibition of NF-kB results in aging-related delayed pathologies. Metformin (Met), an NF-kB inhibitor, significantly reduces both NF-kB activation and expression in aging and cancer. This in vitro study, therefore, was set out to assess the effects of 5mT MF in 50 Hz frequency and Met treatment on the viability and proliferation of aged mouse NIH/3T3 fibroblasts and expression of RELA/p65, matrix metalloproteinases MMP2 and MMP9, and E-cadherin (CDH1) genes. The trypan blue exclusion assay was used to determine cell viability and the BrdU incorporation assay to determine cell proliferation. The MMP-2/9 protein analysis was carried out by immunocytochemistry, NF-kB activity by ELISA and the expressions of targeted genes by qRT-PCR methods. Four doses of Met (500 uM, 1 mM, 2 mM and 10 mM) suppressed both the proliferation and viability of fibroblasts exposed to the MF in a dose-dependent pattern, and the peak inhibition was recorded at the 10 mM dose. Met reduced the expression of NF-kB, and MMP2/9, elevated CDH1 expression and suppressed NF-kB activity. These findings suggest that Met treatment suppresses the carcinogenic potential of 50 Hz MFs in aged mouse fibroblasts, possibly through modulation of NF-kB activation and epithelial-mesenchymal transition modulation.

Steroid receptor coactivator 1 promotes human hepatocellular carcinoma invasiveness through enhancing MMP-9.

SRC-1 functions as a transcriptional coactivator for steroid receptors and various transcriptional factors. Notably, SRC-1 has been implicated in oncogenic roles in multiple cancers, including breast cancer and prostate cancer. Previous investigations from our laboratory have established the high expression of SRC-1 in human HCC specimens, where it accelerates HCC progression by enhancing Wnt/beta-catenin signalling. In this study, we uncover a previously unknown role of SRC-1 in HCC metastasis. Our findings reveal that SRC-1 promotes HCC metastasis through the augmentation of MMP-9 expression. The knockdown of SRC-1 effectively mitigated HCC cell metastasis both in vitro and in vivo by suppressing MMP-9 expression. Furthermore, we observed a positive correlation between SRC-1 mRNA levels and MMP-9 mRNA levels in limited and larger cohorts of HCC specimens from GEO database. Mechanistically, SRC-1 operates as a coactivator for NF-κB and AP-1, enhancing MMP-9 promoter activity in HCC cells. Higher levels of SRC-1 and MMP-9 expression are associated with worse overall survival in HCC patients. Treatment with Bufalin, known to inhibit SRC-1 expression, significantly decreased MMP-9 expression and inhibited HCC metastasis in both in vitro and in vivo settings. Our results demonstrated the pivotal role of SRC-1 as a critical modulator in HCC metastasis, presenting a potential therapeutic target for HCC intervention.

Matrix metalloproteinases in kidney homeostasis and diseases: an update.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases with important roles in kidney homeostasis and pathology. While capable of collectively degrading each component of the extracellular matrix, MMPs also degrade nonmatrix substrates to regulate inflammation, epithelial plasticity, proliferation, apoptosis, and angiogenesis. More recently, intriguing mechanisms that directly alter podocyte biology have been described. There is now irrefutable evidence for MMP dysregulation in many types of kidney disease including acute kidney injury, diabetic and hypertensive nephropathy, polycystic kidney disease and Alport syndrome. This updated review will detail the complex biology of MMPs in kidney disease.

NF-κB/RelA signaling in secretoglobin progenitors mediates plasticity and MMP-induced barrier disruption in house dust mite-induced allergic asthma.

The mechanisms how aeroallergens induce sensitization are incompletely understood. The house dust mite (HDM) Dermatophagoides pteronyssius (Der p) is a ubiquitous aeroallergen that represents a major cause of allergic rhinitis and asthma. Herein, we tested whether HDM-induced aeroallergen exposure sensitivity is caused by the innate-immune response in small airway epithelial cells. HDM exposure is a rapid activator of NF-κB/RelA in the Secretoglobin (Scgb1a1+) lineage associated with upregulation of NF-κB/RelA-dependent markers of epithelial plasticity. To determine the effect of epithelial NF-κB signaling, NF-κB was depleted in a tamoxifen (TMX)-inducible Scgb1a1-CreERTM mouse within a CL57B/L6 background. Corn oil or TMX-treated/RelA-depleted [RelA knockdown (KD)] mice were repetitively exposed to airway HDM challenges to induce airway hyperresponsiveness (AHR). Strikingly, we observed that HDM induces hallmarks of epithelial plasticity through upregulation of the mesenchymal core factors SNAI1 and ZEB1 and production of metalloproteinase (MMP)9 that are RelA-dependent. Downstream, HDM-induced mucous metaplasia, Th2 polarization, allergen sensitivity, and airway hyperreactivity were all reduced in the RelA-depleted mice. Mechanistically, HDM-induced functional and structural barrier disruption was dependent on RelA signaling and associated with active MMP secretion into the bronchoalveolar lavage fluid. To establish the role of MMP2/9 in barrier disruption, we observe that a small-molecule MMP inhibitor (SB-3CT) blocked HDM-induced barrier disruption and activation of plasticity in naïve wild-type (WT) mice. Loss of functional barrier was associated with MMP disruption of zona occludens (ZO)-1 containing adherens junctions. Overall, this data indicates that host innate signaling in the Scgb1a1+ progenitors is directly linked to epithelial plasticity, MMP9 secretion, and enhanced barrier permeability that allows allergen penetration, sensitization producing allergic asthma (AA) in vivo. We propose that maintenance of epithelial integrity may reduce allergic sensitization and AA.NEW & NOTEWORTHY Allergic asthma from house dust mite (HDM) allergy causes substantial morbidity. This study examines the dynamic changes in small airway epithelial cells in a mouse model of HDM exposure. Our findings indicate that NF-κB/RelA signaling mediates matrix metalloproteinase production, disrupting the epithelial barrier resulting in allergic sensitization. Our findings bring new insight into mechanisms for epithelial cell-state change in the allergen response, creating a potential therapeutic pathway for maintaining barrier function in asthma.

Interaction of MMP-9 in the active phase of Graves' disease with and without ophthalmopathy.

Thyroid eye disease (TED) is expressed as orbital inflammation, and serum levels of several proinflammatory cytokines have been studied among patients with Graves' disease (GD) with and without TED; however, a more sensitive and specific marker for the different phases of GD and TED is still lacking. Seventeen active TED, 16 inactive TED, 16 GD without TED, and 16 healthy controls were recruited. Serum IL-17A, MMP-2, MMP-3, and MMP-9 were measured by multiplex bead assay. TED hormone and eye parameters were evaluated, and their relationship with cytokine levels was analyzed. Serum MMP-9 was higher in active TED than healthy controls, whereas IL-17A was lower among these patients than in GD without TED and healthy controls. No differences were found in MMP-3 and MMP-2 concentrations. MMP-9 levels were lower in patients with inactive TED who underwent radioactive iodine (RAI) therapy and those on levothyroxine replacement. MMP-9 levels were elevated in patients on methimazole. A negative correlation was found between age at assessment and time of follow-up with MMP-9 levels in inactive TED. Free T3 and ophthalmometry values were positively correlated with MMP-9 in the GD without TED and inactive TED groups, respectively. In conclusion, serum MMP-9 was increased in patients with active TED and was related to the RAI treatment, longer follow-up time, and higher ophthalmometry in patients with inactive TED, as well as thyroid function in GD without TED. MMP-9 may be involved in both the active phase of TED and the active phase of inflammation related to GD.NEW & NOTEWORTHY Our study addresses clinical aspects of specific ophthalmological examination and serum cytokine concentrations of patients with Graves' disease (GD) with and without ophthalmopathy. Our findings suggest that MMP-9 may be involved in the active phase of ophthalmopathy and in the active phase of GD. The central question is whether MMP-9 is a potential target for future treatments.

Resveratrol differentially affects MMP-9 release from neurons and glia; implications for therapeutic efficacy.

Resveratrol, a naturally occurring polyphenol that activates sirtuin 1 (SIRT1), has been shown to reduce overall levels of matrix metalloprotease-9 (MMP-9) in cerebrospinal fluid (CSF) samples from patients with Alzheimer's dementia (AD). Depending on the site of release, however, MMP-9 has the potential to improve or impair cognition. In particular, its release from microglia or pericytes proximal to the blood brain barrier can damage the basement membrane, while neuronal activity-dependent release of this protease from glutamatergic neurons can instead promote dendritic spine expansion and long-term potentiation of synaptic plasticity. In the present study, we test the hypothesis that resveratrol reduces overall MMP-9 levels in CSF samples from patients with APOE4, an allele associated with increased glial inflammation. We also examine the possibility that resveratrol reduces inflammation-associated MMP release from cultured glia but spares neuronal activity-dependent release from cultured cortical neurons. We observe that resveratrol decreases overall levels of MMP-2 and MMP-9 in CSF samples from AD patients. Resveratrol also reduces CSF levels of tissue inhibitor of metalloproteinases-1 (TIMP-1), glial-derived protein that restricts long-term potentiation of synaptic transmission, in individuals homozygous for APOE4. Consistent with these results, we observe that resveratrol reduces basal and lipopolysaccharide (LPS)-stimulated MMP and TIMP-1 release from cultured microglia and astrocytes. In contrast, however, resveratrol does not inhibit release of MMP-9 from cortical neurons. Overall, these results are consistent with the possibility that while resveratrol reduces potentially maladaptive MMP and TIMP-1 release from activated glia, neuroplasticity-promoting MMP release from neurons is spared. In contrast, resveratrol reduces release of neurocan and brevican, extracellular matrix components that restrict neuroplasticity, from both neurons and glia. These data underscore the diversity of resveratrol's actions with respect to affected cell types and molecular targets and also suggest that further studies may be warranted to determine if its effects on glial MMP release could make it a useful adjunct for AD- and/or anti-amyloid therapy-related damage to the blood brain barrier.

MMP-2-mediated Scube2 degradation promotes blood-brain barrier disruption by blocking the interaction between astrocytes and endothelial cells via inhibiting Sonic hedgehog pathway during early cerebral ischemia.

We have previously demonstrated a rapid secretion of matrix metalloproteinase-2 (MMP-2) in the ischemic brain. Since Scube2 can interact with Sonic hedgehog (Shh) to maintain blood-brain barrier (BBB) integrity via regulating the interaction between brain capillary endothelial cells (ECs) and perivascular astrocytes, and it is also a substrate of MMP-2, we hypothesized that the secreted MMP-2 could degrade Scube2 and contribute to ischemic BBB disruption. Using an in vitro ischemic model of 90-min oxygen-glucose deprivation/3-h reoxygenation (OGD/R) and an in vivo mouse stroke model of 90-min middle cerebral artery occlusion (MCAO) with 3-h reperfusion, we established an important role of MMP-2-mediated Scube2 degradation in early ischemic BBB disruption. Exposure of C8-D1A cells and bEnd.3 cells to OGD/R increased MMP secretion in both cells, and C8-D1A cells appeared to secrete more MMPs than bEnd.3 cells. Co-IP and double-immunostaining revealed that Scube2 co-localized well with MMP-2 in C8-D1A cells and could be pulled down by MMP-2 antibodies. In MCAO mice, Scube2 protein showed a drastic reduction in ischemic brain tissue, which was accompanied by suppressed expression of Shh and its downstream molecules. Of note, specific knockdown of astrocytic Scube2 with AAV-shScube2 augmented MCAO-induced Shh suppression and exacerbated BBB leakage and inflammatory reactions in the ischemic brain. Last, incubation of bEnd.3 cells with conditioned medium derived from OGD-treated C8-D1A cells led to a significant inhibition of the Shh pathway in bEnd.3 cells and degradation of VE-cadherin and ZO-1. Inhibition of MMP-2 with SB-3CT or over-expression of Scube2 with plasmids in C8-D1A cells alleviated the above effect of C8-D1A cells-derived conditioned medium. Taken together, our data indicate that ischemia-induced secretion of MMP-2 may contribute to early BBB disruption in ischemic stroke via interrupting the shared Scube2-Shh pathway between brain capillary ECs and perivascular astrocytes.

Phagocytosis of aggrecan-positive perineuronal nets surrounding motor neurons by reactive microglia expressing MMP-9 in TDP-43Q331K ALS model mice.

Perineuronal nets (PNNs) are extracellular matrix structures that surround excitable neurons and their proximal dendrites. PNNs play an important role in neuroprotection against oxidative stress. Oxidative stress within motor neurons can act as a trigger for neuronal death, and this has been implicated in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). We therefore characterised PNNs around alpha motor neurons and the possible contributing cellular factors in the mutant TDP-43Q331K transgenic mouse, a slow onset ALS mouse model. PNNs around alpha motor neurons showed significant loss at mid-stage disease in TDP-43Q331K mice compared to wild type strain control mice. PNN loss coincided with an increased expression of matrix metallopeptidase-9 (MMP-9), an endopeptidase known to cleave PNNs, within the ventral horn. During mid-stage disease, increased numbers of microglia and astrocytes expressing MMP-9 were present in the ventral horn of TDP-43Q331K mice. In addition, TDP-43Q331K mice showed increased levels of aggrecan, a PNN component, in the ventral horn by microglia and astrocytes during this period. Elevated aggrecan levels within glia were accompanied by an increase in fractalkine expression, a chemotaxic protein responsible for the recruitment of microglia, in alpha motor neurons of onset and mid-stage TDP-43Q331K mice. Following PNN loss, alpha motor neurons in mid-stage TDP-43Q331K mice showed increased 3-nitrotyrosine expression, an indicator of protein oxidation. Together, our observations along with previous PNN research provide suggests a possible model whereby microglia and astrocytes expressing MMP-9 degrade PNNs surrounding alpha motor neurons in the TDP-43Q331K mouse. This loss of nets may expose alpha-motor neurons to oxidative damage leading to degeneration of the alpha motor neurons in the TDP-43Q331K ALS mouse model.