ABSTRACT
BACKGROUND: Membrane-type matrix metalloproteinase 5 (MT5-MMP) deficiency in the 5xFAD mouse model of Alzheimer's disease (AD) reduces brain neuroinflammation and amyloidosis, and prevents deficits in synaptic activity and cognition in prodromal stages of the disease. In addition, MT5-MMP deficiency prevents interleukin-1 beta (IL-1ß)-mediated inflammation in the peripheral nervous system. In this context, we hypothesized that the MT5-MMP/IL-1ß tandem could regulate nascent AD pathogenic events in developing neural cells shortly after the onset of transgene activation. METHODS: To test this hypothesis, we used 11-14 day in vitro primary cortical cultures from wild type, MT5-MMP-/-, 5xFAD and 5xFAD/MT5-MMP-/- mice, and evaluated the impact of MT5-MMP deficiency and IL-1ß treatment for 24 h, by performing whole cell patch-clamp recordings, RT-qPCR, western blot, gel zymography, ELISA, immunocytochemistry and adeno-associated virus (AAV)-mediated transduction. RESULTS: 5xFAD cells showed higher levels of MT5-MMP than wild type, concomitant with higher basal levels of inflammatory mediators. Moreover, MT5-MMP-deficient cultures had strong decrease of the inflammatory response to IL-1ß, as well as decreased stability of recombinant IL-1ß. The levels of amyloid beta peptide (Aß) were similar in 5xFAD and wild-type cultures, and IL-1ß treatment did not affect Aß levels. Instead, the absence of MT5-MMP significantly reduced Aß by more than 40% while sparing APP metabolism, suggesting altogether no functional crosstalk between IL-1ß and APP/Aß, as well as independent control of their levels by MT5-MMP. The lack of MT5-MMP strongly downregulated the AAV-induced neuronal accumulation of the C-terminal APP fragment, C99, and subsequently that of Aß. Finally, MT5-MMP deficiency prevented basal hyperexcitability observed in 5xFAD neurons, but not hyperexcitability induced by IL-1ß treatment. CONCLUSIONS: Neuroinflammation and hyperexcitability precede Aß accumulation in developing neural cells with nascent expression of AD transgenes. MT5-MMP deletion is able to tune down basal neuronal inflammation and hyperexcitability, as well as APP/Aß metabolism. In addition, MT5-MMP deficiency prevents IL-1ß-mediated effects in brain cells, except hyperexcitability. Overall, this work reinforces the idea that MT5-MMP is at the crossroads of pathogenic AD pathways that are already incipiently activated in developing neural cells, and that targeting MT5-MMP opens interesting therapeutic prospects.
Subject(s)
Alzheimer Disease , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/toxicity , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Astrocytes/metabolism , Disease Models, Animal , Matrix Metalloproteinases/metabolism , Mice , Mice, Transgenic , Neuroinflammatory Diseases , Neurons/metabolismABSTRACT
We previously discovered the implication of membrane-type 5-matrix metalloproteinase (MT5-MMP) in Alzheimer's disease (AD) pathogenesis. Here, we shed new light on pathogenic mechanisms by which MT5-MMP controls the processing of amyloid precursor protein (APP) and the fate of amyloid beta peptide (Aß) as well as its precursor C99, and C83. We found in human embryonic kidney cells (HEK) carrying the APP Swedish familial mutation (HEKswe) that deleting the C-terminal non-catalytic domains of MT5-MMP hampered its ability to process APP and release the soluble 95 kDa form (sAPP95). Catalytically inactive MT5-MMP variants increased the levels of Aß and promoted APP/C99 sorting in the endolysosomal system, likely through interactions of the proteinase C-terminal portion with C99. Most interestingly, the deletion of the C-terminal domain of MT5-MMP caused a strong degradation of C99 by the proteasome and prevented Aß accumulation. These discoveries reveal new control of MT5-MMP over APP by proteolytic and non-proteolytic mechanisms driven by the C-terminal domains of the proteinase. The targeting of these non-catalytic domains of MT5-MMP could, therefore, provide new insights into the therapeutic regulation of APP-related pathology in AD.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Protein Precursor/metabolism , Matrix Metalloproteinases, Membrane-Associated/metabolism , Peptide Fragments/metabolism , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/metabolism , Animals , Cell Line , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , ProteolysisABSTRACT
Processing of amyloid beta precursor protein (APP) into amyloid-beta peptide (Aß) by ß-secretase and γ-secretase complex is at the heart of the pathogenesis of Alzheimer's disease (AD). Targeting this proteolytic pathway effectively reduces/prevents pathology and cognitive decline in preclinical experimental models of the disease, but therapeutic strategies based on secretase activity modifying drugs have so far failed in clinical trials. Although this may raise some doubts on the relevance of ß- and γ-secretases as targets, new APP-cleaving enzymes, including meprin-ß, legumain (δ-secretase), rhomboid-like protein-4 (RHBDL4), caspases and membrane-type matrix metalloproteinases (MT-MMPs/η-secretases) have confirmed that APP processing remains a solid mechanism in AD pathophysiology. This review will discuss recent findings on the roles of all these proteinases in the nervous system, and in particular on the roles of MT-MMPs, which are at the crossroads of pathological events involving not only amyloidogenesis, but also inflammation and synaptic dysfunctions. Assessing the potential of these emerging proteinases in the Alzheimer's field opens up new research prospects to improve our knowledge of fundamental mechanisms of the disease and help us establish new therapeutic strategies.
ABSTRACT
Accumulating evidence indicates that microglial TLR2 and TLR4 play a significant role in nociception. Experiments were conducted to evaluate the contribution of TLR2 and TLR4 and their adaptor molecules to neuropathy and their ability to amplify opioid effectiveness. Behavioral tests (von Frey's and cold plate) and biochemical (Western blot and qRT-PCR) analysis of spinal cord and DRG tissue were conducted after chronic constriction injury (CCI) to the sciatic nerve. Repeated intrathecal administration of LPS-RS (TLR2 and TLR4 antagonist) and LPS-RS Ultrapure (TLR4 antagonist) attenuated allodynia and hyperalgesia. Biochemical analysis revealed time-dependent upregulation of mRNA and/or protein levels of TLR2 and TLR4 and MyD88 and TRIF adaptor molecules, which was paralleled by an increase in IBA-1/CD40-positive cells under neuropathy. LPS-RS and LPS-RS Ultrapure similarly influenced opioid analgesia by enhancing the effectiveness of buprenorphine but not morphine. Summing up, in light of their upregulation over the course of pain, both TLR2 and TLR4 may indeed play a significant role in neuropathy, which could be linked to the observed activation of IBA-1/CD40-positive cells. Blockade of TLR2 and TLR4 produced analgesia and enhanced buprenorphine's effectiveness, which suggests that they may be a putative target for future pharmacological pain relief tools, especially for opioid rotation, when the effect of morphine is tolerated.
Subject(s)
Analgesics, Opioid/administration & dosage , Buprenorphine/administration & dosage , Neuralgia/metabolism , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolism , Animals , Disease Models, Animal , Ganglia, Spinal/metabolism , Hyperalgesia/etiology , Hyperalgesia/prevention & control , Lipopolysaccharides/administration & dosage , Male , Neuralgia/complications , Neuralgia/prevention & control , Rats , Rats, Wistar , Sciatic Nerve/injuries , Spinal Cord/metabolism , Toll-Like Receptor 2/antagonists & inhibitors , Toll-Like Receptor 4/antagonists & inhibitorsABSTRACT
Currently, the low efficacy of antinociceptive drugs for the treatment of neuropathic pain is a major therapeutic problem. Here, we show the potential role of interleukin (IL)-18 signaling in this phenomenon. IL-18 is an important molecule that performs various crucial functions, including the alteration of nociceptive transmission in response to neuropathic pain. We have studied the changes in the mRNA and protein levels (qRT-PCR and Western blot analysis, respectively) of IL-18, IL-18-binding protein (IL-18BP) and the IL-18 receptor (IL-18R) over time in rats following chronic constriction injury (CCI) of the sciatic nerve. Our study demonstrated that the spinal levels of IL-18BP were slightly downregulated at days 7 and 14 in the rats subjected to CCI. In contrast, the IL-18 and IL-18R mRNA expression and protein levels were elevated in the ipsilateral spinal cord on days 2, 7 and 14. Moreover, in rats exposed to a single intrathecal administration of IL-18BP (50 and 100 ng) 7 or 14 days following CCI, symptoms of neuropathic pain were attenuated, and the analgesia pursuant to morphine and buprenorphine (0.5 and 2.5 µg) was enhanced. In summary, the restoration of the analgesic activity of morphine and buprenorphine via the blockade of IL-18 signaling suggests that increased IL-18 pathway may account for the decreased analgesic efficacy of opioids for neuropathic pain.
Subject(s)
Analgesics, Opioid/pharmacology , Buprenorphine/pharmacology , Intercellular Signaling Peptides and Proteins/pharmacology , Interleukin-18/metabolism , Morphine/pharmacology , Neuralgia/metabolism , Analgesics, Opioid/administration & dosage , Analgesics, Opioid/therapeutic use , Animals , Astrocytes/drug effects , Astrocytes/metabolism , Buprenorphine/administration & dosage , Buprenorphine/therapeutic use , Cells, Cultured , Drug Synergism , Intercellular Signaling Peptides and Proteins/administration & dosage , Intercellular Signaling Peptides and Proteins/metabolism , Interleukin-18/genetics , Male , Morphine/administration & dosage , Morphine/therapeutic use , Neuralgia/drug therapy , Neurons/drug effects , Neurons/metabolism , Rats , Rats, Wistar , Receptors, Interleukin-18/genetics , Receptors, Interleukin-18/metabolism , Spinal Cord/cytology , Spinal Cord/metabolismABSTRACT
An interesting research and therapeutic problem is the reduced beneficial efficacy of opioids in the treatment of neuropathic pain. The present study sought to investigate the potential role of IL-1 family members in this phenomenon. We studied the time course of changes in IL-1alpha, IL-1beta, IL-1 receptor type I and IL-1 receptor antagonist mRNA and protein levels experienced by rats after chronic constriction injury (CCI) of the sciatic nerve using qRT-PCR and Western blot analysis. In CCI-exposed rats, spinal levels of IL-1alpha mRNA were slightly downregulated on the 7th day, and protein levels were not changed on the 7th and 14th days. Levels of IL-1 receptor antagonist and IL-1 receptor type I were slightly upregulated in the ipsilateral part of the spinal cord on the 7th and 14th days; however, protein levels were not changed at those time points. Interestingly, we observed that IL-1beta mRNA and protein levels were strongly elevated in the ipsilateral part of the dorsal spinal cord on the 7th and 14th days following CCI. Moreover, in rats exposed to a single intrathecal administration of an IL-1 receptor antagonist (100 ng i.t.) on the 7th and 14th day following CCI, symptoms of neuropathic pain were attenuated, and the analgesic effects of morphine (2.5 µg i.t.) and buprenorphine (2.5 µg i.t.) were enhanced. In summary, restoration of the analgesic activity of morphine and buprenorphine by blockade of IL-1 signaling suggests that increased IL-1beta responses may account for the decreased analgesic efficacy of opioids observed in the treatment of neuropathy.
Subject(s)
Analgesics, Opioid/therapeutic use , Buprenorphine/therapeutic use , Interleukin 1 Receptor Antagonist Protein , Morphine/therapeutic use , Neuralgia/drug therapy , Analgesics, Opioid/pharmacology , Animals , Buprenorphine/pharmacology , Hyperalgesia/drug therapy , Hyperalgesia/metabolism , Interleukin 1 Receptor Antagonist Protein/genetics , Interleukin 1 Receptor Antagonist Protein/metabolism , Interleukin-1alpha/genetics , Interleukin-1alpha/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Male , Morphine/pharmacology , Neuralgia/metabolism , RNA, Messenger/metabolism , Rats, Wistar , Sciatic Nerve/injuries , Spinal Cord/drug effects , Spinal Cord/metabolism , Treatment OutcomeABSTRACT
Neuropathic pain treatment remains a challenge because pathomechanism is not fully understood. It is believed that glial activation and increased spinal nociceptive factors are crucial for neuropathy. We investigated the effect of parthenolide (PTL) on the chronic constriction injury to the sciatic nerve (CCI)-induced neuropathy in rat. We analyzed spinal changes in glial markers and M1 and M2 polarization factors, as well as intracellular signaling pathways. PTL (5 µg; i.t.) was preemptively and then daily administered for 7 days after CCI. PTL attenuated the allodynia and hyperalgesia and increased the protein level of IBA1 (a microglial/macrophage marker) but did not change GFAP (an astrocyte marker) on day 7 after CCI. PTL reduced the protein level of M1 (IL-1ß, IL-18, and iNOS) and enhanced M2 (IL-10, TIMP1) factors. In addition, it downregulated the phosphorylated form of NF-κB, p38MAPK, and ERK1/2 protein level and upregulated STAT3. In primary microglial cell culture we have shown that IL-1ß, IL-18, iNOS, IL-6, IL-10, and TIMP1 are of microglial origin. Summing up, PTL directly or indirectly attenuates neuropathy symptoms and promotes M2 microglia/macrophages polarization. We suggest that neuropathic pain therapies should be shifted from blanketed microglia/macrophage suppression toward maintenance of the balance between neuroprotective and neurotoxic microglia/macrophage phenotypes.
Subject(s)
Analgesics/administration & dosage , Macrophages/drug effects , Microglia/drug effects , Neuralgia/metabolism , Sciatic Neuropathy/metabolism , Sesquiterpenes/administration & dosage , Analgesics/therapeutic use , Animals , Cell Polarity/drug effects , Cells, Cultured , Chronic Disease , Hyperalgesia/drug therapy , Hyperalgesia/metabolism , Macrophages/metabolism , Male , Microglia/metabolism , Neuralgia/drug therapy , Rats , Rats, Wistar , Sciatic Neuropathy/drug therapy , Sesquiterpenes/therapeutic use , Signal Transduction/drug effectsABSTRACT
The mechanism involved in the development of diabetic neuropathy is complex. Currently, it is thought that chemokines play an important role in this process. The aim of this study was to determine how the level of some chemokines from the CXC subfamily varies in diabetic neuropathy and how the chemokines affect nociceptive transmission. A single intraperitoneal (i.p.) injection of streptozotocin (STZ; 200 mg/kg) resulted in an increased plasma glucose. The development of allodynia and hyperalgesia was measured at day 7 after STZ administration. Using Antibody Array techniques, the increases in CXCL1 (KC), CXCL5 (LIX), CXCL9 (MIG), and CXCL12 (SDF-1) protein levels were detected in STZ-injected mice. No changes in CXCL11 (I-TAC) or CXCL13 (BLC) protein levels were observed. The single intrathecal (i.t.) administration of CXCL1, CXCL5, CXCL9, and CXCL12 (each in doses of 10, 100, and 500 ng/5 µL) shows their pronociceptive properties as measured 1, 4, and 24 hours after injection using the tail-flick, von Frey, and cold plate tests. These findings indicate that the chemokines CXCL1, CXCL5, CXCL9, and CXCL12 are important in nociceptive transmission and may play a role in the development of diabetic neuropathy.
Subject(s)
Chemokines/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetic Neuropathies/metabolism , Animals , Behavior, Animal , Blood Glucose/analysis , Blood Glucose/metabolism , Body Weight , Chemokine CXCL1/metabolism , Chemokine CXCL12/metabolism , Chemokine CXCL5/metabolism , Chemokine CXCL9/metabolism , Diabetic Neuropathies/pathology , Disease Models, Animal , Gene Expression Profiling , Hyperalgesia , Inflammation , Injections, Intraperitoneal , Male , Mice , Nociception , StreptozocinABSTRACT
BACKGROUND: Neuropathic pain is clinically challenging because it is resistant to alleviation by morphine. The nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways may be involved in the development of neuropathic pain. The aim of our study was to examine the influence of a chronic, intrathecal administration of parthenolide (PTL, inhibitor of NF-κB) and U0126 (inhibitor of MEK1/2) on nociception and morphine effectiveness in a rat model of neuropathy. METHODS: The chronic constriction injury of the sciatic nerve in Wistar rats was performed. PTL and U0126 were injected chronic intrathecally and morphine was injected once at day 7. To evaluate allodynia and hyperalgesia, the von Frey and cold plate tests were used, respectively. The experiments were carried out according to IASP rules. Using qRT-PCR we analyzed mRNAs of µ-(mor), δ-(dor) and κ-(kor)-opioid receptors in the lumbar spinal cord after drugs administration. RESULTS: The administration of PTL and U0126 decreased allodynia and hyperalgesia and significantly potentiated morphine effect. The mor, dor and kor mRNAs were down-regulated 7 days after injury in the ipsilateral spinal cord. The PTL and U0126 significantly up-regulated the mRNA levels of all opioid receptors. The levels of mor and dor mRNAs were much higher compared to those in naïve, but only the kor levels returned to control values. CONCLUSIONS: These results indicate that the inhibition of the NF-κB pathway has better analgesic effects. Both inhibitors similarly potentiate morphine analgesia, which parallels the up-regulation of both mor and dor mRNAs expression spinal levels of the model of neuropathy.