Alzheimer’s Disease is the most common neurodegenerative disease, and is highly prevalent in the elderly population. The signature of this debilitating disease is the formation of amyloid-β (Aβ) oligomers. These oligomers that gradually appear with old age aggregates into insoluble amyloid plaque that can be found throughout the neural tissue. The soluble form of these oligomers is linked to cell toxicity and general neurodegenerative symptoms found in Alzheimer’s patients. Aβ oligomers are formed by the cleavage of the amyloid precursor protein (APP) via β-secretase and γ-secretase. As discussed in our course, Aβ oligomers have been associated with faulty insulin signaling in the brain (Duarte et al., 2012). Impairment of the insulin signaling pathway has been linked to cognitive defects in Alzheimer’s patients (de la Monte, 2009). Aβ oligomers bind to the membrane surface, which removes the insulin receptors (IRs) at the synapses and leads to stimulation of pro-apoptotic pathways activated by c-Jun N-terminal kinase (JNK) pathway. This activation phosphorylates insulin substrate-1 (IRS1) at Serine residue 636 (pIRS1-Ser636), which makes IRS1 inactive and suppresses insulin.
To counter this, the insulin signaling pathway down regulates oligomeric binding sites. Normal insulin function results in the binding of insulin to its receptor, which activates IRS1 by phosphorylating at Tyrosine residue 465 (pIRS1-Tyr465)(Kaminari et al.). This phosphorylation activates cell survival Akt/PKB kinase (Akt), which promotes insulin’s neurotrophic effects. In this state, Akt phosphorylates/ inactivates Glycogen Synthase Kinase-3β (GSK-3β), which is an enzyme associated with the hyper-phosphorylation of the Tau protein (Kaminari et al.). This is another hallmark characteristic of Alzheimer’s Disease.
Matrix metalloproteinase 9 (MMP-9), is a metalloproteinase, a family of zinc bound proteolytic enzymes that are linked to the remodeling of the extracellular matrix (Egeblad et al., 2002). MMP-9 has been associated with the configuration of synaptic connections. It has also been shown to breakdown Aβ in vivo and in vitro, which leads to decreased levels of neurotrophic factors like BNDF (Kaminari et al.). In their paper, “Overexpression of matrix metalloproteinase-9 (MMP-9) rescues insulin-mediated impairment in the 5XFAD model of Alzheimer’s disease”, Kaminari et al. conducted a series of experiments testing the effect of MMP-9 on the insulin signaling pathway of Alzheimer’s Disease 5XFAD (insulin impairment), TgMMP-9, 5XFAD/TgMMP-9, and wild type mice models .
First, they looked over the phosphorylation of IRS1 at Serine-636 (pIRS1-Ser636) in all mice models. Protein samples from primary and 3 month old hippocampal cell lysates showed that 5XFAD mice had the highest increase levels of pIRS1-Ser636 compared to wild type mice. Conversely, this was not observed in MMP-9 overexpressing mice in which pIRS1-Ser636 levels were reduced to standard levels in 5XFAD/TgMMP-9 and TgMMP-9 mice. They also observed a significant decrease in activating phosphorylation levels of IRS1 at Tyrosine-465 (pIRS1-Tyr465), which is consistent with insulin resistance associated with pIRS1-Ser636. Immunofluorescence labeling of hippocampal neurons from 5XFAD mice revealed levels of pIRS1-Tyr465 were reduced by 50% compared to the wild type mice. However, in the presence of MMP-9, 5XFAD/TgMMP-9 and TgMMP-9 mice’s levels of pIRS1-Tyr465 phosphorylation stayed within normal range. These findings proved that overexpression of MMP-9 enhanced IRS1 activation.
Next, the researchers observed the effects of MMP-9 in signaling kinases downstream of IRS1. They examined phosphorylation levels of Akt at Serine 473 (pAkt) and GSK-3β-pSer9 (pGSK-3β) in primary hippocampal neurons and in hippocampal extracts of 3 month-old mice. Hippocampal pAKT and pGSK-3β levels observed in 5XFAD mice declined significantly compared to wild type mice. Overexpression of MMP-9 in other mice models showed opposite results. The decrease in Akt and GSK-3β phosphorylation was not observed. This proved that overexpression of MMP-9 increased the phosphorylation levels of Akt and GSK-3β.
Following this, Kaminari et al. examined the effects of overexpression of MMP-9 on the activation of TrkB via Tyrosine phosphorylation, which oversees the activation of IRS1. Westernblots of hippocampal cultures showed that in 5XFAD mice, phosphorylation of TrkB was reduced compared to the wild type model while TgMMP-9 and 5XFAD/TgMMP-9 both maintained normal phosphorylation levels. Hippocampal cultures also revealed that 5XFAD mice had significantly lower levels of the neurotrophic factor, BNDF. This was the opposite in TgMMP-9 and 5XFAD/TgMMP-9 mice. In addition to this, the researchers gave a dose of BNDF to the primary cultured cells of 5XFAD mice to observe pIRS1-Tyr465 and pGSK3-β levels. A significant increase in pIRS1-Tyr465 and pGSK3-β levels were observed compared to untreated cells. These findings confirmed that IRS1 activation is regulated by BDNF.
In their last series of experiments, Kaminari et al. looked at the effects of overexpression of MMP-9 on the phosphorylation of JNK. JNK is a crucial stress-activated kinase in peripheral insulin resistance. It is also involved in IRS1 Serine phosphorylation. In 5XFAD mice, JNK phosphorylation levels were significantly increased compared to wild type mice. In 5XFAD/TgMMP-9 mice, where there was overexpression of MMP-9, JNK phosphorylation levels stayed at normal range and cell apoptosis was lower than in 5XFAD mice. These findings confirmed that overexpression of MMP-9 reduced hippocampal phosphorylation of JNK and significantly reduced occurrences of cell apoptosis.
These sets of experiments have opened a door to a treatment option worth testing for Alzheimer’s Disease patients who also have Type 2 Diabetes. Overexpressing MMP-9 in neural tissue may be a treatment worth pursuing.
Figure 1. Kaminari et al. proposed mechanism for MMP-9’s neuroprotective function in inhibiting amyloid β mediated impairment of the insulin survival pathway. Aβ oligomers stimulates TNFR signaling, which is linked to pro-apoptotic pathways. This activates KNJ kinase, which results in IRS1-Ser636 phosphorylation and GSK-3β activation. This leads to insulin resistance. MMP-9 reduces Aβ peptide accumulation, which effectively blocks Aβ oligomers from negatively affecting insulin signaling, and contributes to increased BDNF levels. BDNF binds to TrkB receptors, which induces Akt activation.
1. Kaminari, A., Giannakas, N., Tzinia, A. & Tsilibary, E. Overexpression of matrix metalloproteinase-9 (MMP-9) rescues insulin-mediated impairment in the 5XFAD model of Alzheimer’s disease. Scientific Reports 7, (2017).