Effects of matrix metalloproteinase-9 on insulin survival pathways in Alzheimer’s disease Introduction Defective brain insulin signaling has been suggested to contribute to the cognitive deficits in patients with Alzheimer’s disease (AD). Although a connection between AD and diabetes has been suggested, the mechanism by which insulin resistance in the brain arises in individuals with AD remains to be elucidated. One of the hallmarks of AD is the abnormal accumulation of amyloid-beta (Αβ) peptide, the oligomeric form of which is believed to be primarily responsible for cell toxicity and neuronal dysfunction in various experimental models of AD, as well as in AD patients. Interestingly, insulin signaling provides a physiological defense mechanism against oligomer-induced synapse loss. Aim of the study We have previously generated transgenic mice that overexpress the enzyme matrix metalloproteinase 9 (MMP-9), an enzyme critically involved in neuronal plasticity. MMP9 appears to have a neuroprotective role by possessing α-secretase-like activity which gives rise to increased sAPPα, by interfering with Aβ formation and by enhancing neuronal plasticity in mice models (3, 4). To elucidate the role of MMP-9 on insulin resistance in AD, we will examine possible alterations in the proteins involved in the insulin pathway, such as insulin receptor (IR) and insulin receptor substrate -1 and -2 (IRS1, IRS2), as well as in proteins involved in diabetic pathology, such as nephrin. Figure 4. By examining the expression levels of the survival-associated nephrin in primary cultures by RT-PCR and confocal imaging we also observed an increase in nephrin expression in transgenic animals, compared to control mice, with a more pronounced effect in 5xFAD/TgMMP-9 mice. Data from preliminary experiments revealed that: There is no difference in the expression levels of the insulin-survival pathway proteins IR, IRS1/2 in any of our transgenic animals, compared to wild type animals in brain homogenates of 1 year of age. This is in agreement with the literature, where changes in the total levels are observed after the age of 12 months(1). There was an increase of pIRS-1pSer636 in 5xFAD mice which is possibly attributed to the increase of Αβ oligomers. Phosphorylation in serine 636 has been shown to result in insulin resistance in brain tissues of AD patients, as well as in hippocampal cultures(1, 2). Interestingly, this effect was reduced in the presence of MMP-9 In differentiated hippocampal neurons, we observed an increase in the total levels of IRS1 of 5xFAD/TgMMP-9 and TgMMP-9, followed by a downstream increase of phosphorylated Akt, which indicates that the insulin survival pathway is triggered, possibly via the action of MMP-9, compared to wild types and 5xFAD animals. RT-PCR and confocal imaging showed that the expression levels of survival-associated nephrin in primary cultures are increased in nephrin expression in all transgenic animals, compared to wild type mice. Further investigation is required to elucidate the role of MMP-9 with regards to the insulin survival pathway. Discussion Materials & Methods We established primary hippocampal cultures from wild type (WT) mice, as well as from mice that overexpress MMP9 in the CNS (TgMMP-9), mice models of AD that overexpress APP (5xFAD) and mice that overexpress both proteins (5xFAD/TgMMP-9). After differentiation, the cells were lysed for western blot or were used for immunofluorescence staining. Alternatively, total RNA from primary hippocampal cells was collected for RT-PCR. Moreover, we performed western blot in 1 year old mouse brain homogenates of the same genotypes. Primary cultures Primary hippocampal cells of P0-1 mice were cultured in serum-free medium on poly-D- lysine covered plates or coated coverslips. Results brain homogenates (12 months) Figure 1. By western blot analysis, no difference was observed in the expression levels of IR or IRS1/2 in TgMMP-9, 5XFAD and 5XFAD/TgMMP-9, compared to controls. C.. A) Generation of TgMMP9 via microinjection. B) Amyloidogenic pathway /non-amyloidogenic pathway in AD. C) A portion of the insulin survival pathway and the possible role of MMP-9. B. Figure 3. An increase in the total levels of IRS1 in the 5xFAD/TgMMP-9 but no change in the 5xFAD model, compared to the WT in primary hippocampal cells. Concomitantly, an increase in pAkt was observed. pAKT AKT Figure 2. An increase in pIRS-1pSer636 levels in 5xFAD mice was observed, compared to the WT that appears reduced in 5xFAD/TgMMP9 and TgMMP9 mice. An increase in pAKT was observed in the 5xFAD/TgMMP9 and TgMMP9 mice, whereas no change was observed in the 5xFAD mice, compared to the WT.. References 1. Bomfim, T.R., et al., J Clin Invest. 122, Talbot, K, et al , Fragkouli, A., et al., J Neurochem. 121, Talamagas, A.A., et al., Neurobiol Dis. 28, This work was supported by a grant from the Greek Secretariat for Research and Development (EXCELLENCE: 164) Acknowledgments