MMP9 modulation improves specific neurobehavioral deficits in a mouse model of Alzheimer's disease.

BACKGROUND: Matrix metallopeptidase 9 (MMP9) has been implicated in a variety of neurological disorders, including Alzheimer's disease (AD), where MMP9 levels are elevated in the brain and cerebrovasculature. Previously our group demonstrated apolipoprotein E4 (apoE4) was less efficient in regulating MMP9 activity in the brain than other apoE isoforms, and that MMP9 inhibition facilitated beta-amyloid (Aß) elimination across the blood-brain barrier (BBB) METHODS: In the current studies, we evaluated the impact of MMP9 modulation on Aß disposition and neurobehavior in AD using two approaches, (1) pharmacological inhibition of MMP9 with SB-3CT in apoE4 x AD (E4FAD) mice, and (2) gene deletion of MMP9 in AD mice (MMP9KO/5xFAD) RESULTS: Treatment with the MMP9 inhibitor SB-3CT in E4FAD mice led to reduced anxiety compared to placebo using the elevated plus maze. Deletion of the MMP9 gene in 5xFAD mice also reduced anxiety using the open field test, in addition to improving sociability and social recognition memory, particularly in male mice, as assessed through the three-chamber task, indicating certain behavioral alterations in AD may be mediated by MMP9. However, neither pharmacological inhibition of MMP9 or gene deletion of MMP9 affected spatial learning or memory in the AD animals, as determined through the radial arm water maze. Moreover, the effect of MMP9 modulation on AD neurobehavior was not due to changes in Aß disposition, as both brain and plasma Aß levels were unchanged in the SB-3CT-treated E4FAD animals and MMP9KO/AD mice compared to their respective controls. CONCLUSIONS: In total, while MMP9 inhibition did improve specific neurobehavioral deficits associated with AD, such as anxiety and social recognition memory, modulation of MMP9 did not alter spatial learning and memory or Aß tissue levels in AD animals. While targeting MMP9 may represent a therapeutic strategy to mitigate aspects of neurobehavioral decline in AD, further work is necessary to understand the nature of the relationship between MMP9 activity and neurological dysfunction.

Apolipoprotein E isoforms differentially regulate matrix metallopeptidase 9 function in Alzheimer's disease.

Apolipoprotein E (APOE) has been shown to influence amyloid-ß (Aß) clearance from the brain in an isoform-specific manner. Our prior work showed that Aß transit across the blood-brain-barrier was reduced by apoE4, compared to other apoE isoforms, due to elevated lipoprotein receptor shedding in brain endothelia. Recently, we demonstrated that matrix metallopeptidase 9 (MMP-9) induces lipoprotein receptor proteolysis in an apoE isoform-dependent manner, which impacts Aß elimination from the brain. The current studies interrogated the relationship between apoE and MMP-9 and found that apoE impacted proMMP-9 cellular secretion from brain endothelia (apoE2 < apoE3 = apoE4). In a cell-free assay, apoE dose-dependently reduced MMP-9 activity, with apoE4 showing a significantly weaker ability to inhibit MMP-9 function than apoE2 or apoE3. Finally, we observed elevated MMP-9 expression and activity in the cerebrovasculature of both human and animal AD brain specimens with an APOE4 genotype. Collectively, these findings suggest a role for apoE in regulating MMP-9 disposition and may describe the effect of apoE4 on Aß pathology in the AD brain.

Neuronal Spleen tyrosine kinase (SYK) mediates cytokine release in Transgenic Tau P301S mice organotypic brain slice cultures.

The Spleen Tyrosine Kinase (SYK) is known for its involvement in B-cell and T-cell signaling, modulating the peripheral immune response. We have previously shown that SYK is overactive in the brains of human Alzheimer's Disease (AD) patients, as well as mouse models of AD and tauopathy including Tg Tau P301S mice. More specifically, SYK activation occurs mainly in neurons in human AD brain specimens and mouse models of AD and colocalizes with tau pathogenic species, suggesting it could play a role in AD pathobiology. To assess the possible contribution of SYK to the inflammatory response induced by tau pathology, we analyzed cytokine production in organotypic brain slices cultures from Tg Tau P301S mice and wild-type littermates. Organotypic brains slices from Tau P301S mice produce more cytokines than brain slices from wild-type littermates while SYK inhibition completely antagonizes cytokine production from Tg Tau P301S brain slices. Interestingly, LPS exacerbates the production of pro-inflammatory cytokines in Tg Tau P301S brain sections compared to wild-type organotypic sections while SYK inhibition alleviates the release of pro-inflammatory cytokines induced by LPS. Given that SYK is mainly activated in neurons in Tg Tau P301S mice and not in glial cells, these data suggest that neuronal SYK contributes to the neuroinflammation triggered by the tau pathology. SYK represents an attractive target for regulating the underlying neuroinflammatory component induced by tau pathology.

Distinct Signaling Pathways Regulate TREM2 Phagocytic and NFκB Antagonistic Activities.

Several genetic variants of the Triggering Receptor Expressed on Myeloid Cells-2 (TREM2) have been shown to increase the risk of developing Alzheimer's disease (AD) supporting a role of microglia and immune cells in the pathobiology of AD. We have employed an ectopic model of TREM2 and DAP12 expression in HEK293 cells to study selectively TREM2 dependent signaling and phagocytic functions and evaluated the effects of some of the TREM2 mutations associated with AD. We show that shedding of the TREM2 N-terminal domain does not affect the inhibition of NFκB activation induced by TREM2 while it completely blocks phagocytosis suggesting that TREM2 anti-inflammatory properties can be mediated by the TREM2 C-terminal fragment while the phagocytic activity requires the full-length receptor. In addition, we confirm in that model that apolipoprotein E (APOE) is a ligand for TREM2 and triggers TREM2 signaling. In particular, we show that APOE4 stimulates spleen tyrosine kinase (SYK) activation more potently than APOE2 in a TREM2 dependent manner. Interestingly, TREM2 appears to antagonize NFκB activation induced by phorbol ester but is unable to prevent TNFα induction of NFκB activation suggesting that TREM2 antagonizes inflammatory events triggered downstream of PKC. TREM2 mutations drastically impact TREM2 phagocytosis as well as its ability to antagonize NFκB activation and notably prevent the activation of the PI3K/AKT pathway observed with wild-type TREM2. Overall our data suggest that TREM2 dependent phagocytosis requires an activation of the SYK/PI3K/AKT/PLCγ pathways while the suppression of NFκB activation by TREM2 is independent of SYK, PI3K, and PLCγ activities. This model of ectopic TREM2-DAP12 co-expression appears suitable to study TREM2 signaling as several biological functions of TREM2 and TREM2 mutations that have been previously described in myeloid and microglial cells were also replicated in this model.

Spleen tyrosine kinase (SYK) blocks autophagic Tau degradation in vitro and in vivo.

Spleen tyrosine kinase (SYK) plays a major role in inflammation and in adaptive immune responses and could therefore contribute to the neuroinflammation observed in various neurodegenerative diseases. Indeed, previously we have reported that SYK also regulates ß-amyloid (Aß) production and hyperphosphorylation of Tau protein involved in these diseases. Moreover, SYK hyperactivation occurs in a subset of activated microglia, in dystrophic neurites surrounding Aß deposits, and in neurons affected by Tau pathology both in individuals with Alzheimer's disease (AD) and in AD mouse models. SYK activation increases Tau phosphorylation and accumulation, suggesting that SYK could be an attractive target for treating AD. However, the mechanism by which SYK affects Tau pathology is not clear. In this study, using cell biology and biochemical approaches, along with immunoprecipitation and immunoblotting, quantitative RT-PCR, and ELISAs, we found that SYK inhibition increases autophagic Tau degradation without impacting Tau production. Using neuron-like SH-SY5Y cells, we demonstrate that SYK acts upstream of the mammalian target of rapamycin (mTOR) pathway and that pharmacological inhibition or knockdown of SYK decreases mTOR pathway activation and increases autophagic Tau degradation. Interestingly, chronic SYK inhibition in a tauopathy mouse model profoundly reduced Tau accumulation, neuroinflammation, neuronal and synaptic loss, and also reversed defective autophagy. Our results further suggest that the SYK up-regulation observed in the brains of individuals with AD contributes to defective autophagic clearance leading to the accumulation of pathogenic Tau species. These findings further highlight SYK as a therapeutic target for the treatment of tauopathies and other neurodegenerative proteinopathies associated with defective autophagic clearance.

Alzheimer's disease pathological lesions activate the spleen tyrosine kinase.

The pathology of Alzheimer's disease (AD) is characterized by dystrophic neurites (DNs) surrounding extracellular Aß-plaques, microgliosis, astrogliosis, intraneuronal tau hyperphosphorylation and aggregation. We have previously shown that inhibition of the spleen tyrosine kinase (Syk) lowers Aß production and tau hyperphosphorylation in vitro and in vivo. Here, we demonstrate that Aß-overexpressing Tg PS1/APPsw, Tg APPsw mice, and tau overexpressing Tg Tau P301S mice exhibit a pathological activation of Syk compared to wild-type littermates. Syk activation is occurring in a subset of microglia and is age-dependently increased in Aß-plaque-associated dystrophic neurites of Tg PS1/APPsw and Tg APPsw mice. In Tg Tau P301S mice, a pure model of tauopathy, activated Syk occurs in neurons that show an accumulation of misfolded and hyperphosphorylated tau in the cortex and hippocampus. Interestingly, the tau pathology is exacerbated in neurons that display high levels of Syk activation supporting a role of Syk in the formation of tau pathological species in vivo. Importantly, human AD brain sections show both pathological Syk activation in DNs around Aß deposits and in neurons immunopositive for pathological tau species recapitulating the data obtained in transgenic mouse models of AD. Additionally, we show that Syk overexpression leads to increased tau accumulation and promotes tau hyperphosphorylation at multiple epitopes in human neuron-like SH-SY5Y cells, further supporting a role of Syk in the formation of tau pathogenic species. Collectively, our data show that Syk activation occurs following Aß deposition and the formation of tau pathological species. Given that we have previously shown that Syk activation also promotes Aß formation and tau hyperphosphorylation, our data suggest that AD pathological lesions may be self-propagating via a Syk dependent mechanism highlighting Syk as an attractive therapeutic target for the treatment of AD.