Astrocyte-secreted TIMP-1 binds to CD63 and differentially phosphorylates Akt in protecting neurons and promoting cognitive recovery in 5xFAD mice.

Abstract

Astrocytes respond dramatically to pathological alterations in the brain including Alzheimer's disease (AD) through a highly heterogeneous process called astrocyte reactivity. Astrocyte reactivity is characterized by changes in morphological, transcriptional and biochemical levels. The reactive astrocytes secrete a plethora of cytokines that regulate neuronal health through disease stages. Recently, we showed that oligomeric Aβ1-42 (Aβ) treatment to primary astrocytes induced an early reactivity and detected tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) as the major candidate in the astrocyte secretome that promoted neuronal viability against Aβ toxicity. Moreover, in Aβ-injected rats, TIMP-1 recovered cognitive functions (Brain Behav Immun. 2020 87:804-819). However, the mechanisms underlying TIMP-1's neuroprotective role remained to be revealed which we aim to achieve through our present study. Primary cortical neurons were co-treated with Aβ and recombinant TIMP-1, and Akt phosphorylation at different sites and downstream protein levels were checked by western blotting and reconfirmed by immunofluorescence studies. Co-localization and immunoprecipitation studies were performed to determine TIMP-1's binding partner/s. Subsequently, TIMP-1 was stereotactically injected in 6-month old 5xFAD mice and cognitive behaviors were assessed through locomotion, passive avoidance, fear conditioning and elevated plus maze tests. Immunohistochemical analyses and western blotting were employed to analyze pathway inductions in vivo. In neurons, TIMP-1 activated Akt by phosphorylating at two different residues: S473 and Thr308. Akt S473 phosphorylation ameliorated impaired autophagy flux and downregulated Aβ-induced pro-apoptotic protein levels by inhibiting FoxO3a in cultured neurons and in cortex and hippocampus of 5xFAD mouse. Subsequently, CD63 was detected as the binding partner of TIMP-1 on the neuronal surface. TIMP-1 treatment recovered the cognitive deficits in 5xFAD mice. Synaptic proteins, SNAP-25 and PSD-95, expressions were increased following TIMP-1 treatment in 5xFAD. This was consistent with Akt thr308 phosphorylation and downstream GSK3β phosphorylation-mediated inhibition, implicated in improving synaptic plasticity. Data show that TIMP-1 regulates differential phosphorylation of Akt that is potentially central to regulating autophagy, apoptotic pathways and synaptic plasticity in models of AD resulting in cognitive recovery. We highlight that TIMP-1 mediates its neuroprotective role by binding to CD63 and propose it as a primary candidate in astrocyte-secreted cytokine-mediated AD therapy.