Anatabine Attenuates Tau Phosphorylation and Oligomerization in P301S Tau Transgenic Mice

Abstract

We have previously shown that the natural alkaloid anatabine displays some anti-inflammatory and Alzheimer amyloid (Aβ) lowering properties in the central nervous system associated with reduced STAT3 and NFkB activation. We investigated here the impact of a chronic oral treatment with anatabine in a model of tauopathy. We found that anatabine reduces the incidence of paralysis and abnormal hind limb extension reflex while improving rotarod performances in P301S mutant human Tau transgenic mice (Tg Tau P301S) suggesting that anatabine delays the disease progression in this model of tauopathy. Analyzes of brain and spinal cord homogenates reveal that anatabine reduces tau phosphorylation at multiple pertinent Alzheimer's disease (AD) epitopes and decreases the levels of pathological tau conformers/oligomers in both detergent soluble and insoluble fractions. Pathological tau species reduction induced by anatabine was accompanied by decreased Iba1 expression suggesting a diminution of microgliosis in the brain and spinal cord of Tg Tau P301S mice. In addition, we found that anatabine administration increases phosphorylation of the inhibitory residue (Ser9) of glycogen synthase kinase-3β, a primary tau kinase, associated with AD pathology, providing a possible mechanism for the observed reduction of tau phosphorylation. These data support further exploration of anatabine as a possible disease modifying agent for neurodegenerative tauopathies and, in particular AD, since anatabine also displays Aβ lowering properties.

The Spleen Tyrosine Kinase (Syk) Regulates Alzheimer Amyloid-β Production and Tau Hyperphosphorylation

“…Potential cytotoxicity of the different treatments was routinely evaluated using the cytotoxicity detection kit (Roche Diagnostics), and no significant toxicity was observed for the different treatments (data not shown). Following the treatments with nilvadipine enantiomers, A␤ 40 and A␤ 42 were analyzed in the culture medium by using commercially available sandwich ELISAs (Invitrogen) according to recommendations of the manufacturer. Following the treatments with a dose range of BAY61-3606, A␤38, A␤ 40 , and A␤ 42 were quantified by electrochemiluminescence using multiplex A␤ assays according to the manufacturer's recommendations (Meso Scale Discovery, MD).…”

“…Briefly, brains were homogenized at 4°C in MPER reagent (Thermo Fisher Scientific Inc.) containing 1 mM PMSF and 1ϫ protease/phosphatase inhibitors mixture (Thermo Fisher Scientific Inc.) and centrifuged at 15,000 ϫ g for 30 min at 4°C. The supernatant-containing detergent-solubilized A␤ was collected and denatured in 5 M guanidine for 1 h before being diluted with the sample diluent provided in the A␤ ELISA kits and assessed for A␤ 40 and A␤ 42 according to the manufacturer's protocol. The pellet containing detergentinsoluble material was resuspended in 5 M guanidine and diluted with the sample diluent provided in the A␤ ELISA kits before assaying for A␤.…”

“…The pellet containing detergentinsoluble material was resuspended in 5 M guanidine and diluted with the sample diluent provided in the A␤ ELISA kits before assaying for A␤. Protein concentrations were measured in the guanidine extracts using the BCA method (Invitrogen), and results of brain A␤ 40 and A␤ 42 were calculated in picograms/mg of protein.…”

“…A␤ is generated by the sequential proteolytic cleavage of APP by ␤-and ␥-secretase. The ␥-secretase cut is imprecise and leads to the formation of A␤ peptides of different size, mainly A␤ 38 , A␤ 40 , and A␤ 42 . Soluble A␤ oligomers are now widely recognized as pathogenic because they have been shown to inhibit synaptic function, to cause synaptic degeneration, to induce Tau hyperphosphorylation, and to impair memory (8 -11).…”

“…Vehicle-treated Tg Tau P301S received an intraperitoneal injection of 50% DMSO in PBS (vehicle used to dissolve (Ϫ)-nilvadipine) for 10 consecutive days. Tau phosphorylation was quantified by Western blots using the following Tau antibodies: AT8 (Thermo Fisher Scientific Inc., IL) and PHF-1 (41) as we described previously (42). BAY61-3606 (dissolved in PBS) was administered intraperitoneally at a dosage of 2 mg/kg of body weight to 25-week-old Tg Tau P301S mice for 5 consecutive days.…”