rTMS Modulation of Behavioral and Biological Measures in 3xTg-AD Mice

Description

Abstract: Background/Objectives: The biological basis for behavioral manifestations of Alz-heimer’s disease remains unclear. Behavioral disinhibition, an overlooked manifestation of Alz-heimer’s disease, can result in substantial caregiver burden, and lacks effective management. This study expands upon previous work investigating disinhibited behavior in Alzheimer’s disease and a potential treatment of increasing brain-derived neurotrophic factor (BDNF) with rTMS. Methods: 47 3xTg-AD (Alzheimer’s) and 52 B6 (wildtype) mice were administered with ANA12 (an antagonist of TrkB receptor) or Vehicle (saline) and then rTMS or Sham treatment daily. After 14 days of treatments and injections, mouse behavior was assessed under various behavioral cognitive tests. Mice were then perfused, and brain samples were processed for histology and protein assays. Brain homogenates were analyzed for BDNF and its downstream signaling mol-ecules. Results: Open field testing demonstrated that 3xTg-AD mice traveled less total distance than B6 mice. 3xTg-AD-Sham mice injected with ANA12 were the only group to travel signifi-cantly less distance than B6-ANA12-Sham or B6-Vehicle-Sham mice (p<0.05), while 3xTg-AD-rTMS mice (irrespective of injection) were not significantly different from any group, indicating some corrective influence from rTMS. 3xTg-AD mice had significantly greater meas-ured levels of BDNF and TrkB than the wild type mice. Conclusions: Treatment of Alzheimer’s disease using rTMS positively affects elements of disinhibition, but not all behavioral abnormali-ties. rTMS shifts 3xTg-AD open field behavioral test measures, eliminating and generating sig-nificant differences between untreated 3xTg-AD and B6 genotypes. Despite its benefit, further in-vestigation of rTMS as a treatment for Alzheimer’s disease as well as its biological underpin-nings are needed.

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This study included 99 female mice (52 B6 and 47 3x) aged 12 months. rTMS was applied at 10 Hz for 10 minutes for a total of 6000 pulses per day over 14 days (intensity ~ 12mT). Sham mice were treated the same as the rTMS groups with a coil attached to the coil support, but no stimulation was delivered. ANA12 (Sigma) in 1% DMSO and sterile PBS or Vehicle control (1% DMSO in PBS) was injected intraperitoneally at 0.5mg/kg 2 hours prior to treatment. The day after the completion of the 14-day rTMS treatment, mice completed the Open Field Task (OFT) in a 50x50cm arena for 10min. 24 hours after completion, vaginal smears were taken to assess estrus cycle and brains were collected after perfusion. Hippocampi were homogenized in 400μL cold Syn-Per synaptic Protein Extraction Re-agent (Thermo Scientific) containing 1x Protease and Phosphatase inhibitor tablets (Roche). The synaptic and cytosolic fractions were used for Western Blot for TrkB and downstream proteins (AKT, ERK, PLCγ) . Briefly, samples were diluted in tris-buffered saline and denatured by boiling at 95°C for 5min with 1:40 mercaptoethanol. Electrophoresis was run on precast gels (BioRad) at 100V and proteins transferred via the Transblot Turbo System (BioRad) prior to blocking (Rockland). Membranes were incubated overnight with the following antibodies: TrkB (1:500, BD), ERK1/2 (1:1000, Santa Cruz), AKT (1:500, Cell Signaling), and PLCγ (1:500, R&D). Cofilin (1:2000, Sigma), and β-actin (1:1000, ThermoFisher) were used for the standard loading controls. The Full Fractions of the homogenate were diluted 1:10 in sample dilution buffer and measured in an enzyme-linked immunosorbent assay (ELISA) for BDNF analysis (R&D). A dual staining procedure for Aβ and phosphorylated-tau was conducted with Aβ (di-lution 1:1200, Cell Signaling) and AT8 (dilution 1:1200, ThermoFisher) antibodies. The slices were then incubated with the primary antibodies overnight at 4°C, rinsed in PBS, and subsequently exposed to secondary antibodies 1:500 (ThermoFisher) for 1 hour at room temperature. DAPI 1:1000 was introduced during the final rinse. Quantification of plaques and tangles was performed using ImageJ software in the dorsal cortex, ventral cortex, septal nucleus, hippocampus, and subiculum and normalized to the total area of each region examined.

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