Augmenting BDNF/TrkB signaling has been demonstrated to be a promising strategy for reversing cognitive deficits in preclinical models of Alzheimer disease (AD). significant and selective increase in the density of thin spines in CA1 of lesioned mice, without affecting mushroom or stubby spines. These findings suggest chronic upregulation of TrkB signaling with 7,8-DHF may be an effective and practical strategy for improving function in AD, even after substantial neuronal loss has occurred. Introduction The interaction between brain-derived neurotrophic factor (BDNF) and its high-affinity receptor, TrkB, initiates downstream signaling that is critical for plasticity and memory [1]C[4]. Recent studies have highlighted the potential of targeting BDNF/TrkB signaling for the treatment of neurodegenerative diseases such as Alzheimer disease (AD) [5]C[8]. Such work has demonstrated that TrkB activation improves cognition, and this improvement is associated with increased synapse density. Furthermore, the effects of TrkB activation on cognition are typically independent of any effect on A or tau pathology [5], [6]. While promising, the efficacy of BDNF/TrkB-based therapies has yet to be demonstrated in a mouse model with severe neuronal and synaptic loss, such as occurs in AD [9], [10]. The loss of neurons is a prominent and early feature of AD, and this loss correlates with cognitive deficits more closely than amyloid load [9], [11], [12]. The hippocampal formation is particularly vulnerable, and in the late stages of the disease loss of neurons in the CA1 and layer II of the entorhinal cortex reaches 80C90% [13]. Even in mild AD, A-443654 there is already a loss of over half of neurons in CA1 and layer II of the entorhinal cortex [10]. Patients can have significant plaque and tangle pathology without detectable cognitive impairment, and only once substantial neuronal loss develops do patients begin to show signs of dementia [10]. Since significant synaptic and neuronal loss has already occurred by the time symptoms manifest and AD is diagnosed, it may be useful to promote the restoration of lost synapses by targeting signaling pathways such as BDNF/TrkB. Since BDNF has poor blood-brain barrier penetration and a short half life [14], TrkB has typically been targeted by locally upregulating BDNF through viral overexpression or direct protein infusion [6], [7], [15]. While these methods have provided a convincing proof of principle, other delivery methods should be developed to make clinical translation more feasible. Specific small molecule TrkB agonists have been recently developed that can be administered systemically and initiate TrkB activation with equal potency to BDNF [16], [17]. Treatment with these drugs has the benefit of being far less invasive than viral or recombinant protein delivery, and allows the chronic targeting KLHL22 antibody of multiple affected brain regions. One recently identified TrkB agonist, 7,8-dihydroxyflavone (DHF), has shown promising protective effects in mouse models of aging, Parkinson disease, and AD [8], [17]C[19]. In the A-443654 current study, we investigate whether 7,8-DHF treatment can also reverse functional deficits related to severe neuronal loss using CaM/Tet-DTA mice. The CaM/Tet-DTA model harbors a tetracycline-inducible transgene system that selectively kills neurons of the hippocampus and cortex, and produces behavioral deficits in cognitive domains subserved by these regions [20]C[22]. The pattern and degree of neuronal loss in this model emulates that found in AD [9], [13], [23]C[25]. Furthermore, for the current study we modified the CaM/Tet-DTA model to carry a Thy1-GFP-M transgene, which expresses GFP stochastically in CA1 pyramidal neurons [26] and allows analysis of the impact of 7,8-DHF on dendritic spine remodeling. Results Generating GFP-expressing CaM/Tet-DTA Mice To facilitate the analysis of the impact of 7,8-DHF on dendritic spines, we modified CaM/Tet-DTA mice to also carry a Thy1-GFP-M transgene, which produces a stochastic and sparse pattern of labeling in CA1 pyramidal neurons A-443654 (Fig. 1D) [26]. Tet-DTA+/+ mice were bred with A-443654 Thy1-GFP-M+/?, and subsequent generations were genotyped by Southern blot and interbred until animals that were homozygous for both transgenes were produced. Tet-DTA+/+/Thy1-GFP-M+/+ and CaM-tTA+/? colonies were maintained separately and then crossed to produce CaM-tTA+/?/Tet-DTA+/?/Thy1-GFP-M+/? or CaM-tTA?/?/Tet-DTA+/?/Thy1-GFP-M+/? mice for experiments. Figure 1 CaM/Tet-DTA mice develop neuronal loss in Alzheimer-disease related brain areas. To ensure that the presence of GFP or new background strain elements did not disrupt the functioning of the CaM/Tet-DTA lesion system, hippocampal and cortical lesions were induced in 3-month-old CaM-tTA+/?/Tet-DTA+/?/Thy1-GFP-M+/? (CaM/Tet-DTA) mice by replacing mouse chow containing doxycycline, a tetracycline analog, with standard chow. Tet-DTA+/?/Thy1-GFP-M+/? littermates were also taken off doxycycline chow, and since they do not carry CaM-tTA these animals served.