Early BDNF treatment ameliorates cell loss in the entorhinal cortex of APP transgenic mice

Abstract

Brain-derived neurotrophic factor (BDNF) improves molecular, cellular, and behavioral measures of neural dysfunction in genetic models of Alzheimer's disease (Blurton-Jones et al., 2009; Nagahara et al., 2009). However, BDNF treatment after disease onset has not been reported to improve neuronal survival in these models. We now report prevention of neuronal loss with early life BDNF treatment in mutant mice expressing two amyloid precursor protein (APP) mutations associated with early-onset familial Alzheimer's disease. APP transgenic mice underwent lentiviral BDNF gene delivery into the entorhinal cortices at age 2 months and were examined 5 months later. BDNF-treated mice exhibited significant improvements in hippocampal-dependent contextual fear conditioning compared with control-treated APP mice (p < 0.05). Stereological analysis of entorhinal cortical cell number demonstrated ∼20% reductions in neuronal number in layers II-VI of the entorhinal cortex in untreated APP mutant mice compared with wild-type mice (p < 0.0001), and significant amelioration of cell loss by BDNF (p < 0.001). Moreover, BDNF gene delivery improved synaptophysin immunoreactivity in the entorhinal cortex and, through anterograde BDNF transport, in the hippocampus (p < 0.01). Notably, BDNF did not affect amyloid plaque numbers, indicating that direct amyloid reduction is not necessary to achieve significant neuroprotective benefits in mutant amyloid models of Alzheimer's disease.

Figure 1.

BDNF improves hippocampal-dependent learning. Transgenic mice that receive Lenti-BDNF vector injections into the entorhinal cortex (APP-BDNF) exhibit improvement in a hippocampal-dependent contextual fear conditioning task. A, WT exhibit ∼40% freezing in response to a tone paired with a shock. Transgenic mice that receive control, Lenti-GFP vector injections into the entorhinal cortex (APP-GFP) exhibit a significant reduction in freezing, indicating impaired learning of the hippocampal-dependent task. Transgenic mice that receive BDNF injections into the entorhinal cortex (APP-BDNF) exhibit significant improvement in contextual fear conditioning relative to APP-GFP mice and do not differ significantly from WT mice. *p < 0.05, APP-GFP mice versus WT or APP-BDNF mice. B, On a version of the fear-conditioning task that does not depend on hippocampal circuitry, cued fear conditioning, there are no differences between groups (ANOVA, p = 0.4).

Figure 2.

Gene delivery into entorhinal cortex. GFP immunoreactivity shows transduced cells in the entorhinal cortex of (A) WT control mice, (B) GFP-treated APP transgenic mice injected with the GFP lentivirus, and (C) BDNF-treated APP transgenic mice injected with a BDNF-GFP lentivirus. BDNF expression is not visible in neurons in the entorhinal cortex of (D) WT or (E) APP-GFP mice, but (F) markedly elevated in BDNF-treated APP transgenic mice. Scale bar, 100 μm.

Figure 3.

Transport of BDNF protein from entorhinal cortex to hippocampus. A, GFP-labeled axons of entorhinal cortical neurons are detected by GFP immunolabeling in terminal regions of hippocampus, including stratum lacunosum-moleculare (L-M) of CA fields and outer molecular layer (O) layer of dentate gyrus. B–D, BDNF immunoreactivity shows comparable levels of endogenous BDNF levels in (B) WT mice and (C) APP-GFP mice. D, In contrast, BDNF levels are markedly elevated in the lacunosum-moleculare layer (L-M) and outer molecular layer (O) of the dentate gyrus in APP-BDNF mice. G, Granule cell layer; I, inner molecular layer. Scale bar, 100 μm.

Figure 4.

BDNF ELISA. A, Lenti-BDNF gene delivery in APP transgenic mice (APP-BDNF) resulted in significant elevations of BDNF levels compared with APP transgenic mice that received Lenti-GFP (APP-GFP) and WT mice that received Lenti-GFP (WT-GFP) or vehicle control (WT-Sham). *p < 0.01. B, Standard curve of known quantities of BDNF.

Figure 5.

BDNF ameliorates loss of synaptophysin. A, Synaptophysin immunolabeling in WT, APP-GFP, and APP-BDNF mice. Labeling is generally less dense and less intense in APP-GFP mice, quantified in B. Scale bar, 10 μm. B, Quantification of synaptophysin immunoreactivity reveals uniform reductions in cortical and hippocampal regions, but not striatum, in APP-GFP mice. Notably, cortical regions therapeutically exposed to BDNF, the entorhinal cortex, and hippocampal dentate gyrus showed significant amelioration of reductions in synaptophysin labeling. *p < 0.05.

Figure 6.

AAV2-BDNF gene delivery does not reduce amyloid plaque formation. A, Amyloid plaques in the CA3 region of APP-GFP mice detected with 4G8 antibody. B, Quantification reveals an absence of plaques in the brain of WT mice and significant increases in the hippocampus of both APP-GFP and APP-BDNF mice. *p < 0.001. Scale bar, 100 μm.