Medeiros Destynie, Ayala-Baylon Karen, Egido-Betancourt Hailey, Miller Eric, Chapleau Christopher A, Robinson Holly A, Phillips Mary L, Yang Tao, Longo Frank, Li Wei, Pozzo-Miller Lucas
bioRxiv. 2023 Nov 17:2023.11.09.566435. doi: 10.1101/2023.11.09.566435.
Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in methyl-CpG-binding protein-2 (MECP2), encoding a transcriptional regulator of many genes, including brain-derived neurotrophic factor (Bdnf). BDNF mRNA and protein levels are lower in RTT autopsy brains and in multiple brain regions of Mecp2-deficient mice, and experimentally increasing BDNF levels improve atypical phenotypes in Mecp2 mutant mice. Due to the low blood-brain barrier permeability of BDNF itself, we tested the effects of a brain penetrant, small molecule ligand of its TrkB receptors. Applied in vitro, LM22A-4 increased dendritic spine density in pyramidal neurons in cultured hippocampal slices from postnatal day (P) 7 male Mecp2 knockout (KO) mice as much as recombinant BDNF, and both effects were prevented by the TrkB receptor inhibitors K-252a and ANA-12. Consistent with its partial agonist activity, LM22A-4 did not affect spine density in CA1 pyramidal neurons in slice cultures from male wildtype (WT) mice, where typical BDNF levels outcompete its binding to TrkB. To identify neurons of known genotypes in the "mosaic" brain of female Mecp2 heterozygous (HET) mice, we treated 4-6-month-old female MeCP2-GFP WT and HET mice with peripheral injections of LM22A-4 for 4 weeks. Surprisingly, mutant neurons lacking MeCP2-GFP showed dendritic spine volumes comparable to that in WT controls, while MeCP2-GFP-expressing neurons showed larger spines, similar to the phenotype we described in symptomatic male Mecp2 KO mice where all neurons lack MeCP2. Consistent with this non-cell-autonomous mechanism, a 4-week systemic treatment with LM22A-4 had an effect only in MeCP2-GFP-expressing neurons in female Mecp2 HET mice, bringing dendritic spine volumes down to WT control levels, and without affecting spines of MeCP2-GFP-lacking neurons. At the behavioral level, we found that female Mecp2 HET mice engaged in aggressive behaviors significantly more than WT controls, which were reduced to WT levels by a 4-week systemic treatment with LM22A-4. Altogether, these data revealed differences in dendritic spine size and altered behaviors in Mecp2 HET mice, while providing support to the potential usefulness of BDNF-related therapeutic approaches such as the partial TrkB agonist LM22A-4.
雷特综合征（RTT）是一种神经发育障碍，由甲基-CpG结合蛋白2（MECP2）突变引起，MECP2编码一种转录调节因子，可调节包括脑源性神经营养因子（Bdnf）在内的许多基因。在RTT尸检大脑和Mecp2基因敲除小鼠的多个脑区中，BDNF的mRNA和蛋白质水平较低，通过实验提高BDNF水平可改善Mecp2突变小鼠的非典型表型。由于BDNF本身的血脑屏障通透性较低，我们测试了一种可穿透大脑的BDNF TrkB受体小分子配体的作用。在体外实验中，LM22A-4使出生后第7天（P7）雄性Mecp2基因敲除（KO）小鼠海马切片培养物中锥体神经元的树突棘密度增加，其效果与重组BDNF相当，而TrkB受体抑制剂K-252a和ANA-12可阻断这两种作用。与其部分激动剂活性一致，LM22A-4对雄性野生型（WT）小鼠切片培养物中CA1锥体神经元的树突棘密度没有影响，因为在野生型小鼠中，正常水平的BDNF会竞争性抑制LM22A-4与TrkB的结合。为了在雌性Mecp2杂合（HET）小鼠的“镶嵌”大脑中识别已知基因型的神经元，我们对4-6月龄的雌性MeCP2-GFP野生型和杂合小鼠进行外周注射LM22A-4，持续4周。令人惊讶的是，缺乏MeCP2-GFP的突变神经元的树突棘体积与野生型对照组相当，而表达MeCP2-GFP的神经元则表现出更大的树突棘，这与我们在有症状的雄性Mecp2基因敲除小鼠中描述的表型相似，即所有神经元均缺乏MeCP2。与这种非细胞自主机制一致，对雌性Mecp2杂合小鼠进行4周的全身LM22A-4治疗仅对表达MeCP2-GFP的神经元有作用，可使树突棘体积降至野生型对照水平，而不会影响缺乏MeCP2-GFP的神经元的树突棘。在行为水平上，我们发现雌性Mecp2杂合小鼠的攻击性行为明显多于野生型对照组，而通过4周的全身LM22A-4治疗可将其攻击性行为减少至野生型水平。总之，这些数据揭示了Mecp2杂合小鼠在树突棘大小上的差异和行为改变，同时也为BDNF相关治疗方法（如部分TrkB激动剂LM22A-4）的潜在有效性提供了支持。
