The impact of mild episodic ketosis on microglia and hippocampal long-term depression in 5xFAD mice

In Alzheimer’s disease, dysfunctional microglia possess abnormal immunometabolic features that cause neuronal/synaptic damage and aggravate pathology. A critical question is how to reverse or fine-tune abnormal microglial metabolism towards beneficial immunometabolic outcomes. A major metabolic intervention strategy to raise circulating ketone levels for health benefits, such as by consumption of a ketogenic diet 1, fasting, or other approaches collectively called ketotherapeutics, has raised a great deal of interest, but its effects on microglia are not well understood. Our previous in vitro study showed that β-hydroxybutyrate (BHB), a major ketone body, reverses multiple pathological features of amyloid-β oligomer (AβO)-activated human microglia. In the current study, we tested the in vivo effects of BHB on microglia and synaptic plasticity in the 5xFAD Alzheimer’s disease mouse model. To capture the metabolic impact of BHB on microglia, we employed a “subacute” 1-week regimen of daily intraperitoneal injection of BHB (250 mg/kg), which induced brief and mild episodic (daily) ketosis. This short regimen was able to mitigate pro-inflammatory microglia activation linked to NLRP3 inflammasome formation, and reduce brain amyloid-β deposition by enhancing phagocytosis. Remarkably, this regimen mitigated the deficits of hippocampal long-term depression but not long-term potentiation, and this effect was linked to suppression of the inflammasome-generated cytokine IL-1β. Our results suggest that short-term BHB treatment may ameliorate microglial abnormalities and microglia-regulated synaptic deficits in Alzheimer’s disease. Because beneficial results were achieved with mild episodic BHB elevation alone without diet restriction and without the need of feeding a KD, our results have significant implications to human ketotherapeutics. As KDs are known for poor compliance and low sustainability due to their restrictive nature, our study opens the possibility for alternative ketogenic approaches that are less restrictive, potentially safer, and easier for compliance than a KD, such as short-term BHB injections or dietary ketone esters, a translatable form of induced ketosis. In this study, we tested the effects of BHB on microglia in vivo using 5xFAD mice, which harbor five familial mutations of APP and PSEN1 genes and show robust Aβ production and Aβ‐associated microglia activation and neuroinflammation. we employed a “subacute” 1-week regimen of daily injection of BHB, which induces episodic ketosis similar to that induced by daily strenuous exercise, exercise or fasting. The subacute course was expected to allow a metabolic shift to develop. Freshly isolated microglia was use for RNAseq and analysis.阿尔茨海默病中，功能失调的小胶质细胞（microglia）具有异常的免疫代谢特征，可引发神经元/突触损伤并加重病理进程。当前亟需解决的关键问题是，如何逆转或微调异常的小胶质细胞代谢，以实现有益的免疫代谢结局。提升循环酮体水平的主流代谢干预策略（如生酮饮食（ketogenic diet, KD）¹、禁食或其他统称为酮体疗法（ketotherapeutics）的手段）已引发广泛关注，但此类手段对小胶质细胞的作用机制尚未明确。我们此前的体外研究显示，β-羟基丁酸（β-hydroxybutyrate, BHB）——一种主要的酮体——可逆转淀粉样β寡聚体（amyloid-β oligomer, AβO）激活的人小胶质细胞的多种病理特征。在本研究中，我们于5xFAD阿尔茨海默病小鼠模型中检测了BHB对小胶质细胞及突触可塑性的体内作用。为捕捉BHB对小胶质细胞的代谢影响，我们采用了“亚急性”1周给药方案：每日腹腔注射BHB（250 mg/kg），该方案可诱导短暂且轻度的间歇性（每日）酮症。该短期干预能够减轻与NLRP3炎性小体（NLRP3 inflammasome）形成相关的促炎小胶质细胞激活，并通过增强吞噬作用减少脑内淀粉样β沉积。值得注意的是，该方案可改善海马长时程抑郁的功能缺陷，但对长时程增强无显著影响，这一效应与抑制炎性小体产生的细胞因子IL-1β密切相关。我们的研究结果表明，短期BHB治疗或可改善阿尔茨海默病中的小胶质细胞异常及小胶质细胞调控的突触功能缺陷。由于仅通过轻度间歇性BHB升高即可获得有益结局，无需饮食限制，也无需喂食生酮饮食，因此本研究结果对人类酮体疗法具有重要指导意义。鉴于生酮饮食因限制性过强而存在依从性差、可持续性低的缺陷，本研究为限制性更低、潜在更安全且更易依从的替代生酮手段提供了可能，例如短期BHB注射或膳食酮酯——一种可转化的诱导酮症形式。本研究中，我们使用携带APP和PSEN1基因5个家族性突变的5xFAD小鼠检测了BHB对体内小胶质细胞的影响，该模型可稳定产生大量Aβ并出现Aβ相关的小胶质细胞激活与神经炎症。我们采用“亚急性”1周每日注射BHB的方案，该方案可诱导类似于每日剧烈运动、常规运动或禁食的间歇性酮症。亚急性干预有望促使代谢发生定向转变。我们使用新鲜分离的小胶质细胞进行RNA测序（RNAseq）及相关分析。