探討CPEB2所調控之呼吸生理

Abstract

CPEB2是一個能結合到核醣核酸並調控轉譯作用的蛋白。為瞭解CPEB2在生理上的功能，我建立CPEB2的基因剔除小鼠模式，並發現大部分Cpeb2剔除的新生鼠會在出生約三天內死亡。為找出新生鼠死亡原因，我針對調控呼吸及循環系統的重要器官進行分析。我發現CPEB2大量地表現在腦幹的部位。腦幹是一個維持生命所必需的腦區，其主要功能為調控呼吸及心跳。利用體積變化偵測掃描儀(Whole body plethysmography)測量後發現Cpeb2剔除的新生小鼠有呼吸異常和窒息的現象。進一步檢查腦幹的呼吸中樞以及橫膈肌的神經肌肉接合處(Neuromuscular junctions)後，都未發現任何型態或是功能上的異常。我發現Cpeb2剔除的新生小鼠在迷走神經背運動核(Dorsal motor nucleus of vagus)中乙醯膽鹼轉化酶(ChAT)的轉譯活性增加，使得副交感神經過度活化，進而造成支氣管收縮。我發現若只在小鼠膽鹼能神經元(Cholinergic neurons)中剔除Cpeb2，同樣能造成新生小鼠窒息以及成鼠支氣管過度收縮的現象。此外，不論是全身性或專一性地在膽鹼能神經元將Cpeb2剔除，給予吸入性抗膽鹼能的氣管擴張劑都能減緩新生鼠的窒息現象。總結，我的發現證明了副交感神經過度活化進而造成支氣管收縮可能是造成Cpeb2剔除新生鼠呼吸異常和死亡率增加的原因。 另外，我也發現存活下來的Cpeb2剔除新生鼠，成年時期會出現肺泡擴張及類似肺氣腫的特徵。檢測肺功能後發現Cpeb2剔除成鼠肺臟的彈性和呼吸道阻力都有降低的趨勢，而肺部的動態順應性(Dynamic compliance)則有增加的現象。在不同發育時期的Cpeb2剔除鼠，也被發現其彈性纖維不正常地堆積在肺泡間。此外，肺泡發育時負責製造彈性纖維的肌成纖維細胞(Myofibroblasts)也同樣不正常地座落在肺泡間。已知彈性纖維的形成在肺泡發育時期扮演十分重要的角色，因此我假設: Cpeb2剔除小鼠可能因為彈性纖維不正常地堆積在肺泡間而造成Cpeb2剔除鼠形成了類似肺氣腫的特徵。然而，CPEB2蛋白是如何藉由轉譯過程來調控彈性纖維的形成目前仍然在研究中。

Cytoplasmic polyadenylation element binding protein 2 (CPEB2) is an RNA-binding protein and translational regulator. To understand the physiological function of CPEB2, we generated CPEB2 knock-out (KO) mice and found that most died within 3 days after birth. CPEB2 is highly expressed in the brainstem, which controls vital functions, such as breathing. Whole-body plethysmography revealed that KO neonates had aberrant respiration with frequent apnea. Nevertheless, the morphology and function of the respiratory rhythm generator and diaphragmatic neuromuscular junctions appeared normal. We found that upregulated translation of choline acetyltransferase in the CPEB2 KO dorsal motor nucleus of vagus resulted in hyperactivation of parasympathetic signaling-induced bronchoconstriction, as evidenced by increased pulmonary acetylcholine and phosphorylated myosin light chain 2 in bronchial smooth muscles. Specific deletion of CPEB2 in cholinergic neurons sufficiently increased the apnea frequency in neonatal pups and airway hyper-reactivity in adult mice. Moreover, inhalation of an anticholinergic bronchodilator reduced apnea episodes in global and cholinergic CPEB2-KO mice. Together, the elevated airway constriction induced by cholinergic transmission in KO neonates may account for the respiratory defect and mortality. Moreover, we observed that the survived adult KO mice developed emphysema-like phenotype with enlarged distal airspace. Using a forced oscillatory system, we found KO mice showed impaired lung functions, including decreased airway elastance and resistance as well as increased dynamic compliance. Another evident defect in CPEB2-KO lungs at different developmental stages is the abnormal deposition of elastic fibers. Moreover, the elastin-producing cells, myofibroblasts, at the alveolar stage was also mislocalized. Because proper elastogenesis plays an important role in alveolarization during lung development, we hypothesize that the emphysematous phenotype in global CPEB2 KO mice probably resulted from abnormal deposition of elastic fibers. Whether and how CPEB2-mediated translation regulates elastic fiber deposition are currently under investigation.