神經生長素1基因變異對成年小鼠嗅覺上皮構造及神經細胞新生的影響

Abstract

嗅覺系統與情感和記憶功能的關係很密切，所以當嗅覺功能異常時，會導致情感的不正常，可能與情緒性的疾病有相關性，例如精神分裂症。先前的研究的確指出，精神分裂症病人會出現異常的嗅覺功能，例如受到干擾的氣味探索，氣味記憶和氣味辨別；也已有學者利用核磁共振掃描發現精神分裂病人的嗅球體積減少。已知Neuregulin1 (NRG1，神經生長素1)基因是精神分裂症的易感性基因之一。NRG1與它的受體，ErbBs，被認為在神經系統發育上扮演重要的角色。本實驗的目的是探討NRG1基因的變異，對小鼠嗅覺功能及嗅覺上皮神經細胞新生的影響。我們觀察八週大的NRG1+/+（野生型）小鼠與NRG1 +/- (變異型)小鼠的行為。之後它們接受腹腔bromodeoxyuridine (BrdU)注射，150毫克/公斤，每天一劑連續三天；在第4天 (BrdU-4D) ， 第10天(BrdU-10D) ， 第21天 (BrdU-21D) 以及第28天 (BrdU-28D) ，我們將動物麻醉灌流，取出它們的嗅覺上皮進行免疫細胞化學染色。 實驗結果如下 1. 行為實驗結果: NRG1變異小鼠體重比野生型輕約9.2%。開放性空間行為測驗顯示，變異小鼠在中央及周邊停留的時間和行走距離與野生型相似。根據嗅不同氣味的習慣/去習慣測試，與野生型相比，變異小鼠幾乎無法辨識不同味道與兩個不同鼠籠的墊料氣味。這些結果指出NRG1基因活性是維持正常嗅覺的重要因素。 2. 形態免疫染色結果: (A) 嗅覺上皮的背側區 (i) BrdU-4D和BrdU-10D NRG 1變異小鼠的BrdU正染色細胞數目比對應組野生型小鼠多94.6%和65.8%，但BrdU-21D和BrdU-28D NRG 1變異小鼠的含BrdU細胞數目與野生型類似。 (ii) 與野生型比較，NRG 1變異小鼠的doublecortin(DCX)染色細胞，即神經母細胞(neuroblast)的數目減少45.6%; olfactory marker protein(OMP)，一種成熟嗅覺接受神經細胞(olfactory receptor neuron)的標誌蛋白的染色面積與強度減少28.9%與18.6%；calretinin (CR)，一種鈣離子結合蛋白，染色細胞數量減少37.6%。 (iii) 變異小鼠的cleaved caspase 3染色細胞數目比野生型增加75.8%。 (B)嗅覺上皮中隔區 (i) BrdU-4D，BrdU-10D和BrdU-28D NRG1變異小鼠的BrdU染色細胞數目比野生型增加62.1%，54.5%和54.6%，但BrdU-21D NRG1變異小鼠沒有改變。 (ii) 與背側區類似，NRG1變異小鼠DCX染色細胞數目比野生型減少31.5%和cleaved caspase 3染色細胞數目增加38.5%，雖然OMP與CR表現與野生型相似。 (iii) 電子顯微鏡觀察發現，與野生型比較，變異小鼠嗅覺上皮底層細胞的核膜孔洞數量呈現上升趨勢，與嗅覺接受神經細胞的纖毛從其基底部單支突起，而非野生型的多支突起。 我們的染色結果也證實嗅覺上皮存在NRG1與ErbB 2接受器蛋白質，此指出NRG1可能會透過與ErbB 2結合導致上述特定細胞表現的改變。根據以上結果我們推測，NRG1基因的變異導致NRG1蛋白質異常，可能使小鼠嗅覺上皮含ErbB 2細胞的下游的訊息路徑發生改變，使細胞增生數目上升，但也使細胞存活下降，未成熟與成熟神經細胞數目也減少。變異小鼠嗅覺接受神經細胞纖毛不正常的超微結構，可能與其不正常的行為和細胞新生與凋亡增加有關係。NRG1基因活性顯然會影響神經細胞的增生，分化和存活，故我們推論其在精神分裂症病理機制扮演重要角色。

Olfactory system is closely associatied with emotion and memory. Olfactory malfunction may lead to abnormal emotion, and this may play roles in mood-related disease, such as schizophrenia. Previous studies indicate that abnormal olfactory functions are common symptoms of schizophrenics, which include disturbed identification, discrimination, memory, and detection of odors. As shown by MRI scans, reduced bulb volumes were seen in schizophrenic patients. Neuregulin 1 (NRG1) is thought to be one of the susceptibility genes of schizophrenia. Neuregulin 1 (NRG1) and its receptors, ErbBs, may play important roles in the development of nervous system. Thus, the goal of this experiment was to examine the effects of the mutation of NRG1 gene on the olfaction and olfactory epithelium (OE) neurogenesis. Following behavioral observation 8 week-old wildtype (WT, NRG1+/+) and mutant (Mut, NRG1+/-) mice were intraperitoneally injected with bromodeoxyuridine (BrdU), 150 mg/kg, once daily for 3 consecutive days. On the 4th day (BrdU-D4), 10th day (BrdU-D10), 21st day (BrdU-D21) and 28th day (BrdU-D28) the mice were subjected to preparation for immunocytochemistry on their OE. Our results are as follows. 1. Behavior test results: NRG1 mutant mice showed decreased average body weight by about 9.2%. As revealed from the open field test, the distance and duration traveled by mutant mice were similar in the central and peripheral parts of the field respectively to that of WT. The olfactory habituation/dishabituation test revealed that, mutant mice were unable to discriminate different odors and litter odors. 2. Morphological results: (A) In the dorsal region of OE (i) The numbers of BrdU-positive cells were increased by 94.6% and 65.8% in the BrdU-D4 and BrdU-D10 mutant mice, compared to WT, whereas BrdU-D21 and BrdU-D28 mutant mice were similar to WT. (ii) Mutant mice contained 45.6% less number of the doublecortin (DCX) , a marker for neuroblasts, positive cells than that of WT. (iii) The levels of the olfactory marker protein (OMP) for mature olfactory receptor neurons (ORNs) were decreased by 28.9% and 18.6% in its expression area and OD in the Mutant mice. (iv) OE of mutant mice had 37.6% lower number of calretinin (CR) positive cells. (v) Mutant mice had 75.8% more number of cleaved caspase 3 positive cells. (B) In the septal region of OE (i) The numbers of BrdU-positive cells were increased by 62.1%, 54.5% and 54.6% in the BrdU-D4, BrdU-D10 and BrdU-D28 mutant mice, compared to WT, whereas BrdU-D21 mutant mice were similar to WT. (ii) Mutant mice contained 31.5% less number of the doublecortin (DCX) positive cells than that of WT. (iii) Mutant mice had 38.5% more number of cleaved caspase 3 positive cells, whereas OMP and CR expression were similar to WT. (C) Electron microscopy revealed that compared to that of WT, the number of nuclear membrane pores seemed incereased in the mutant basal cell and a single cilium arose from its basal part in the mutant ORN, instead of the multiple cilia arising from one single basal part in WT ORNs. Our results also confirmed the presence of NRG1 and ErbB 2 receptor in OE. These point out that the abnormal NRG1 protein may bind to the ErbB 2, leading to altered intracellular signaling of those specific cells. In the mutant mice, the decreased number and abnormal ultrastructure of olfactory receptor neurons may be related to their abnormal behavior and increased neurogenesis and apoptosis. NRG1 gene activity apparently is critically involved in the proliferation, differentiation and survival of neural cells, and thus may play important roles in the pathogenesis of schizophrenia.