Possible Correlations between Major Depressive Disorder and Neurogenesis

이승주 경희대학교 대학원 2010 국내박사

Rimonabant is a cannabinoid receptor 1 antagonist, and is used to treat anorexia and obesity. However, it has been suggested that rimonabant may act as a depressant. In the present study, we investigated the depressive effects of rimonabant using behavioral and biochemical methods. A single treatment with rimonabant (10 mg/kg, p.o) reduced immobility duration in the forced swimming test to a level similar to that observed for the tricyclic antidepressant, imipramine (15 mg/kg, i.p.). However, mice treated with rimonabant for 2 weeks did not show any significant reductions in immobility duration versus vehicle-treated controls. To investigate why the antidepressant effect of rimonabant disappeared after extended treatment, we carried out 5-bromo-2-deoxyuridine (BrdU) and doublecortin (DCX) immunohistochemistry assay. Numbers of BrdU-immunoreactive cells were not significantly changed after administering rimonabant (10 mg/kg, p.o) for 2 weeks in the hippocampal dentate gyrus (DG), but interestingly, numbers of DCX-immunopositive cells in the DG were significantly reduced after 2 weeks of rimonabant treatment at doses of 1 or 10 mg/kg/day compared with vehicle-treated controls (P < 0.05). These results suggest that sub-chronic treatments with rimonabant inhibit cell proliferation in DG, and that a lack of antidepressant-like activity may be related to a reduction in cell proliferation in this region. The aim of this study is to identify the effects of water extracts of Allium macrostemon Bunge (AM-W), a traditional herb, on the depressive behaviors in mice using the forced swimming test. AM-W (100 and 200 mg/kg, p.o.) reduced the immobility duration in forced swimming test in a dose-dependent manner. To elucidate the mode of action of the antidepressant?like effects of AM-W, new born cells in the subgranular zone and the granule cell layer were analyzed using 5-bromo-2-deoxyuridine (BrdU) incorporation immunostaining. AM-W with sub-chronic administration (100 or 200 mg/kg, p.o, for 14 days) increases the number of BrdU-incorporating cells. The percentage of BrdU-incorporating cells co-localized with NeuN was significantly increased by the AM-W administration (100 or 200 mg/kg, P < 0.05). Moreover, the expression levels of brain-derived neurotrophic factor (BDNF) which is reported to be related with neurogenesis was significantly increased in the hippocampus by the administration of AM-W on immunohistochemistrty and western blot analysis. These results suggest that AM-W would be a good antidepressant, and that its effects would be related with its positive effects on neurogenesis and BDNF expression. Throughout these studies, the alteration of hippocampal neurogenesis is closely related with depressive-like or antidepressant-like behaviors. 우울증은 정서, 인지, 자율신경계 및 내분비 기능의 장애를 동반하는 심각한 질환이다. 최근 보고에 따르면 이러한 우울증의 발병과 치료에 해마에서의 neurogenesis가 매우 중요한 역할을 한다고 알려져 있다. 본 연구에서는 CB1 antagonist인 rimonabant와 利氣藥 (이기약)인 해백 (&#34212;白, Allium macrostemon)의 물추추출 (AM-W)의 생쥐에서의 우울증 관련 행동에 대한 효과를 forced swimming test 또는 tail suspension test를 이용하여 확인하였고, 그 행동의 변화와 neurogenesis 간의 상관관계를 분석하였다. Forced swimming test를 실시한 결과, rimonabant의 단회 투여 (10 mg/kg, p.o)에 의해서 양성대조군으로 사용한 imipramine (15 mg/kg, i.p.) 수준만큼 유의성 있는 부동시간 (immobility duration)의 감소가 관찰되었다. 그러나 rimonabant를 14일 동안 연속적으로 투여하였을 때 대조군과 비교하여 유의성 있는 부동시간의 감소는 관찰되지 않았다. Rimonabant의 항우울 활성이 연속 투여에 의해 소실되는 원인을 규명하기 위하여 5-bromo-2-deoxyuridine (BrdU) 및 doublecortin (DCX) immunohistochemistry assay를 실시하였다. 그 결과, 14일 동안의 rimonabant 투여에 의해서 해마의 치이랑에서의 BrdU포함 세포수는 변화가 없었다. 그러나, 흥미롭게도, 14일 투여 후 rimonabant 1 및 10 mg/kg/day의 용량에서 치이랑에서의 DCX-양성 세포수가 유의성 있게 감소하였다. 이를 통해서 rimonabant가 치이랑에서의 neurogenesis를 억제하는 것을 확인하였고, rimonabant의 항우울 활성의 소실이 감소된 neurogenesis에 관련되어 있을 것이라 사료된다. 또한 AM-W의 항우울 활성을 측정한 결과, forced swimming test 및 tail suspension test에서 AM-W (200 mg/kg, p.o.) 의 단회 투여에 의해 유의성 있는 부동시간의 감소가 관찰되었다. 또한 AM-W를 14일 간 투여 후 forced swimming test를 실시하여, AM-W (100 or 200 mg/kg, p.o.)의 투여가 용량 의존적으로 부동시간을 감소시킴을 확인하였다. AM-W의 항우울 활성의 기전을 확인하기 위하여 BrdU 및 DCX immunohistochemistry assay, BrdU/NeuN multi-labeling을 이용하여 해마에서의 neurogenesis를 확인하였다. AM-W의 14일 투여에 의해 해마의 치이랑에서의 BrdU포함 세포수 및 DCX-양성 세포수가 모두 대조군과 비교하여 유의성 있게 증가하였고, BrdU/NeuN multi-labeling 시험하여 BrdU와 NeuN이 공존하는 세포의 비율 또한 유의성 있게 증가하였다. 뿐만 아니라 immunohistochemistrty and western blot analysis를 통해서 해마에서 neurogenesis 및 항우울 활성과 관련되어있는 brain-derived neurotrophic factor (BDNF)의 분비가 AM-W에 의해 증가됨이 관찰되었다. 이 결과로 AM-W의 항우울 효과는 BDNF의 분비 또는 neurogenesis 와 관련 되어 나타난다는 사실을 알 수 있다. 따라서 AM-W는 우울증에 대한 치료제로 개발될 수 있을 것이다. 본 연구를 통하여, 해마에서의 neurogenesis의 변화가 우울증 양태 혹은 우울증 치료와 밀접한 관련이 있고, 치료의 목표로 연구 가능성에 대하여 시사하는 바가 크다고 사료된다.

The role of TREK-1 channel for adult hippocampal neurogenesis in depression mouse model

Kim, Seung-Chan 고려대학교 대학원 2023 국내박사

장기증강(LTP) 및 장기억압(LTD)의 유도를 통한 쥐의 치상(dentate gyrus) 부위의 선조세포(progenitor cells)의 증식(proliferation) 및 생존(survival)에 미치는 효과에 관한 연구

천성근 아주대학교 일반대학원 2007 국내박사

The dentate gyrus (DG) is among the few areas in the mammalian brain where production of new neurons continues in the adulthood. Although its functional significance is not completely understood, several lines of evidence suggest the role of DG neurogenesis in learning and memory. Considering that long-term potentiation (LTP) is a prime candidate for the process underlying hippocampal learning and memory, these results raise the possibility that LTP and neurogenesis are closely related. Here, I investigated whether or not LTP induction in the afferent pathway triggers enhanced proliferation of progenitor cells in the DG. LTP was induced by tetanic stimulation in perforant path-DG synapses in one hemisphere, and the number of newly generated progenitor (BrdU-labeled) cells in the DG was quantified. Compared with the control hemisphere (stimulated with low-frequency pulses), the LTP-induced hemisphere contained a significantly higher number of newly generated progenitor cells. When CPP, an NMDA receptor antagonist, was administered, tetanic stimulation neither induced LTP nor enhanced progenitor cell proliferation, indicating that NMDA receptor activation, rather than tetanic stimulation per se, is responsible for enhanced progenitor proliferation in the control animal. These results show that tetanic stimulation of perforant path sufficient to induce LTP increases progenitor proliferation in adult DG. In addition, it indicates that progenitor proliferation is affected by an NMDA receptor-dependent manner. Futhermore, I investigated whether LTP triggers survival of proliferating progenitor cells. Through the immunohistochemical staining methods, the effect of survival 3 weeks after LTP induction was investigated. There were also significant enhancements of BrdU-labeled cells in the LTP-induced hemispheres compared to LFS-stimulated. Most of these BrdU-labeled cells were double-labeled with NeuN, a neuronal marker. It indicates that LTP induction can trigger also survival new born cells. Long-term depression (LTD) is the phenomenon of a weakening of synaptic efficacy, and it is the contrary concept with LTP. Therefore, I did more experiments to investigate whether induction of LTD has any effect on proliferation of progenitor cells. LTD was induced by stimulating afferent fibers with low-frequency stimulation (900 pulses), and then the fluorescent staining was performed. There was no significant difference between LTD-induced hemispheres and baseline LFS-stimulated, indicating LTD unlike LTP has no effect on proliferation. It is possible to suggest that LTP may not affect proliferating process in short-term period. Because LTD is the weakening of synaptic efficacy, it doesn’t need any proliferation-related process unlike LTP. From those concepts, I did more experiments to investigate whether LTD induction can trigger survival of new born cells in the long-term period. It is on going now. Through the analysis of those data, it is possible to distinguish the effect of LTP and LTD on neurogenesis. In the analysis of experiment data, I found an interesting result. There was a significant increase of clusters of progenitor cells in the LTP-induced hemispheres compared to LFS-stimulated. It indicates that quiescent stem cells was activated by LTP, and then those cells were accelerated to divide, finally the proliferation of progenitor cells were enhanced. Meanwhile, there was no significant difference in the number of clusters after LTD induction. It is possible to suppose that LTD couldn’t trigger powerful stimulation to make quiescent stem cells to be activated, and then it couldn’t affect enhancement of proliferation of progenitor cells but also number of clusters. In the conclusion, LTP can trigger not only proliferation but survival of progenitor cells, but LTD has no effect on proliferation. It can be concluded that the enhancement of proliferation is due to the activation of quiescent stem cells. 쥐의 치상 (dentate gyrus) 부위는 포유류의 뇌 부분에서, 성체기에도 새로운 뉴런의 생성 (neurogenesis)이 계속되는 특이한 부분 중의 하나이다. 이러한 새로운 뉴런의 생성에 있어서, 그 기능적인 중요성은 아직 완전히 이해되고 있지 않으나, 학습 (Learning) 및 기억 (memory) 에 있어 그러한 치상에서의 뉴런의 생성의 중요성이 몇 가지 증거들로 인해 보여지고 있는 실정이다. 장기증강 (LTP) 현상이 해마 (hippocampus)의 학습 및 기억의 가장 유력한 기전으로 알려져 있는 점과 관련해서, 이 논문에서는 장기증강과 새로운 뉴런의 생성이 밀접히 관련이 있을 것이라는 가능성을 높이는 증거를 보였다. 여기서, 본 저자는 해마에 있어서 학습 및 기억의 가장 유력한 기전으로 알려져 있는 장기증강을 구심성 신경 경로 (afferent pathway)를 자극하여 유도함으로써, 그러한 장기증강이 치상에서의 전구세포들 (progenitor cells)의 증식 (proliferation)을 향상시킬 수 있는지를 살펴보았다. 뇌의 한쪽 부위에 강직성 자극 (tetanic stimulation)을 가함으로써 장기증강을 유도하였으며, 치상에서의 새로이 생성된 전구세포들의 수를 헤아렸다. 대조구 (LFS-stimulated hemisphere)와 비교해 봤을 때, 장기증강이 유도된 반구의 등쪽 (dorsal) 뿐만 아니라 배쪽 (ventral) 해마부위에서 상당히 높은 수의 새로이 생성된 전구세포들이 관찰되었다. 글루탐산염 수용체 길항제 (NMDA receptor antagonist)인 CPP를 처리하였을때는, 강직성 자극을 가하였음에도 불구하고 장기증강뿐만 아니라 전구세포들의 증식을 향상시키지 못했는데, 이러한 결과들을 통해서 강직성 자극 그 자체에 의해서라기 보다는 글루탐산염 수용체의 활성이 전구세포의 증식을 향상시킴을 알 수 있다. 본 연구에서는 장기증강을 유도하기에 충분한 강직성 자극을 관통부위 (perforant path)에 가함으로써, 글루탐산염 수용체에 의존적인 방식으로 성체의 치상부위에서 전구세포의 증식을 증가시킬 수 있음을 보였다. 또한 장기증강을 유도하고 3주 후에 전구세포들의 생존에 미치는 효과를 관찰하였을 때, 장기증강을 유도한 한쪽 부위에서 저빈도 자극(low-frequency stimulation)을 가한 다른 한쪽 부위와는 달리 상당히 높은 수의 BrdU에 표지된 세포의 수가 관찰됨을 알 수 있었다. 이렇게 증가한 BrdU에 표지된 세포들은 또한 형광 염색을 통한 관찰결과 성숙한 뉴런의 표지로 사용되는 NeuN으로 함께 염색됨을 알 수 있었다. 이러한 결과를 통해서 장기증강의 유도만으로 전구세포의 증식뿐만 아니라 생존을 증가시키는 효과가 있음을 알 수 있다. 장기억압(long-term depression, LTD)은 시냅스의 효능을 약화시키는 것으로 장기증강의 반대 개념으로 알려져 있다. 따라서 본 연구자는 추가 실험을 통해 이러한 장기억압이 장기증강과는 달리 어떠한 영향을 미치는지를 알아보고자 하였다. 장기증강과 달리 뇌의 한쪽 부위에 900 펄스의 저빈도 자극을 가함으로써 장기억압 현상을 유도한 뒤, 염색을 통한 세포의 수적 증가를 관찰한 결과, 선조세포의 증식에 있어서 대조구와 뚜렷한 차이가 관찰되지 않았다. 이러한 결과는 장기증강과 달리 장기억압은 짧은 시간 동안의 유도를 통해서는 어떠한 선조세포의 증식에도 그리 큰 영향을 미치지 않음을 볼 수 있었다. 그런 연유로, 조금 더 오랫동안 장기억압의 영향을 미칠 수 있는 선조세포의 생존에 관계하는 효과를 관찰하였고, 현재 많은 수의 데이터를 확보하여 분석을 실행 중이다. 이러한 실험의 결과를 분석을 통해서 매우 흥미로운 점을 발견하였는데, 장기증강을 유도한 부위에서 대조구와는 달리 뚜렷이 증가한 선조세포의 군집이 관찰된다는 것이었다. 이것은 무활동성 줄기세포(quiescent stem cell)가 장기증강에 의해서 활성을 띠게 되어 분열이 가속화 되어 선조세포의 증식이 증가하게 되었음을 시사하는 것이다. 또한 그러한 차이가 장기억압에서는 관찰되지 않았는데, 장기억압은 무활동성 줄기세포가 활성을 띨 수 있을 만큼의 강력한 자극을 이끌어 내지 못하는 이유로 선조세포의 증식에 아무런 영향을 주지 못했을 것이란 추측이 가능하다. 위의 결과들을 종합해서 생각해 볼 때, 장기증강은 선조세포의 증식 및 생존에도 큰 영향을 미치는 반면, 그 반대적인 기능을 가지고 있는 장기억압은 어떤 영향도 미치지 않음을 알 수 있다. 또한 그런 차이가 무활동성 줄기세포를 활성시킴으로써 일어나는 것 또한 추측할 수 있다.

(The) effects of Scutellaria baicalensis on spatial memory and neurogenesis in the dentate gyrus of the hippocampus of adult rats

이선화 경희대학교 대학원 2010 국내박사

Background and purpose-Thousands of new neurons are continuously produced in the dentate gyrus of the hippocampus of adult mammalian brain. There are many factors regulating neurogenesis (environmental stimuli, physical activity, hippocampal-dependent learning, medications and pathologic stimuli) but a little have been studied about the effects of herbal medicine on neurogenesis. Thus we investigated the effects of ethanol extracts of Scutellaria baicalensis (EESB) on spatial memory and neurogenesis in the dentate gyrus of the hippocampus in Sprague-Dawley adult rats. Methods- Rats were divided into four groups (CON, SB50, SB100, SB200) and EESB were administrated orally once a day for six days in SB treated groups, saline was administrated to CON in the same way. To investigate the spatial memory and learning in rats, radial-arm maze test was performed on the 16th day. After behavior test, neurogenesis was assessed with immunohistochemistry of BrdU (5-bromo-2’-deoxyuridine) single staining and immunofluorescence of BrdU double staining with the glial marker GFAP (glial fibrillary acidic protein) and the neuronal marker NeuN (neuron-specific nuclear protein). Results- The decrease in escape latency of radial arm maze test was detected in SB200. EESB treatment increased the number of BrdU-labeled cells in the dentate gyrus of the hippocampus in all SB treated rats (SB50, SB100, SB200). Confocal microscopic analysis showed that the number of BrdU-positive cells (n/mm3) co-localized with NeuN within the GCL (6150 &#61617; 152.5) increased significantly in SB100 contrasted to CON (1067± 470.7). Approximately 97% of the BrdU-labelled cells in SB100 co-labeled with the neuronal marker NeuN in the GCL. In the SGZ, there were significant increases in the percentage of BrdU-NeuN double labeled cells in 100SB (89.5%) and 200SB (82.5%) contrasted to CON (42.25%). The number of BrdU -positive cells double labeled with GFAP within the SGL increased significantly in SB 200 (16145 &#61617; 2089) contrasted to CON (6446 &#61617; 341.7). The percentage of BrdU+GFAP+ /BrdU+ in the dentate gyrus was about 46% in CON. This ratio changed to 59% in SB50, to 56% in SB100, to 78% in SB200. Conclusion- Administration of 200mg/kg of EESB was effective on cell proliferation, the revelation of the subsets of astrocytes and differenciation into mature neurons in the dentate gyrus of the hippocampus, and resulted in improvement of spatial memory and learning in adult rats. This results suggest that EESB can promote neurogenesis in the dentate gyrus of the hippocampus of adult rats. Keywords ; Scutellaria baicalensis, adult neurogenesis, hippocampus, dentate gyrus, BrdU (5-bromo-2’-deoxyuridine), NeuN (neuron-specific nuclear protein), GFAP (glial fibrillary acidic protein) 실험배경과 실험목적 : 성장한 포유동물의 뇌에서는 끊임없이 새로운 neuron들이 생성되고 있으며 생성부위는 주로 hippocampus의 dentate gyrus와 lateral ventricle의 subventricular zone이다. 새롭게 생긴 neuron들은 증식하고 분화하는 과정을 거쳐 성숙한 neuron이 된다. neurogenesis에 영향을 미치는 요인으로는 운동, 환경, 약물, 스트레스, 호르몬, 외상 등 여러가지가 있으며 지금까지 이와 관련된 많은 연구가 이루어져왔다. 우리는 한약재중 항염, 항알레르기, 항미생물의 주요작용을 가지고 있고 최근에는 신경계 보호작용이 보고되고 있는 황금의 에탄올추출물을 이용하여 성인쥐 hippocampus의 dentate gyrus에서의 신경세포재생과 공간기억능력의 향상여부를 연구하고자 한다. 실험방법 : 성인쥐 (Sprague-Dawley rats, n = 44, 180 ± 10 g, 6주)를 대조군 (생리식염수투여군, n=11)과 SB50 (황금에탄올추출물 50mg/kg 투여군, n=11), SB100 (황금에탄올추출물 100mg/kg 투여군, n=11), SB200 (황금에탄올추출물 200mg/kg 투여군, n=11)의 총 4군으로 나누어 하루1회, 총6일간 황금에탄올추출액을 경구투여하였다. 그리고 면역화학염색법을 위하여 5-Bromodeoxyuridine (BrdU)를 황금추출액투여 30분전, 하루1회, 총4일간 피하주사하였다. 마지막 한약투여 10일후 성인쥐의 공간지각능력을 알아보기위하여 팔미로실험을 시행하였다. 행동실험 다음날, 쥐는 희생되었고 새로운 신경세포의 증식과 분화여부를 알아보기위하여 5-Bromodeoxyuridine (BrdU)를 이용한 면역화학염색법, BrdU와 GFAP (glial fibrillary acidic protein), neuron-specific nuclear protein(NeuN)를 이용한 면역형광염색법을 시행하였다. 실험결과 : 팔미로실험에서는 SB200에서 팔미로안의 설탕용액을 모두 찾아먹는데 걸리는 시간이 유의성있게 단축되었다 (CON 231.7±22.59, SB50 202.1±27.33, SB100 202.3±22.62, SB200 151.5±14.49). 면역화학염색법중 BrdU단일염색에서는 황금추출물투여 모든군 (SB50, SB100, SB200)에서 BrdU에 염색된 세포의 수가 유의성있게 증가하였고 (CON 36.20± 4.173, SB50 49.88&#61617; 2.911, SB100 54.62&#61617; 3.985, SB200 53.40&#61617; 4.339), 이는 새로운 세포의 증식을 의미한다. BrdU와 NeuN의 이중형광염색군에서는 hippocampus의 dentate gyrus 내부인 granular cell layer (GCL)의 SB100에서 이중염색된세포수의 유의성있는 증가가 관찰되었다 (CON, 1064 ± 470.7, SB100 6150 &#61617; 1525). Dentate gyrus 전체에서 볼 때는 SB100, SB200에서 이중염색된세포수가 유의성있게 증가하였다 (CON 4278 ± 1152, SB100 12415±2110, SB200 9992 ± 920.0). 그리고 dentate gyrus 전체에서 BrdU 총 염색군에 대한 NeuN과의 이중염색된 수의 비율이 황금투여군 전체에서 유의성있게 높이 나타났다 (CON 42%, 50SB 72%, SB100 92%, SB200 86%). 여기서 이중염색된 세포는 새로생성된 뉴런이 성숙한 뉴런으로 분화되었음을 의미한다. BrdU와 GFAP의 이중형광염색에서는 hippocampus의 dentate gyrus 내부의 subgranular zone (SGZ)에서의 SB200에서 GFAP와 이중염색된 세포수의 유의성있는 증가가 관찰되었다 (CON 6446 ± 341.7, SB200 16145 &#61617; 2089). 이는 dentate gyrus의 subgranular zone에서 neurogenesis를 의미하는 astrocyte의 유의성 있는 발현을 나타내는 결과이다. 결론 : 황금에탄올추출물 투여 (100mg/kg, 200mg/kg)는 성인쥐 hippocampus의 dentate gyrus에서의 신경세포재생을 증가시켰고 공간지각능력의 향상을 가져왔다. 황금의 신경계보호작용은 최근 지속적으로 연구되어오고 있으며, 본 실험은 BrdU, NeuN, GFAP등의 neuronal marker를 사용, 면역화학염색법과 면역형광염색법을 시행하여 성인쥐에서의 신경세포재생을 보여주는 유의성있는 연구결과로, 향후 알츠하이머질환이나 파킨슨씨병 등 뇌의 퇴행성병변이나 뇌손상의 치료에 응용할 수 있을것으로 기대한다.

Roles of Stress-related Factors in Embryonic Neurogenesis

Jyhyun Ahn 서울대학교 대학원 2015 국내박사

Stress exposure during development can lead to severe neurological diseases in infants and children. Many genes are known to be related to stress induction, but their roles in brain development have not been clearly elucidated. To rapidly screen for biologically meaningful factors involved in brain development, I first selected several genes that are known to be up-regulated in the fetal brain during prevalent stressed conditions such as prenatal infection and alcohol exposure, including nuclear factor (erythroid-derived 2)-like 2 (Nrf2), glycogen synthase kinase 3 (GSK3) α, GSK3β, interferon-gamma (IFN-γ), and sterol-C4-methyl oxidase-like gene (SC4MOL). These genes were overexpressed in primary embryonic day (E) 14.5 murine neural progenitor cells (NPCs) using retroviral vectors. The percentage of neuronal cells was measured by TuJ1 expression. Using this method, GSK3β and IFN-γ were found to have strong negative effects on neurogenesis, and they were further characterized in vitro and in vivo using various molecular techniques. GSK3 is known as an important regulator during the proliferation and differentiation of NPCs, but the roles of the isoforms of this molecule (GSK3α and GSK3β) have not been clearly defined. Thus, the functions of GSK3α and GSK3β in the context of neuronal differentiation of NPCs were characterized. Treatment of primary NPCs with a GSK3 inhibitor (SB216763) resulted in an increase in the percentage of TuJ1-positive immature neurons, suggesting an inhibitory role of GSK3 in embryonic neurogenesis. Downregulation of GSK3β expression increased the percentage of TuJ1-positive cells, whereas the knock-down of GSK3α appeared to have no effect. Mutant GSK3β (Y216F) failed to suppress neuronal differentiation, indicating that the kinase activity of GSK3β is important for this regulatory function. Similar results were obtained in vivo when a retroviral vector expressing GSK3β was delivered to E9.5 mouse brains. In addition, SB216763 was found to block the rapamycin-mediated inhibition of neuronal differentiation of NPCs. Taken together, these data demonstrate that GSK3β, but not GSK3α, negatively controls the neuronal differentiation of NPCs and that GSK3β may act downstream of the mTORC1 signaling pathway. IFN-γ is one of the critical cytokines released by host immune cells upon infection. Despite the important role(s) of IFN-γ in host immune responses, there have been no in vivo studies of the effects of IFN-γ on brain development, and the results obtained from many in vitro studies have been controversial. Treatment of E14.5 murine NPCs with IFN-γ resulted in a decrease in the percentage of TuJ1-positive immature neurons but an increase in the percentage of Nestin-positive NPCs. Similar results were obtained in vivo. Treatment of NPCs with a JAK inhibitor or the knock-down of STAT1 expression abrogated the IFN-γ-mediated inhibition of neurogenesis. Interestingly, the expression of one of proneural genes, Neurogenin2 (Neurog2) was inhibited dramatically upon IFN-γ treatment, and cells overexpressing Neurog2 did not respond to IFN-γ. Both IFN-γ treatment and overexpression of the constitutively active form of STAT1 reduced the Neurog2 promoter activity by nearly half. These results suggest that IFN-γ inhibits the neuronal differentiation of NPCs by negatively regulating the expression of Neurog2 partially at the promoter level via the JAK/STAT1 pathway. In this thesis work, I identified GSK3β and IFN-γ as negative controllers of neuronal differentiation, which act downstream of the mTORC1 signaling pathway and upstream of the JAK/STAT1 pathway, respectively. This is the first study to clearly distinguish the roles of GSK3 isoforms in the context of neuronal differentiation and to investigate the effects of IFN-γ on embryonic neurogenesis in vivo. The findings from this thesis may provide insights into the mechanism of action of different stress-related factors during the early period of brain development, especially the initiation of neurogenesis and the possible consequences of congenital stress exposure.

Effects of glucocorticoid on the amygdala-dependent fear memory and hippocampal neurogenesis

이은정 서울대학교 대학원 2013 국내박사

Glucocorticoid (GC) is a steroid hormone playing diverse roles which enable organisms to respond to and to cope with environmental changes such as stress. The secretion of GC is primarily governed by the hypothalamus-pituitary-adrenal (HPA) axis, a major neuroendocrine circuit in the stress response system. Maternal stress-elicited elevation of GC has programming effects on the long-lasting modification of the HPA axis and brain functions in offspring. However, the prolonged impact of maternal stress on emotional learning remains largely unknown. In addition to stress responsiveness and early life programming, another apparent characteristic of GC is its robust circadian rhythm. However, the significance of the functional GC rhythm in the brain function is not well understood yet. Recently, our group generated transgenic mice with adrenal-specific knockdown of canonical clock protein BMAL1 (A-BMKD), which showed attenuated circadian GC rhythm under constant darkness (Son et al., 2008). In Chapter 1, I intended to investigate how maternal stress affects fear memory in the amygdala, a major target of stress in the brain. In Chapter 2, using A-BMKD transgenic mouse line, I investigated the effect of circadian GC oscillation on hippocampal neurogenesis implicating in cognition deficits and mood disorders in Chapter 2. 1. In Chapter 1, I examined whether maternal stress influences on the amygdala-related learning processes. Maternally stressed mice exhibit normal fear memory acquisition as well as synaptic NMDA receptor expression in the amygdala; however, fear memory consolidation and the activation of related signaling cascades are significantly attenuated. In accordance with these behavioral aspects, maintenance of long-term potentiation (LTP) evoked in the thalamo-lateral amygdala pathway is significantly attenuated in amygdalar slices from maternally stressed animals, though basal synaptic properties and LTP induction were unaffected in these slices. Interestingly, administration of GC immediately after training or LTP induction restores impaired memory consolidation and LTP maintenance, indicating the weakened aversive memory-enhancing effect of GC in maternally stressed mice. Moreover, the membrane-impermeable form of GC mimics the restorative effect in maternally stressed animals, implying the involvement of a nongenomic mechanism. Taken together, it appears that maternal stress causes dysregulation of amygdala-dependent fear memory in adult offspring by an impairment of amygdalar synaptic plasticity in association with reduced nongenomic action of GC on long-term memory formation. 2. In Chapter 2, I focused on hippocampal neurogenesis in A-BMKD transgenic mice under constant darkness for 7 days. The number of newborn neurons in the hippocampus was significantly declined in the transgenic mice. These mice showed depressive mood states, impaired safety memory, and rapidly shutting off stress reactivity, which are known to be regulated by hippocampal neurogenesis. Moreover, enhanced rhythmic translocation of activated glucocorticoid receptor (GR) into hippocampal nucleus by binding to GC was observed against hypo-GC secretion with attenuation rhythm in TG mice. In accordance with this finding, transcript levels of brain-derived neurotrophin factor (BDNF) and its receptor TrkB, which are known to be essential for adult neurogenesis and regulated by GC negatively, were reduced in the hippocampus of A-BMKD transgenic mice. Interestingly, a daily oscillation of plasma GC restored by rhythmic intake of drinking water containing GC recovered not only neurogenesis but also gene expression of BDNF and TrkB in the hippocampus, suggesting that BDNF-related signal cascade is one of candidates responsible for the impaired hippocampal neurogenesis in the transgenic mice. Taken together, GC levels as well as its rhythmic secretion are important for maintaining normal neurogenesis associated with gene expression of BDNF and TrkB in the hippocampus. In conclusion, GC has a critical role in fear memory formation via nongenomic action in the amygdala and the functional circadian rhythm of GC is required for maintaining adult neurogenesis in the hippocampus.

EFFECTS OF SEXUAL ACTIVITY ON THE HIPPOCAMPAL NEUROGENESIS OF ADULT CD-1 MICE

김종인 건국대학교 대학원 2013 국내박사

Adult neurogenesis can be influenced by a variety of factors. Stress is one of the most potent inhibitors of hippocampal neurogenesis. Stress responses on adult hippocampal neurogenesis are affected differently by environmental factors including social interaction. The sexual behavior between males and females has been suggested to influence neurogenesis and enhance hippocampal cell proliferation. However, its mechanism of action, possible changes in stress state, and effects on learning and memory remain uncertain. The current study examined the influence of sexual activity on the neurological response in adult male mice and the function of sexual experience relative to recognition memory in stress state. Changes of the expression of neurotrophic and transcription factors were assessed in reference to stress and/or sexual activity. The survival of newly generated cells and their rate of differentiation into neurons were determined in the hippocampus of chronically stressed and/or sexually experienced mice. Finally, to evaluate the possibility of whether sexual experience alters adult hippocampal function, learning and memory in a recognition memory task were tested. Brain-derived neurotrophic factor (BDNF) mRNA and mature form BDNF (matBDNF) protein expressions in the hippocampus were increased more by repeated sexual activity, and a similar pattern of changes of TrkB mRNA, fullTrkB protein, and p-TrkB protein was evident. On the contrary, the expression levels of proBDNF and truncBDNF in hippocampus were significantly decreased by consecutive daily sexual activity. Expressions of matBDNF, fullTrkB, and p-TrkB, as well as CREB mRNA were markedly decreased by stress. Despite of such effect of stress on the hippocampus, BDNF, TrkB, and CREB mRNA were elevated in stressful situation by prior sexual activity, and similar aspects were also found in the matBDNF and fullTrkB expressions. In case of p-TrkB, moreover, stress could also not reduce the protein expression under prior sexual experience. The results indicate that prolonged sexual experience enhances cell survival and neuronal differentiation related to hippocampal neurogenesis, and counteracts the suppressing effect of chronic stress in adult male mice. Furthermore, recognition memory task, as assessed by NORT, is also improved by repeated sexual activity. The results demonstrated that sexual interaction increases the expression of brain-derived neurotrophic factor, tyrosine kinase B, and cAMP response element-binding. Furthermore, the results support the views that sexual interaction could be helpful for buffering the adult hippocampal neurogenesis against the suppressing actions of chronic stress and its effect has potential to influence on recognition memory. As a general conclusion, it is expected that sexual interaction could be helpful for buffering the adult hippocampal neurogenesis and recognition memory function against the suppressing actions of chronic stress.

Molecular Mechanisms Whereby Dietary Restriction Protects Neurons And Stimulates Neurogenesis

이재원 University of Kentucky 2002 해외박사

Dietary restriction (DR) increases the lifespan of a variety of species and wards off many age-related diseases. However, the molecular mechanisms underlying the neuroprotective actions of DR are not known. The present dissertation research focused on investigating the molecular mechanisms involved in the neuroprotective effects of DR. In addition, the impacts of DR on neurogenesis in the adult hippocampus were examined. The regulation of endogenous adrenocorticosteroid receptors in the brain was examined in rats maintained on control and DR feeding regimens to determine whether DR affects the responsiveness of neurons to glucocorticoids. The results suggest that DR down-regulates potentially damaging glucocorticoid receptor-mediated transcription, while maintaining neurotrophic mineralocorticoid receptor-mediated transcription. To determine whether DR results in a cellular stress response in neurons, levels of several candidate proteins were measured in brain tissues from control and DR rats. DR increased the expression of BDNF and NT-3 in several different populations of neurons in the brain. When adult rats and mice were maintained on DR regimen, there was a greater number of newly generated cells in the dentate gyrus of the hippocampus, apparently as the result of increased cell survival. The majority of newborn cells in the granular layer of the hippocampus exhibited neuronal phenotypes indicating that DR enhances hippocampal neurogenesis. Direct evidence of the role of BDNF in the enhancement of hippocampal neurogenesis resulting from DR was obtained in studies of heterozygous BDNF knockout mice. Lastly, the effects of DR on the responses of neural stem cells, microglia, and astrocytes, to seizure-induced hippocampal damage were determined to better understand the functional roles of the hippocampal neurogenesis. Collectively, the findings from this research have revealed molecular mechanisms underlying the neuroprotective actions of DR and have shown, for the first time that neural stem cells in the brain can be affected by diet. These findings suggest that DR may be an effective approach for the prevention of neurodegenerative diseases and the enhancement of neural plasticity in the adult brain.

뇌졸중 후 신경발생에 미치는 미세아교세포의 영향

서진주 차의과학대학교 대학원 2013 국내석사

뇌졸중은 크게 뇌혈관이 막혀서 생기는 허혈성 뇌졸중(뇌경색, Ischemia stroke)과 터져서 생기는 출혈성 뇌졸중(뇌출혈, Hemorrhage stroke)으로 나뉜다. 이 중 뇌경색이 더 많이 발병을 한다. 뇌졸중이 걸린 후에는 신경줄기세포의 빠른 분열과 손상부위로의 이동 그리고 신경세포로의 분화가 일어난다. 이 현상들은 뇌졸중을 회복시키는 것에 기여 하는 것으로 알려져 있다. 따라서 neurogenesis를 더 증진 시킬 수 있는 무언가를 찾으면 치료제로써 개발될 가능성이 아주 높다. 그래서 우리는 neurogenesis를 일으키는 무언가를 찾고자 했다. 먼저 뇌에 존재 하고 있는 세포들이 뇌졸중이 걸린 후에 어떤 변화가 일어나는지 확인했다. 머리에는 뇌에서 중요한 면역역할을 하는 세포로서 미세아교세포(microglia)가 존재 하고 이는 여러 형태의 뇌 손상에서 활성화 된다. 이 미세아교세포는 염증이 발생하면 그 수가 급격히 증가를 하는데 뇌졸중이 발병을 하고 나서 보이는 BrdU+한 세포의 증가와 감소 패턴이 미세아교세포랑 매우 흡사하다. 따라서 우리는 미세아교세포랑 신생세포생성 간에 관계가 있는지 확인을 하였고 더 나아가서 SDF1α가 미세아교세포에서 neurogenesis에 중요한 역할을 하는 것인지 알아냈다. There is two major types of stroke; ischemic stroke and hemorrhagic stroke. Ischemic stroke is the most common form of stroke and occurs when there is an abrupt interruption of blood flow to the brain. Stroke is increased neuroblst in the subventricular zone(SVZ) area and generation of mature striatal neuron and also migration to the damaged area. This effect is contributed to stroke damage recovery. So, we found neurogenesis factor. First, we researched cells that present in the brain and observed any changes after stroke. Microglia are immune cells of central nervous system(CNS), which are activated in response to injury and diseases. Increased the microglia cells by occur of immflamation So, that it is very similar, pattern of the increase and decrease BrdU+cell, after ischemic stroke. Therefore, we investigated between microglia and newborn cells. Furthermore, we found SDF1α what important role the incresed neurogenesis in microglia Keywords : microglia, ischemia stroke, neurogenesis, SDF1α

Identification the skin-brain connection: Effects of UV exposure and dietary supplements on cognitive function and neurogenesis

윤경노 서울대학교 대학원 2024 국내박사

피부-뇌 연결은 신체의 가장 큰 기관인 피부와 뇌의 복잡한 신경망 사이의 복잡하고 역동적인 상호 작용을 나타낸다. 이러한 양방향 통신 시스템은 외부 자극을 신경학적 반응으로 변환하는데 중추적인 역할을 하며, 이를 통해 뇌 건강의 생리적, 심리적 측면 모두에 영향을 미친다. 햇빛의 기본 구성 요소인 자외선은 피부에 심각한 영향을 미치고 결과적으로 뇌 내 일련의 신경반응을 유발하는 광범위한 환경 자극으로 작용한다. 피부에 자외선 노출은 학습 및 기억, 중독성 행동, 해마 신경발생과 같은 뇌 기능에 영향을 미칠 수 있다. 이러한 변화는 학습과 기억 형성에 중추적인 역할을 하는 해마 기능과 밀접한 관련이 있다. 자외선 노출이 뇌 기능에 미치는 영향은 이전 연구에서 조사되었지만 자외선에 의한 인지 장애를 담당하는 특정 신경 전달 물질 매개 메커니즘은 아직 밝혀지지 않았다. 1장에서는 장기적인 자외선 노출이 인지 기능에 해로운 영향을 미친다는 사실을 발견했다. 또한, 자외선이 신경발생을 손상시키고, 염증을 증가시키며, 뇌의 뇌혈액장벽을 손상시킴으로써 신경줄기세포의 증식, 분화 및 생존을 저해하였다. 따라서, 장기적인 자외선 노출은 인지 장애를 유발할 수 있음을 나타낸다. 자외선에 인한 인지 장애를 유도하는 특정 신경 전달 물질 매개체의 메커니즘을 밝히기 위해 액체 크로마토그래피/질량 분석법을 사용하여 28개의 신경전달물질을 분석하였고 그 결과, 장기적인 자외선 노출이 혈액을 포함한 중추 신경계(피질 및 시상하부) 및 말초 기관 (피부 및 부신)에서 도파민 수치가 유의하게 상승한 사실을 발견하였다. 또한, 도파민 수용체 D1/D5 SCH 23390이 자외선으로 유도된 해마 기억력 결핍과 손상된 신경 발생을 다시 회복했다. 시상하부 유전자 분석에 따르면, 장기간 자외선에 노출된 쥐는 도파민성 신경 세포 분화 경로와 관련된 유전자가 상향 조절한 반면, SCH 23390 처리 후 이러한 경로 내의 유전자는 하향 조절했다. 더욱이, 말초 도파민 주사는 자외선으로 인한 인지 변화를 모방했다. 이처럼 자외선에 의한 해마 기억 손실 및 신경 발생 손상과 같은 신경 행동학적 변화는 도파민이 주된 영향일 수 있다. 또한, 장기간 자외선을 조사한 생쥐의 해마 조직을 전사체 분석한 결과, 신경면역 증가 및 BBB 관련 유전자가 감소되었다. 또한, 세포 유형 분석에서 미세아교세포에 증가하였다. 더욱이, 면역형광염색에서는 자외선을 조사한 쥐의 해마에서 Iba1 양성 미세아교세포의 수가 증가하였다. 또한, 70kDa FITC-dextran 누출이 관찰되었다. 이러한 결과는 자외선 이 미세아교세포를 활성화시켜 신경염증을 증가시키고, 혈액내장벽을 손상시킨다는 것을 시사한다. 이 포괄적인 연구는 장기적인 자외선 조사가 도파민 수치 증가, 신경 염증 및 뇌혈액장벽 손상으로 인지 기능 장애를 유발한다는 강력한 증거를 제공한다. 인지 장애의 주요 위험 요소인 노화는 연령 관련 신경병리의 축적이며, 이는 해마 신경 발생의 감소에 중요한 역할을 한다. BDNF는 인지 능력을 유지하고 강화하는 데 중요한 역할을 하는 신경영양인지로, 연령에 따른 BDNF의 감소가 나타나며 이는 여러 인지 변화와 관련이 있다. 인지 기능을 개선하기 위해 BDNF을 증가시키는 효과적인 영양소를 식별하면 뇌 건강을 유지하는 데 도움이 될 수 있다. 과거 우리 실험실에서 토마토 레몬 추출물이 피부에서의 BDNF 합성을 증가시켜 혈중 농도를 증가시키고, 뇌에 작용하여 인지기능에 영향을 준다는 사실을 규명하였다. 2장에서는 토마토와 레몬 에탄올 추출물(각각 TEE와 LEE)의 효과와 노화된 쥐의 인지 기능 및 신경 발생 강화에 대한 시너지 효과를 조사한다. 또한, TEE와 LEE의 시너지 효과를 뒷받침하는 분자 메커니즘을 조사했다. TEE, LEE 또는 TEE+LEE를 12개월령 마우스에 9주 동안 경구 투여하였다. TEE 또는 LEE의 단일 투여는 각각 NOR test와 DCX 면역조직화학적염색을 사용하였고 그 결과, 노화된 쥐의 인지 기능과 신경 발생이 증가하는 경향을 확인하였다. TEE + LEE 혼합물을 투여한 후 노화된 쥐의 인지 기능과 신경발생의 상당한 향상이 관찰되었으며, 이는 시너지 효과를 나타냈다. N-methyl-D-aspartate receptor 2B postsynaptic density protein 95, and brain-derived neurotrophic factor (BDNF) levels, and TrkB/extracellular signal-regulated kinase (ERK) phosphorylation의 인산화도 개별 추물물에 비해 TEE + LEE 혼합물 투여 후 상승적으로 증가했습니다. 결론적으로, TEE와 LEE 단독처리와 비교하여 TEE+LEE 혼합물 처리는 BDNF/TrkB/ERK 신호 전달 경로를 통해 노화된 마우스의 인지 기능, 신경 발생 및 시냅스 가소성을 시너지적으로 향상시켰다. 1장에서는 만성 UV 조사가 도파민과 신경염증의 증가 및 BBB 손상을 통해 인지 기능에 대한 부정적인 영향을 조사하였으며, 2장에서는 노화된 쥐의 TEE + LEE 혼합물이 BDNF 관련 신호 전달 경로를 통해 인지 기능 및 신경 발생을 향상시키는 영향에 대해 연구하였다. 이러한 발견은 인지 기능에 영향을 미치는 요인 및 인지 장애의 기저 메커니즘에 대한 우리의 이해에 종합적으로 기여한다. The skin-brain connection represents a complex and dynamic interplay between the body's largest organ, the skin, and intricate neural networks of the brain. This bidirectional communication system plays a pivotal role in translating external stimuli into neurological responses, thereby influencing both the physiological and psychological aspects of brain health. Ultraviolet radiation, a fundamental component of sunlight, acts as a pervasive environmental stimulus that profoundly influences the skin and consequently triggers a cascade of neural responses within the brain. UV exposure of the skin can affect brain functions such as learning and memory, addictive behavior, and hippocampal neurogenesis. These changes are closely associated with hippocampal function, which plays a pivotal role in learning and memory formation. The effects of UV exposure on brain function have been investigated in previous studies, but the specific neurotransmitter-mediated mechanisms responsible for UV radiation-induced cognitive impairment remain elusive. Chapter 1 aimed to elucidate the specific neurotransmitter-mediated mechanisms underlying UV-induced cognitive impairment. Chronic UV irradiation significantly impaired cognitive function and elevated dopamine (DA) levels in both the central nervous system (cortex and hypothalamus) and peripheral organs (skin and adrenal gland) in a mouse model. The DA receptor D1/D5 antagonist, SCH 23390, ameliorated UV-induced hippocampal memory deficits and impaired neurogenesis. Hypothalamic gene analysis showed that UV-irradiated mice had upregulated genes linked to dopaminergic neuron differentiation pathways, whereas post-SCH 23390 treatment downregulated the genes within these pathways. Furthermore, peripheral dopamine injections mimicked UV-induced cognitive alterations, suggesting that dopamine-mediated changes may contribute to cognitive impairment. In addition, transcriptome analysis revealed that significantly downregulated genes in UV-irradiated mice are enriched in neuroimmune-related and BBB-related signaling pathways. Cell-type analysis showed enrichment of differentially expressed genes in microglia, and immunofluorescence imaging confirmed an increased number of Iba1-positive microglia in the hippocampi of UV-irradiated mice. Furthermore, the observation of 70kDa FITC-dextran leakage indicated damage to the blood-brain barrier. These results strongly suggest that chronic UV irradiation induces cognitive dysfunction through mechanisms that involve increased dopamine levels, neuroinflammation, and BBB damage. Aging is a primary risk factor for cognitive impairment and is associated with the accumulation of age-related neuropathologies that affect hippocampal neurogenesis. The decline in brain-derived neurotrophic factor (BDNF), a neurotrophic factor critical for cognitive function, is linked to various cognitive changes associated with aging. Therefore, identifying effective nutrients that can increase BDNF levels may help to maintain and enhance cognitive abilities. In a previous study, we found that tomato and lemon extracts increased BDNF synthesis in the skin, increased BDNF blood concentration, and acted on the brain to affect cognitive function. In Chapter 2, I investigated the effects of tomato and lemon ethanolic extracts (TEE and LEE, respectively) and their possible synergistic effects on the enhancement of cognitive function and neurogenesis in aged mice. The molecular mechanisms underlying the synergistic effects of TEE and LEE were investigated. For the in vivo experiment, TEE, LEE, or their mixture was orally administered to 12-month-old mice for 9 weeks. A single administration of either TEE or LEE improved cognitive function and neurogenesis in aged mice to some extent, as determined by the novel object recognition test and doublecortin immunohistochemical staining, respectively. However, a significant enhancement in cognitive function and neurogenesis in aged mice was observed after the administration of the TEE + LEE mixture, which had a synergistic effect. N-methyl-D-aspartate receptor 2 B postsynaptic density protein 95, brain-derived neurotrophic factor (BDNF) levels, and TrkB/extracellular signal-regulated kinase (ERK) phosphorylation also synergistically increased after administration of the mixture compared with those in the individual treatments. Therefore, compared to their separate treatments, treatment with the TEE+LEE mixture synergistically improved the cognitive function, neurogenesis, and synaptic plasticity in aged mice via the BDNF/TrkB/ERK signaling pathway. In conclusion, Chapter 1 explored the adverse effects of chronic UV irradiation on cognitive function by increasing dopamine levels, neuroinflammation, and BBB damage, and Chapter 2 focused on the potential advantages of a TEE + LEE mixture in enhancing cognitive function and neurogenesis via the BDNF-related signaling pathway in aging mice. These findings collectively contribute to our understanding of the factors that influence cognitive function and the mechanisms underlying cognitive impairment.