[보고서]신경손상 후 Progranulin에 매개되는 신경 사멸 및 기능적 회복기전연구

임희영

신경손상 후 Progranulin에 매개되는 신경 사멸 및 기능적 회복기전연구
The mechanism of progranulin-mediated neuronal cell death and functional recovery to damage 원문보기

보고서 정보
주관연구기관	한림대학교 HalLym University
연구책임자	임희영
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2017-12
과제시작연도	2017
주관부처	과학기술정보통신부 Ministry of Science and ICT
등록번호	TRKO202200002471
과제고유번호	1711053182
사업명	개인기초연구(미래부)
DB 구축일자	2022-06-11
키워드	프로그라눌린.그라눌린 펩타이드.신경세포사멸.신경 손상.만성통증.신경 재생.Progranulin.Granulin peptide.Autophagy.Nerve injury.Neuropathic pain.Neurodegeneration.

초록 ▼

□ 연구의 목적 및 내용
Progranulin(PGRN)은 분비성 성장인자인자 다양한 생물학적 과정에 다기능성 단백질로서, 2006년에 유전적 돌연변이가 전두측두엽성 치매(frontotemporal dementia; FTD)를 유발한다고 알려져 그 역할에 대해 주목받고 있다. 활성화된 소신경교세포 및 신경세포에서 PGRN 발현이 보고되어 이는 PGRN이 신경질환 발병기전에 관여한다는 것을 의미하고 있다. 따라서 본 연구에서는 다음과 같은 내용을 규명하고자 하였다..
• in vivo 및 in vitro 신경 손상 후 진행되는 이차적 신경손상을 방지/감쇄하고, 손상된 신경세포 사멸기전과 축삭회복기전과 같은 적응과정에서 PGRN 혹은 granulin peptide들의 생리학적 역할 및 분자적 조절기능 규명
• 비정상적인 특정 단백질의 응집과 축척으로 인해 야기되는 신경 퇴화 및 손상, 그리고 신경 변성으로 야기되는 만성 통증 및 신경퇴행성 질환의 치료 전략연구에 활용할 수 있는 과학적 자료 제시 및 치료표적인자로서의 가치 검증

□ 연구결과
○ 1차년도: 신경세포의 유도성 사멸기작에서 PGRN의 생리학적 역할 규명
⦁ Viral vector system을 이용한 PGRN 과발현 혹은 감쇄발현된 신경세포주 확립
: Lentiviral과 adeno-associated viral vector를 이용한 유전자 조절 전달체 구축
⦁ Rapamycin, 1% Hypoxia, nutrient 결핍에 따른 신경세포사멸시 PGRN과 Autophagy 관련인자들간 상호연관성 확인 및 분석
⦁ 말초신경 손상 모델(신경병증성 통증모델)의 구축 및 확립
: Spared Nerve Injury(SNI)와 Crush Injury(CI) 모델
○ 2차년도: 말초신경손상 모델에서 PGRN 매개성 신경생성 및 기능회복기전 분석연구
⦁ PGRN 결핍 마우스의 SNI를 통한 신경손상 유도 후 세포사멸과정에서 PGRN 효과 확인
⦁ 말초신경 손상모델에서 PGRN 결핍에 따른 행동학 실험을 통한 기능 회복기전 분석
⦁ Crush injury 모델에서 PGRN 과발현시 Autophagic protein 발현량 측정 및 신경재생 측정을 통한 PGRN의 신경보호효과 분석

□ 연구결과의 활용계획
Progranulin의 발현조절을 이용한 신경세포모델 및 신경손상 동물모델을 이용한 본 연구는
• 목적 단백질 발현량 조절을 위한 Viral delivery system을 제작, 일차신경세포에 적용 가능성을 확인하고, 다양한 신경 질환모델에 적용가능한 in vitro system을 구축함.
• 만성 신경질환에서 수반되는 신경세포 사멸 및 기능적 회복 기작에서의 PGRN 혹은 GRN의 생리학적 역할 및 분자적 기능을 규명하여 신경손상을 포함한 다양한 신경퇴행성 질환의 임상연구를 위한 중요한 기초자료를 제공할 수 있음
• 만성 신경퇴화질병의 잠재적인 치료표적 단백질로서의 가능성을 타진할 수 있음.
• 신경계에 관련된 질병의 병리학적 기작 연구를 지속적으로 촉발하게 될 것임.

(출처 : 한글요약문 4p)

Abstract ▼

□ Purpose & contents
Progranulin(PGRN) is a cystein-rich highly glycosylated multifunctional protein that is involved in various biological processes, wound response, inflammation, and tumorigenesis. It has attracted a considerable attention since loss-offunction mutations of PGRN were found to cause familial ubiquitin-positive frontotemporal dementia in 2006. Although its expression is reported in neurons and activated microglia, the physiological functions of PGRN or granulin peptides in chronic neuronal disorders are still not well-understood. Thus this study is aimed to clarify the molecular functions of PGRN or granulin fragments in the adaptation process of nociceptive system, including on neuron death pathway for misfolding protein clearance and functional recovery, after nerve injury using in vivo and in vitro system. It will be provided the scientific data that may contribute to the development of therapeutic strategies against chronic neurodegenerative diseases and verify it as a potential therapeutic target.

□ Result
■ Investigation of PGRN functions in induced cell death of neuronal cell
⦁ Establishment of stable neuronal cells overexpressing PGRN or downregulated by shRNA against Grn mediated viral delivery system
: construction of lenti- or adeno-associated viral vector system
⦁ Examination of interaction between PGRN and autophagic-related proteins (LC3, p62, ATG5 and 4b) in neuronal cells under the various stimulation
: stimulation by rapamycin treatment, nutrient withdrawal, 1% Hypoxia,
⦁ Construction and establishment of peripheral nerve injury model(in vivo)
: Spared Nerve Injury and Crush Injury model as neuropathic pain mode
■ Analsysis of PGRN-mediated functional recovery and nerve regeneration in nerve injury model
⦁ Confirmation of PGRN effect on the neuronal cell death in SNI-operated PGRN knockout mice(PGRN deficient mice)
⦁ Analysis of functional recovery in SNI-operated PGRN knockout mice
⦁ Assessment of PGRN functions in PGRN-overexpressing primery neurons after nerve injury by Crush Injury

□ Expected Contribution
In this funtional study of PGRN in neuronal cell and neuropathic pain model have provided next points:
• A viral delivery system to regulated the epxression of target protein(PGRN) was constructed and employed in vitro system, resulting in its applicability to primary neurons was confirmed.
• The physiological role and functions of PGRN in neuronal cell death, autophagy and functional recovery after nerve injury in chronic neuro-disease were verified. It could be useful scientific data for clinical study of various neurodegenerative diseases.
• The probability of PGRN as a therapeutic target for nerve injury-evoked chronic pain can be explored.

(source: SUMMARY 5p)

목차 Contents

표지 ... 1
목차 ... 2
연구계획 요약문 ... 3
연구결과 요약문 ... 4
한글요약문 ... 4
SUMMARY ... 5
연구내용 및 결과 ... 6
1. 연구개발과제의 개요 ... 6
2. 연구수행 내용 및 결과 ... 6
3. 목표달성도 및 관련분야에의 기여도 ... 21
4. 연구결과의 활용계획 ... 22
5. 주관연구책임자 대표적 연구실적 ... 23
6. 참고문헌 ... 23
7. 연구성과 ... 24
8. 국가과학기술지식정보서비스에 등록한 연구시설‧장비 현황 ... 24
9. 기타사항 ... 24
끝페이지 ... 24

표/그림 (18)

표 Regulation of progranulin expression mediated by lentiviral particles in neuronal cell lines in vitro. (A) Analysis of GFP expression by flow cytometry in SH-SY5Y neuroblastoma cells infected with lentivirus expressing mouse PGRN under neuron-specific promotor or shRNA against GRN. Transduction efficiency was > 90% on average. (B-C) Progranulin mRNA and protein expression in F11 hybridoma cells and SH-SY5Y transduced with control pLL-3.7, or pSyn-PGRN overexpressing viral paticles, or shGrn-LL3.7 progranulin silencing viral particles. β-actin and Hsp90 were used for normalization
표 Schematic gram of PGRN or GRN fragments
표 Survival of F11 hybridoma and SH-SY5Y neuroblastoma cells transduced with silencing (shGrn-LL3.7) and overexpressing (pUm-mGrn-lox3.7) progranulin lentiviral vectors or control viruses (pLL-3.7). (A) Viability of F11 hybridoma cells after transduction of three different shGrn-LL3.7 progranulin silencing vectors assessed per WST viability assay. Asterisks indicate significant differences versus control virus, P < 0.05. (B-C) FACs analysis of cell death in SH-SY5Y neuroblastoma cells after transduction with each lentiviruses; silencing and overexpressing progranulin or control viruses (pLL-3.7). Cells were starved for 72 hours in FCS-free medium. Early apoptotic cells were identified via annexin-V (lower right), dead cells via DNA intercalation of 7-AAD(upper left) and late apoptotic cells were double positive (upper right). For comparison results are summarized in a stacked bar graph(B)
표 Identified autophagic cell death-related proteins in Y2H screening
표 Optimized conditions for induction of autophagy in vitro
표 Coimmunoprecipitation of progranulin with autophagy proteins. A: Immunoprecipitation (IP) with anti-progranulin and subsequent immunoblot analysis of ATG12 in SH-SY5Y neuroblastoma cells without (SH-SY5Y) and with progranulin overexpression (SH-SY5Y-Grn) without/with rapamycin stimulation. B, C, D: IP of progranulin and immunoblot for ATG4b, LC3b and p62/Sqstm1, respectively. E: Immunoprecipitation of p62/Sqstm1 in SH-SY5Y cells and subsequent immunoblot of progranulin. Representative results of at least 3 experiments each
표 Correlation of granulin fragments with autophagy proteins in vitro. Immunoprecipitation (IP) with anti-progranulin and subsequent immunoblot analysis of autophagic-related proteins in SH-SY5Y neuroblastoma cells with progranulin, G-F fragment, C-D-E fragment, and F-B-A fragments overexpression without/with rapamycin stimulation. From top to bottom panel represented IP of progranulin and immunoblot for ATG5, LC3b and p62/Sqstm1, respectively. Representative results of at least 3 experiments each
표 PGRN supports neurite outgrowth from primary adult DRG neurons in vitro. (A) Representative images demonstrate the morphology of neurons transduced with lentivirus particles expressing pUmGrn-lox3.7 (PGRN overexpression), shGrnpLL3.7c (PGRN silencing) or empty vector pLL-3.7. Neurons were immunostained with anti-NF200. (B) Quantitative analysis of the length and number of neurites and the area of the neurites. Only EGFP positive neurons extending neurites of > 2 fold length of the neuronal soma diameter were analyzed. The results are shown as box plots where the line indicates the median, the box the interquartile range, whiskers 5-95th percentile and open dots individual outliers (n = 15-19 neurons per group). *P <0.05, ANOVA with subsequent Bonferroni t-tests
표 Immunofluorescence analysis of the subcellular localization of PGRN and colocalization with autophagosomes in primary sensory neurons. Neurons were transduced with Grn-EGFP fusion construct and detected by anti-EGFP. ATG12, p62/Sqstm1, ATG4b and LC3b identify autophagosomes at different stages. Images are examples of 10-15 neurons of 3-4 different cultures. Scale bar 10μm
표 Immunofluorescence analysis of the subcellular localization of progranulin relative to early endosome (EEA1), Golgi structures (GM130) and the lysosome (LAMP1) in primary DRG neurons of adult mice. Sortilin is integrated into the plasma membrane or localized in early endosomes and was previously found to drag PGRN to multivesicular bodies. Neurons were transduced with Grn-EGFP fusion construct and detected by anti-EGFP immunofluorescence. Progranulin was not found in colocalization with sortilin, EEA1 or GM130 and weakly with LAMP1 after rapamycin stimulation. Scale bar 10μm
표 Survival and autophagy in primary sensory neurons of C57BL6(controls), shGrn-LL3.7 (inactivation) and neuron-specific Grn-overexpression. (A) Survival of naïve DRG neurons depending on Grn expressing level and (B) survival of neurons exposed to 1 μM rapamycin for 48h assessed by counting viable cells. The box represents the interquartile range, the line is the median and whiskers show 5th-95th confidence intervals. Four visual fields were analyzed per culture of each 10 cultures per genotype. (* P < 0.05, *** P < 0.001 (ANOVA)). (C) Immunofluorescence analysis of LC3b immunoreactive autophagosomes in DRG neurons of adult C57BL6(control, CNT), SNS-Grn-OE and Grn-/- mice exposed to different stress conditions. Cells were stimulated with rapamycin (1 μM 48h), hypoxia (1% oxygen for 24h) or glutamine withdrawal for 24 h. Images show representative images of surviving neurons. The experiment was replicated 3-times. Scale bar 10 μm. Four visual fields were counted per culture of each 4 cultures per genotype. Asterisks indicate significant differences *P < 0.05
표 Nerve injury models. In the Spared Nerve Injury (SNI) model two branches of the sciatic nerve, the tibialis and peronaeus, are ligated and cut distally. The third branch, the suralis nerve, remains intact. The model produces robust neuropathic pain like behavior. In the Crush-Model the sciatic nerve is crushed for 25 seconds with blunt forceps. Myelin sheaths remain intact allowing reinnervation of skin and muscles. Sensory and motor functions recover over time. Neuromuscular junctions rebuild within 4-6 weeks. Enhanced nociceptive sensitivity outlasts the peripheral nerve regeneration
표 Expression of PGRN in primary sensory DRG neurons and microglia cells. (A) Co-staining of PGRN (red) and microglia activation marker Iba-1 (green) in the ventral horn of injured mice. (B) In situ hybridization of PGRN mRNA in naïve and co-immunostaining of neuronal specific markers. Unmyelinated neurons were detected with isolectin B4-FITC (IB4) and glutameteric fiber neurons were identified by immunostaining with anti-CGRP. Representative images of n=5 mice. Scale bars: 50μm(A) and 100μm(B)
표 Up-regulation of PGRN expression in spinal cord (SC) and dorsal root ganglia (DRG) after nerve injury. (A) Time course of mRNA levels of PGRN in the L4/L5 DRGs and the L4/5 dorsal horn of the lumbar spinal cord ipsilateral to a sciatic nerve lesion in the SNI model by QRT-PCR (n=3 per group). (B) Western blot analysis of PGRN in mouse SC and DRGs (n=6/ time point) using anti-PGRN (N19); densitometric analysis is shown on the right panel. (C) In situ hybridization of PGRN mRNA in naïve(upper) and injured(lower) mice. Unmyelinated neurons were detected with isolectin B4-FITC (IB4, green) and injured neurons were identified by immunostaining with anti-ATF-3(red). Representative images of n=3 per group(Scale bars: 100μm(left panel), 50μm(right three panel))
표 Autophagy in primary neurons with progranulin deficiency. Primary neuronal cultures were treated with vehicle or rapamycin and analyzed by Western blot and immunostaining. (A) Western Blot of LC3b-I/II in primary E21 cortical neurons of Grn−/− and WT mice(Grn+/+) without/with rapamycin stimulation (1μM for 6h) on day 7 of the culture. (B) Repetition of the experiment shown in (A), with viral-mediated PGRN overexpressing WT neurons. The box plots show quantitative results, the line is the median, boxes show the interquartile range and whiskers minimum to maximum. Results are from the litter of 3 mothers per genotype.(*P<0.05 as indicated, one-way ANOVA and posthoc Bonferroni). (C) Representative image of CytoID® positive neurons and of neurons with typical apoptotic nuclei. Four images were captured per culture to cover N90% of the neurons and images were analyzed with AxioVision (Zeiss) of each 4 cultures per genotype. Asterisks indicate significant differences *P<0.05
표 Late onset of hypernociception and impaired motor recovery in PGRN-deficient mice. Progranulin-deficient (Grn/) mice showed stronger nociceptive sensitivity after nerve injury than wild-type mice. PGRN knockout (Grn/) mice and their wild-type control mice (n=8 per group, four male, four female, 10~12 weeks at the time of surgery) were tested for their mechanical allodynia (A), cold allodynia (B), heat hyperalgesia (C) and motor functions (D) as in Figure 12. Data are means S.E.M. Comparison of the results with ANOVA revealed statistically significant differences between Grn/and wild-type mice for the percentage change of mechanical, cold and heat nociception and for the RotaRod running time as indicated with asterisks (P 0.05)
표 Autophagy after nerve injury and its impact on neuropathic pain. (A) Immunofluorescence analysis of LC3b-EGFP on time course in the DRGs transduced with mPGRN-expressing virus after crush injury of the sciatic nerve. The contralateral side was used as control. Images are presented as blue-to-yellow pseudocolor. Scale bars 50μm, (B) Quantification of calcium flux along the nerve proximal of the lesion in primary sensory neurons transduced with control or PGRN-overexpressing virus. (mean ± SD of n= 3 mice per group)
표 PGRN-associated nerve regeneration after nerve injury

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연합인증

신경손상 후 Progranulin에 매개되는 신경 사멸 및 기능적 회복기전연구
The mechanism of progranulin-mediated neuronal cell death and functional recovery to damage 원문보기