NEW INSIGHTS INTO THE ROLE OF PROGERIN IN CELLULAR PATHOLOGY OF HUTCHINSONGILFORD PROGERIA SYNDROME (HGPS)

Hutchinson-Gilford Progeria Syndrome (HGPS) is a very rare disease
in children showing rapid aging. The average life expectancy of HGPS
patients is 14.5 years and cardiovascular disease is the major cause of the
death. In 2003, a study found that most HGPS patients carry a single base
mutation (1824 C > T) in the exon 11 of LMNA gene that encode A-type
lamins. A-type lamins are one of major components of the nuclear lamina.
In most cases of HGPS, the 1824 C > T mutation in the LMNA gene induces
a cryptic splicing event, resulting in an alternative mRNA slicing isoform
that is translated into a mutant lamin A known as progerin. Progerin is
characterized by a farnesyl group attached at its carboxy terminus, which in
wild-type lamin A is removed by the metalloproteinase ZmpSte24. Because
of the alternative mRNA splicing, progerin lacks 55 amino acid residues at
the C-terminal region, which contains the recognition site of ZmpSte24.
Therefore, progerin is permanently farnesylated and seems to have dominant
negative effects based on current understanding of HGPS.
There is increasing evidence suggesting that permanent farnesylation of
lamin A is associated with pathological symptoms of HGPS. However, how
that progerin functions to cause pathological phenomena at the cellular level
linking to the rapid senescence phenotypes at the overall organismal level is
unknown yet so far. Therefore, it is significant to investigate the molecular
and cellular mechanisms that underline the pathological symptoms of
progerin-driven HGPS. Moreover, studies of progeria may provide a better
understanding of mechanisms driving natural aging processes.
This thesis is to study cellular pathology of HGPS in a cell model.
Using CRIPSR/Cas9-mediated target base editing approach, I have
successfully generated a progerin knock-in cell line in HeLa cells. Next, I
focus on studying the cytokinesis and DNA damage response of progerin
knock-in cells as compared to wild-type HeLa cells. Based on quantitative
analysis of live-cell imaging and immunofluorescence staining of D-tubulin,a cytokinesis marker, I conclude that progerin knock-in cells exhibit
increased cytokinesis arrest and cytokinesis failure, leading to an increase
in polyploidy cells. To further explore the underlying mechanism of
cytokinesis failure, I examined nuclear pore complex (NPC) via
immunofluorescence staining of Nup153, a subunit of NPC. The NPC153
staining appears normal in progerin knock-in cells compared to wild-type
cells. To investigate sensitivity of cells to DNA damage, I have used γ -
radiation to induce DNA double strand break (DSB). Based on quantitative
analysis of γ-H2AX that marks DSB, I found that in progerin knock-in cells,
sensitivity to DNA damage appears increased while DNA repair seems to be
impaired.
Taken all together, in this thesis, I report for the first time that progerin
knock-in cells exhibit defective cytokinesis, which could result in increases
in gene dosage in the cell. In addition, consistent with previous report, I
also found that progerin leads to increases in sensitivity to DNA damage
and impaired DNA repair. These data suggest that progerin impacts on not
only nuclear lamina structure but also other cellular processes such as
cytokinesis as well as DNA damage response. Further studies are required to
provide mechanistic understanding of progerin functions in HGPS.
 

早衰症（ Hutchinson-Gilford Progeria Syndrome, HGPS） 是一种非常
罕见的儿童快速衰老的疾病，患者平均寿命 14.5 岁，由快速衰老引起的心
血管疾病是主要的致死原因。 2003 年，通过基因测序研究发现，大多数早
衰症患者的 LMNA 基因发生了单碱基突变。 LMNA 基因编码 A 型核纤层蛋
白（ lamin A）， A、 B 型核纤层蛋白均是组成核纤层的主要成分。多数早
衰症病例显示，患者 LMNA 基因位于第 11 个外显子的 1824 位点的碱基由
胞嘧啶（ C）突变为胸腺嘧啶（ T）。虽然这个点突变并未导致氨基酸的改
变，但是却引入了一个隐蔽剪切位点，导致产生一个新的 mRNA 剪切体。
该 mRNA 剪切体与正常剪切体相比， 3’-端缺失 150 个碱基，翻译后蛋白 C-
末端进而缺失 50 个氨基酸。 A 型核纤层蛋白的翻译后法尼基化修饰，以及
随后由金属蛋白酶 ZmpSte24 进行的法尼基团的剪切对于其发挥正常功能十
分重要。由于 ZmpSte24 的识别位点位于 A 型核纤层蛋白 C-末端，上述突
变导致缺失的 50 个氨基酸中即含有金属蛋白酶 ZmpSte24 的识别位点，因
此，突变蛋白（也被称为早衰蛋白） （ progerin） 的特点是永久法尼基化。
在早衰症病人体内，早衰蛋白具有显性的负调节作用（ dominant negative） 。
虽然永久法尼基化的早衰蛋白早已认为是早衰症的致病因子之一，但它
在细胞水平上的作用如何与生物整体水平上的快速衰老表型相关，迄今仍是
不解之谜， 关于早衰症致病机制的研究也不十分清楚。此外，对于早衰症的
研究在一定程度上也可以帮助提升对人类自然衰老的认识。根据我们和其他
实验室的前期研究，我们推测早衰蛋白可能通过影响 DNA 损伤修复，并最
终导致基因组不稳定性。本论文旨在构建早衰蛋白敲入（ knock-in）细胞系，
通过对该细胞系的研究，阐明早衰蛋白的细胞学作用和影响，从而促进早衰
症中细胞、 分子病理机制的认识。
关于来源于早衰症患者的细胞、过表达早衰蛋白的正常细胞，以及敲低
或敲除金属蛋白酶 ZmpSte24 的细胞的研究表明，这些细胞最明显的特征之
一是细胞核形态异常，细胞核呈现许多分叶（ blebbing） 。本实验室的前期
研究发现，敲除金属蛋白酶 ZmpSte24（ ZmpSte24-/-） 的小鼠胚胎成纤维细
胞（ MEF） 中包含了大量的多核细胞（ polyploidy cell），而且绝大多数
ZmpSte24-/-细胞都无法完成正常的胞质分裂（ cytokinesis）。在 HeLa 细胞
中敲低 ZmpSte24 后，也观察到细胞胞质分裂失败（ cytokinesis failure） 或
者胞质分裂阻滞（ cytokinesis arrest） ,并伴随细胞凋亡增加的现象。本论文通过 CRISPR/Cas9 介导的单碱基修饰技术，将位于 1824 位点
的碱基由胞嘧啶（ C） 突变为胸腺嘧啶（ T）的敲入，从而构建了表达早衰
蛋白的细胞系。通过转染带有 GFP 标签的 CRISPR、 Cas9 质粒， A549、
AGS、 HeLa 三种细胞系经流式细胞分选将转染之后带有 GFP 荧光单细胞进
行分选，并于 96 孔板中分别培养单克隆细胞，待细胞增殖后，分别对单克
隆细胞株进行 DNA 测序分析和细胞保种。 DNA 测序结果显示，一株 HeLa
细胞带有预期的 LMNA 基因 1824 位点的点突变，提示早衰蛋白敲入的
HeLa 细胞构建成功。 蛋白质水平上，该细胞系中能检测到早衰蛋白的表达，
而在野生型 HeLa 细胞中则检测不到的早衰蛋白的表达。从而进一步确定了
该细胞系构建成功，该细胞系称为早衰蛋白敲入细胞。
基于上述实验室的前期研究结果，我首先研究了早衰蛋白敲入细胞的胞
质分裂过程。通过免疫荧光染色、激光共聚焦荧光显微镜（ Laser scanning
confocal microscope ） 成 像 ， 我 分 析 了 微 管 蛋 白 D 亚 基 标 记 的 中 间 体
（ midbody），以及细胞核。通过分析中间体和多核细胞数目（ polyploidy
cell）推测早衰蛋白对胞质分裂的影响。结果表明，早衰蛋白敲入细胞呈现
明显增多的多核细胞（ polyploidy cell）。经过三次独立的实验，计算多核
细胞占总细胞数目的比例的定量分析结果表明早衰蛋白敲入细胞中多核细胞
增多的现象具有统计差异 （ n = 3, *p< 0.05）。这一结果提示，多核细胞
的增多可能是由于细胞分裂失败所致。此外，微管蛋白D亚基标记的中间体
（ midbody） 作为胞质分裂后期的标志，同样也出现了在早衰蛋白敲入细胞
中明显增多的现象。经过三次独立实验，定量计算含有中间体结构的细胞占
总细胞数目的比例，结果也显示，早衰蛋白敲入细胞中多数细胞处于胞质分
裂后期（ n = 3, *p < 0.05）。
同时，通过使用时间推移活细胞成像（ Time-lapse Live-cell Imaging）
技术，细胞分裂实况动态得以捕捉和记录。对活细胞成像的分析表明，早衰
蛋白敲入细胞中，两个子细胞融合成为一个双核细胞的现象，即胞质分裂失
败（ cytokinesis failure）明显增加，同时还伴随着双核/多核细胞的细胞凋
亡。经过三次独立的活细胞成像实验，对胞质分裂失败的细胞进行了统计，
结果显示，与野生型 HeLa 细胞相比较，早衰蛋白敲入细胞具有显著的胞质
分裂失败现象 （ n = 3, *p < 0.05）。
基于多篇文献的报道，来源于早衰症病人的细胞与正常细胞相比较，存
在 DNA 修复损伤的滞后。在经过相同辐照诱导的 DNA 双链断裂情况下，
HGPS 病人细胞以及敲除金属蛋白酶 ZmpSte24 的 MEF（ ZmpSte24-/-）均
显示的 DNA 损伤修复明显慢于对照组细胞[1][1][1]。也有研究表明,早衰蛋白是影响 DNA 损伤反应的显性负效应物（ dominant-negative effector） [2]。
DNA 双链断裂是最严重的 DNA 损伤形式，γ -H2AX 可作为 DNA 双链断
裂的标志。在本论文中，我也通过辐照诱导的 DNA 双链断裂，以及γ -
H2AX 染色，分析研究了早衰蛋白敲入细胞中的 DNA 损伤情况，从而评估
早衰蛋白对于细胞对 DNA 损伤的敏感性的影响。γ -H2AX 标记的细胞的
DNA 损伤情况分别在 1Gy 的γ -射线辐射后的 3 个时间点，即 10 分钟、 6
小时、 12 小时，进行了分析。结果显示，γ射线辐射后 10 分钟，早衰蛋白
敲入细胞的细胞核内γ -H2AX 信号的数量和荧光强度均高于野生型 HeLa
细胞，表明早衰蛋白敲入细胞对 DNA 损伤可能比野生型细胞更加敏感。射
线辐射后 6 小时以及 12 小时，野生型细胞和早衰蛋白敲入细胞中的γ -
H2AX 信号均显着降低，表明这两种细胞中都已进行了有效的 DNA 损伤修
复。 然而，与野生型细胞相比，早衰蛋白敲入细胞仍然有更高的γ -H2AX
信号，这一结果表明在早衰蛋白敲入细胞中 DNA 的损伤修复可能被减弱。
综上所述，本论文构建早衰蛋白敲入细胞系，并通过对该细胞系的研究，
首次发现早衰蛋白可能影响胞质分裂，导致胞质分裂失败，进而可能导致基
因剂量（ gene dosage）的增加。此外， 早衰蛋白可能增加细胞对 DNA 损伤
的敏感性，并且可能导致 DNA 损伤修复滞后。这些结果表明， 早衰蛋白不
仅参与核纤层结构，还影响其他细胞过程，包括胞质分裂和 DNA 损伤反应。
阐明早衰蛋白的细胞学作用和影响，尚有待进一步研究以促进对早衰症中早
衰蛋白功能的认识。
 

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