Elsevier

DNA Repair

Volume 74, February 2019, Pages 51-62
DNA Repair

Spy1, a unique cell cycle regulator, alters viability in ALS motor neurons and cell lines in response to mutant SOD1-induced DNA damage

https://doi.org/10.1016/j.dnarep.2018.12.005Get rights and content

Highlights

  • Spy1, a unique cell cycle regulator, was decreased in ALS motor neurons.

  • DNA damage response was activated in SOD1G93A-transfected cells.

  • Decreased Spy1 expression activated the DNA damage response in SOD1G93A-transfected cells.

  • Increased Spy1 expression inhibited the DNA damage response in SOD1G93A-transfected cells.

  • Spy1 played a protective role in ALS motor neurons.

Abstract

Increasing evidence indicates that DNA damage and p53 activation play major roles in the pathological process of motor neuron death in amyotrophic lateral sclerosis (ALS). Human SpeedyA1 (Spy1), a member of the Speedy/Ringo family, enhances cell proliferation and promotes tumorigenesis. Further studies have demonstrated that Spy1 promotes cell survival and inhibits DNA damage-induced apoptosis. We showed that the Spy1 expression levels were substantially decreased in ALS motor neurons compared with wild-type controls both in vivo and in vitro by qRT-PCR, western blotting, and Immunoassay tests. In addition, we established that over-expression of human SOD1 mutant G93A led to a decreased expression of Spy1. Furthermore, DNA damage response was activated in SOD1G93A-transfected cells (mSOD1 cells). Moreover, decreased Spy1 expression reduced cell viability and further activated the DNA damage response in mSOD1 cells. In contrast, increased Spy1 expression improved cell viability and inhibited the DNA damage response in mSOD1 cells. These results suggest that Spy1 plays a protective role in ALS motor neurons. Importantly, these findings provide a novel direction for therapeutic options for patients with ALS as well as for trial designs, such as investigating the role of oncogenic proteins in ALS.

Introduction

The maintenance of genomic integrity is a pivotal function for all cells, particularly terminally differentiated cells such as neurons. Repeated DNA damage cannot be avoided during the neuronal lifetime. Thus, neurons have developed numerous overlapping mechanisms to repair damaged DNA [1]. Increasing evidence suggests oxidative DNA damage in amyotrophic lateral sclerosis (ALS) [2], a devastating degenerative motor neuron disease characterized by selective motor neuron degeneration in the brain and spinal cord that leads to progressive muscle weakness, atrophy and paralysis [[3], [4], [5]]. Approximately 90% of ALS cases are sporadic, whereas 10% of patients are familial cases that result from genetic mutations in Cu/Zn superoxide dismutase 1 (SOD1), transactive response (TAR)-DNA binding protein (TARDBP, also referred to as TDP-43), fused in sarcoma (FUS), C9ORF72, or optineurin (OPTN) [6]. Transgenic mouse models that over-express mutant human SOD1 develop an adult-onset paralysis that closely mimics human ALS [7]. Recent studies have established that cellular antioxidant defenses in ALS are insufficient, leading to damaged nucleic acids, proteins and lipids [8].

Several investigators have reported increased oxidative damage to nuclear DNA, measured as the content of 8-hydroxy-2′-deoxyguanosine (8-OHdG), in hSOD1G93A transgenic mouse models [9,10] and ALS patients [[11], [12], [13], [14], [15], [16]]. In addition to oxidative lesions, DNA strand breaks may significantly contribute to the pathology of ALS [[17], [18], [19]]. Accordingly, in SH-SY5Y neuroblastoma cells that over-expressed the mutant G93 A-SOD1 protein, significantly high levels of oxidative DNA damage and DNA strand breaks, increased p53 activity, and a high percentage of apoptotic cells were identified [20]. In vertebrates, two main signaling pathways were activated in response to DNA damage, including the ataxia telangiectasia mutated kinase (ATM)-checkpoint kinase 2 (Chk2) and ataxia telangiectasia and Rad3-related protein kinase (ATR)-checkpoint kinase 1 (Chk1) pathways [21]. Both signaling pathways (ATM-Chk2 and ATR-Chk1 pathways) could activate downstream substrates, including p53 [22,23]. Increased levels of p53 have been identified in ALS patients and animal models [[24], [25], [26]], and p53 triggers apoptosis as a consequence of DNA damage accumulation [27]. Collectively, these data suggested that increased DNA damage in ALS may play an important role in the pathogenesis of ALS. Moreover, several studies have suggested that a persistent DNA damage response is critical for motor neuron survival, and the accumulation of oxidative DNA damage in ALS motor neurons results from impairments in DNA repair activities [28].

Speedy A1 (Spy1), a member of the Speedy/RINGO family, which is a highly conserved family of “cyclin-like” proteins, promotes cell survival and inhibits DNA damage-induced apoptosis [29,30]. Spy1 plays important roles in a broad spectrum of functions, including cell proliferation and cell survival, chromosome decondensation, apoptosis, cell cycle checkpoint activation, tumorigenesis and the DNA damage response [[31], [32], [33], [34]]. Increased Spy1 expression has been implicated in breast cancer [35,36], hepatocellular carcinoma [37], Non-Hodgkin’s Lymphomas [38], multiple myeloma [39], malignant glioma [40,41] and epithelial ovarian cancer [42]. Spy1 was initially presented as a CDK2 activator that enhances cell proliferation [43]. Spy1 was subsequently shown to directly bind to and induce the kinase activity of CDK2 [44].

Further studies have demonstrated that the effect of Spy1 on cell survival was mediated through the suppression of apoptosis by blocking Caspase-3 cleavage in response to UV irradiation [30]. Moreover, Spy1 expression restrains the functional effects of ATR [30], which is also a member of the DNA damage response mediators [45]. In addition, the up-regulation of Spy1 inhibits the activation of checkpoint proteins [30]. Similarly, the association of Spy1 with CDK2 is necessary for its regulation of apoptosis [30]. Subsequent studies have indicated that p53 was required for the anti-apoptotic function of Spy1 [46]. Furthermore, the over-expression of Spy1 as a contributing factor in cancer progression is most likely restricted to p53-positive cells [46].

Together, these results suggest a specific role for Spy1 in the modulation of the DNA damage response. However, the mechanism that underlies the role of Spy1 in the central nervous system response to DNA damage remains unknown. Interestingly, Spy1 is highly expressed in the central nervous system [44,47]. Recent studies have indicated that Spy1 plays a protective effect in terminally differentiated post-mitotic neurons. The results showed that calpain-induced apoptosis through caspase-3 activation following p53 up-regulation was inhibited by the expression of Spy1 in rat hippocampal neurons [48]. However, whether and how Spy1 participates in the pathogenesis of ALS remains unknown. Thus, the aim of the present study was to determine whether Spy1, a critical player in the DNA damage response, affects the survival of motor neurons in ALS. We have determined the expression levels of Spy1 in ALS motor neurons compared with wild-type controls both in vivo and in vitro. We also observe cell viability and DNA damage response by regulating the expression level of Spy1 in ALS motor neurons.

Section snippets

Experimental animals

Transgenic human SOD1G93A mice [7] were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The mice were genotyped using polymerase chain reaction (PCR) amplification of tissue-extracted DNA to identify the presence of the mutant SOD1 as previously described [49]. In order to evaluate the expression of Spy1 in vivo, we used 15 hSOD1G93A-positive mice and 15 age-matched littermate negative mice. Mouse studies were conducted under a protocol approved by the Harbin Medical University

Decreased Spy1 expression was identified in both in vivo and in vitro models of ALS

In the first series of experiments, we determined whether the level of Spy1 had changed between hSOD1G93A-negative and hSOD1G93A-positive mice using immunohistochemistry. Around 130 days of age, the hSOD1G93A-positive mice showed severe body weakness, loss of hind-limb muscle mass, and had high deficit scores of motor dysfunction assessed by five independent behavioral tests (including rotarod performance, postural reflex, balance beam performance, screen grasping and tail suspension behavior)

Discussion

Oxidative DNA damage and p53 activation play major roles in the mechanism of motor neuron death in ALS [63,64]. The atypical CDK activator Spy1 has previously been shown to promote cell survival, inhibit the DNA damage response, and prevent apoptosis [30]; however, its functions in ALS have not been investigated. The current findings indicated that the expression levels of Spy1 were substantially down-regulated in the motor neurons of in vivo mouse models and in vitro cell culture models of ALS

Author contributions

X. Wang and H. Feng designed the study. S. Wang and Y. Wang performed the immunohistological experiments. Y. Yang and T. Wang performed the immunofluorescence cytochemistry analysis. C. Zhang and W. Liang performed the cell viability assessment. X. Wang and X. Yin performed the immunoblotting and immunoprecipitation experiments. D. Shan, J. Zhang and H. Jiang performed cell culture and cell transfection. H. Jiang, G. Dong and Y. Qi performed the qRT-PCR and flow cytometry analyses. X. Wang and

Conflict of interest

The authors disclose no actual or potential conflicts of interest.

Funding

This work was financially supported by grants from the Natural Science Foundation of China (No. 81571227 and No. 81500924) and the Major Program of the Natural Science Foundation of Heilongjiang Province of China (No. ZD201417).

Acknowledgments

The authors would like to thank Professor Sheng-Wang Liu of the Division of Avian Infectious Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences for assistance and guidance in experimental techniques. The authors gratefully acknowledge Dr. Neil Cashman from the University of British Columbia, Canada for providing NSC34 cells and Professor Min Ren of Xuanwu Hospital of Capital Medical University for

References (77)

  • R.F. Gastwirt et al.

    Spy1 expression prevents normal cellular responses to DNA damage: inhibition of apoptosis and checkpoint activation

    J. Biol. Chem.

    (2006)
  • A. Dinarina et al.

    Cell cycle regulation of the mammalian CDK activator RINGO/Speedy A

    FEBS Lett.

    (2009)
  • Q. Ke et al.

    Expression and prognostic role of Spy1 as a novel cell cycle protein in hepatocellular carcinoma

    Exp. Mol. Pathol.

    (2009)
  • D. Lubanska et al.

    The cyclin-like protein Spy1 regulates growth and division characteristics of the CD133+ population in human glioma

    Cancer Cell

    (2014)
  • H.L. Feng et al.

    Combined lithium and valproate treatment delays disease onset, reduces neurological deficits and prolongs survival in an amyotrophic lateral sclerosis mouse model

    Neuroscience

    (2008)
  • H.Q. Jiang et al.

    Guanabenz delays the onset of disease symptoms, extends lifespan, improves motor performance and attenuates motor neuron loss in the SOD1 G93A mouse model of amyotrophic lateral sclerosis

    Neuroscience

    (2014)
  • S.Y. Wang et al.

    Notch pathway is activated in cell culture and mouse models of mutant SOD1-related familial amyotrophic lateral sclerosis, with suppression of its activation as an additional mechanism of neuroprotection for lithium and valproate

    Neuroscience

    (2015)
  • X. Yin et al.

    Downregulated AEG-1 together with inhibited PI3K/Akt pathway is associated with reduced viability of motor neurons in an ALS model

    Mol. Cell. Neurosci.

    (2015)
  • M. Al Sorkhy et al.

    The cyclin-dependent kinase activator, Spy1A, is targeted for degradation by the ubiquitin ligase NEDD4

    J. Biol. Chem.

    (2009)
  • H. Kasai

    Analysis of a form of oxidative DNA damage, 8-hydroxy-2’-deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis

    Mutat. Res.

    (1997)
  • A. Kikuchi et al.

    Systemic increase of oxidative nucleic acid damage in Parkinson’s disease and multiple system atrophy

    Neurobiol. Dis.

    (2002)
  • H. Warita et al.

    Oxidative damage to mitochondrial DNA in spinal motoneurons of transgenic ALS mice

    Brain Res. Mol. Brain Res.

    (2001)
  • S.C. Barber et al.

    Oxidative stress in ALS: a mechanism of neurodegeneration and a therapeutic target

    Biochim. Biophys. Acta

    (2006)
  • R. Lopez-Gonzalez et al.

    Poly(GR) in C9ORF72-Related ALS/FTD compromises mitochondrial function and increases oxidative stress and DNA damage in iPSC-Derived motor neurons

    Neuron

    (2016)
  • F. Coppede

    An overview of DNA repair in amyotrophic lateral sclerosis

    Sci. World J.

    (2011)
  • N. Santamaria et al.

    Intrinsic disorder in proteins involved in amyotrophic lateral sclerosis

    Cell. Mol. Life Sci.

    (2016)
  • J. Sreedharan et al.

    Amyotrophic lateral sclerosis: problems and prospects

    Ann. Neurol.

    (2013)
  • J.P. Taylor et al.

    Decoding ALS: from genes to mechanism

    Nature

    (2016)
  • M.E. Gurney et al.

    Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation

    Science

    (1994)
  • H.N. Noristani et al.

    Brca1 is expressed in human microglia and is dysregulated in human and animal model of ALS

    Mol. Neurodegener.

    (2015)
  • N. Aguirre et al.

    Increased oxidative damage to DNA in an animal model of amyotrophic lateral sclerosis

    Free Radic. Res.

    (2005)
  • D.J. Amante et al.

    Uridine ameliorates the pathological phenotype in transgenic G93A-ALS mice

    Amyotroph. Lateral Scler.

    (2010)
  • H. Blasco et al.

    Panel of oxidative stress and inflammatory biomarkers in ALS: a pilot study

    Can. J. Neurol. Sci.

    (2017)
  • R.J. Ferrante et al.

    Evidence of increased oxidative damage in both sporadic and familial amyotrophic lateral sclerosis

    J. Neurochem.

    (1997)
  • H. Mitsumoto et al.

    Oxidative stress biomarkers in sporadic ALS

    Amyotroph. Lateral Scler.

    (2008)
  • T. Murata et al.

    Increased mitochondrial oxidative damage and oxidative DNA damage contributes to the neurodegenerative process in sporadic amyotrophic lateral sclerosis

    Free Radic. Res.

    (2008)
  • N. Shibata et al.

    Nonoxidative protein glycation is implicated in familial amyotrophic lateral sclerosis with superoxide dismutase-1 mutation

    Acta Neuropathol.

    (2000)
  • L.J. Martin

    Transgenic mice with human mutant genes causing Parkinson’s disease and amyotrophic lateral sclerosis provide common insight into mechanisms of motor neuron selective vulnerability to degeneration

    Rev. Neurosci.

    (2007)
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