Neuronal gene repression in Niemann–Pick type C models is mediated by the c-Abl/HDAC2 signaling pathway

https://doi.org/10.1016/j.bbagrm.2015.11.006Get rights and content

Highlights

  • HDAC2 levels are increased in NPC disease models.

  • c-Abl inhibition prevents the increase of HDAC2 in NPC disease models.

  • Cholesterol and oxidative stress are upstream regulators of the c-Abl/HDAC2 pathway.

  • c-Abl inhibition prevents the HDAC2-dependent gene repression in NPC models.

Abstract

Background

Niemann–Pick type C (NPC) disease is a fatal neurodegenerative disorder characterized by the accumulation of free cholesterol in lysosomes. There are currently no effective FDA-approved treatments for NPC, although in the last years the inhibition of histone deacetylases (HDACs) has emerged as a potential treatment for this disease. However, the molecular mechanisms that deregulate HDAC activity in NPC disease are unknown. Previously our group had shown that the proapoptotic tyrosine kinase c-Abl signaling is activated in NPC neurons. Here, we demonstrate that c-Abl activity increases HDAC2 levels inducing neuronal gene repression of key synaptic genes in NPC models.

Results

Our data show that: i) HDAC2 levels and activity are increased in NPC neuronal models and in Npc1−/− mice; ii) inhibition of c-Abl or c-Abl deficiency prevents the increase of HDAC2 protein levels and activity in NPC neuronal models; iii) c-Abl inhibition decreases the levels of HDAC2 tyrosine phosphorylation; iv) treatment with methyl-β-cyclodextrin and vitamin E decreases the activation of the c-Abl/HDAC2 pathway in NPC neurons; v) in vivo treatment with two c-Abl inhibitors prevents the increase of HDAC2 protein levels in the brain of Npc1−/− mice; and vi) c-Abl inhibition prevents HDAC2 recruitment to the promoter of neuronal genes, triggering an increase in their expression.

Conclusion

Our data show the involvement of the c-Abl/HDAC2 signaling pathway in the regulation of neuronal gene expression in NPC neuronal models. Thus, inhibition of c-Abl could be a pharmacological target for preventing the deleterious effects of increased HDAC2 levels in NPC disease.

Introduction

Neuronal gene repression has been extensively described in several neurodegenerative diseases [1]. Specifically, histone deacetylation plays a fundamental role in neuronal gene repression in neurodegenerative processes [2], [3], [4]. HDACs are a family of enzymes responsible for the regulation of histone deacetylation, which promote greater chromatin compaction making it inaccessible to the transcriptional machinery [5], [6]. HDACs are grouped into four classes and have been implicated in diverse biological processes including cellular function, differentiation, development, apoptosis, and synaptogenesis [7]. However, the roles of individual HDACs in brain function have only been addressed recently [4], [8], [9]. HDAC1, HDAC2, HDAC3 and HDAC8 belong to class I HDACs. Interestingly, HDAC2 is a master regulator of histone deacetylation and gene repression of neuronal genes [1], [3], [4], [10], [11]. Surprisingly, HDAC2 is predominantly expressed in neurons of the adult brain compared to other HDACs of the same class, such as HDAC1, which is expressed in neural stem cells/progenitors and glia [12]. HDAC2 is preferentially recruited on the promoters of neuronal genes triggering neuronal gene repression, cognitive problems and a decrease of synaptic plasticity in the adult brain [4].

Niemann–Pick type C (NPC) disease is a fatal pediatric neurodegenerative lysosomal storage disorder caused by mutations in the NPC1 or NPC2 genes [13], [14]. Both genes encode cholesterol transport proteins and their deficiency leads to the pathogenic accumulation of cholesterol and other lipids (lactosylceramide, glucosylceramide, GM2 and GM3 glycosphingolipids and sphingosine) within the late endosomal/lysosomal compartment [15], [16], [17], [18]. NPC is a systemic disorder characterized primarily by neurodegeneration, especially in the cerebellum and brain cortex, and liver damage [19]. Although the etiology of NPC has been already elucidated, there is no curative treatment for this devastating and fatal disorder yet. Nevertheless, Miglustat (N-butyldeoxynojirimycin; NB-DNJ; Zavesca®, Actelion Pharmaceuticals Ltd.) is currently approved in the European Union (EU), for the treatment of progressive neurological manifestations in patients with NPC [19], [20]. In addition, the cholesterol lowering agent hydroxypropyl-β-cyclodextrin (HP-β-CD) is undergoing a NIH clinical trial in NPC patients [20].

Interestingly, treatment with the class I HDAC inhibitor valproic acid prevents reduction in the expression of neuronal genes in NPC cellular models [21]. In addition, treatment with HDAC inhibitors of classes I and II reduces cholesterol accumulation in NPC human fibroblasts [22]. Based on this evidence, HDAC inhibition has emerged as a potential therapeutic treatment for NPC disease [20], [23], [24]. Although what HDACs are deregulated in NPC is currently unknown, the evidence suggests that a HDAC class I inhibitor could be a useful treatment to prevent neuronal gene repression and cognitive impairment. As previously mentioned, HDAC2 is the main HDAC I expressed in the adult brain. Moreover, HDAC2-overexpressing mice show reduced expression of synaptophysin, decreased synaptic plasticity, fewer dendritic spines and cognitive impairments [4]. In contrast, Hdac2 knockout mice show increased expression of synaptophysin, augmented synaptic plasticity and a higher number of dendritic spines in the CA-1 region, associated with improvement in associative learning compared to wild-type mice [4], [25]. In addition, HDAC2 has been described as a key protein involved in neuronal gene repression in neurodegenerative diseases [3], [4], [10]. Indeed, HDAC2 is increased in mouse models of Alzheimer's disease (AD) and samples from AD patients, whereas HDAC1 or HDAC3 is unchanged [3], [10], [26]. Therefore, HDAC2 is a good therapeutic candidate for NPC disease.

Interestingly, we recently demonstrated that the activation of the tyrosine kinase c-Abl increases HDAC2 levels in AD models causing neuronal gene repression [10]. Our findings show that c-Abl induces HDAC2 phosphorylation on tyrosine 222, increasing its stability and protein levels. This in turn increases histone H3 deacetylation and HDAC2 recruitment to the promoter of key HDAC2 target genes, such as synaptotagmin, NR2a and GluR1, inducing their transcriptional repression in AD models. Our results suggest that c-Abl/HDAC2 signaling is relevant in neurodegeneration [10].

Moreover, recent data show that c-Abl is also involved in HDAC1 regulation by inhibiting its proteasomal degradation [27], suggesting that c-Abl activity controls epigenetic changes of several genes in different cellular contexts.

Interestingly, although AD and NPC have a different etiology, both diseases share some pathological features, including the accumulation of amyloid-β (Aβ), tau pathology and lysosomal dysfunction [28], [29]. Moreover, high cholesterol is a risk factor in AD and carrying the ε4 isoform of apolipoprotein E (ApoE) is a risk factor in both disorders [30], [31], [32]. Accordingly, treatment with HP-β-CD lowered the levels of Aβ42 in a transgenic mouse model of AD, both by reducing Aβ production and enhancing clearance mechanisms [33].

c-Abl has been previously linked to NPC disease pathology. We demonstrated that c-Abl is activated in in vitro and in vivo NPC models, triggering neuronal apoptosis [34]. Interestingly, treatment with the c-Abl inhibitor Imatinib decreases apoptosis of Purkinje cerebellar neurons, improving locomotor abilities and increasing the survival rates of Npc1−/− mice [35]. In addition, c-Abl activity has been implicated in several neurodegenerative disorders such as AD [36], [37], Parkinson's disease [38] and amyotrophic lateral sclerosis [39]. Finally, c-Abl-overexpressing mice show severe neurodegeneration [40].

In this work we analyzed the relevance of the c-Abl/HDAC2 pathway in NPC neuronal models. We found that HDAC2 protein levels and activity are increased in NPC neuronal models and in Npc1−/− mice. Also the pharmacological inhibition of c-Abl or c-Abl deficiency (c-Abl−/−) prevents the increase of HDAC2 levels and activity in NPC neuronal models and decreases the levels of tyrosine phosphorylation of HDAC2. In addition, we found that cholesterol accumulation and oxidative stress are upstream regulators of the c-Abl/HDAC2 pathway in NPC neurons. Furthermore, c-Abl inhibition prevents the recruitment of HDAC2 to the promoter of neuronal genes, triggering an increase in their expression. Our results suggest that c-Abl could be a therapeutic target for preventing HDAC2-mediated neuronal gene repression in NPC disease.

Section snippets

c-Abl mediates the increase in protein levels and repression activity of HDAC2 in NPC neuronal models

To evaluate the relevance of HDAC2 function in the NPC phenotype we first assessed HDAC2 protein levels in NPC neuronal models. We transfected the hippocampal-like cell line HT22 with an shRNA against NPC1 for 48 h. As expected, the decrease in NPC1 protein levels was associated with an increase in intracellular free cholesterol levels that was detected by filipin staining (Fig. 1A). Interestingly, HDAC2 protein levels were increased in this neuronal NPC model (Fig. 1B). Although the inhibition

Discussion

In this work we report the participation of the c-Abl/HDAC2 signaling pathway in epigenetic alterations in NPC neurons. We found that HDAC2 levels and repression activity are increased in NPC neuronal models and also in Npc1−/− mice, and that the pharmacological inhibition of c-Abl or c-Abl deficiency prevents the increase of HDAC2 levels. In addition, we show that cholesterol accumulation and oxidative stress are upstream regulators of the c-Abl/HDAC2 pathway. Furthermore, c-Abl inhibition

Conclusions

Our data show that HDAC2 levels and repression activity are increased in NPC neuronal models. The pharmacological inhibition of c-Abl or the deficiency of c-Abl prevents the increase of HDAC2 protein levels and activity and the recruitment of HDAC2 to the promoter of neuronal genes, triggering an increase in their expression, in NPC neuronal models.

The results of this work suggest that the c-Abl/HDAC2 signaling pathway could also contribute to the pathogenesis of NPC disease, decreasing

Antibodies and reagents

Mouse anti-HDAC2 3F3 ChIP grade (ab51832), rabbit anti-HDAC2 ChIP grade (ab7029) and rabbit anti-HDAC1 (ab184651) were purchased from Abcam (Cambridge, UK). Rabbit anti-acetyl-histone H3 (06-599) was purchased from Millipore (Billerica, USA). Mouse anti-c-Abl (sc-23), rabbit anti-B-tubulin (sc-9104), mouse anti-GAPDH (sc-32,233) were purchased from Santa Cruz Biotechnology (Dallas, USA). Rabbit anti-phospho-c-Abl (C4240), filipin, the ANTI-FLAG M2 affinity gel (A2220), methyl-β-cyclodextrin and

Abbreviations

    NPC

    Niemann–Pick type C

    HDAC2

    histone deacetylase 2

    U18

    U18666A

    Ima

    Imatinib

    MS

    MS275

Competing interests

The authors declare that there are no conflicts of interest.

Author Contributions

Conceived and designed the experiments: P.S.C., M.G-Z., A.R.A., S.Z.; performed the experiments: P.S.C., M.G-Z., L.G-H. M.J.Y.; analyzed the data: P.S.C., M.G-Z., J. M., E. S., A.R.A., S.Z.; contributed reagents/materials/analysis tools: A.D., J.M., E.S.; wrote the paper: P.S.C., M.G-Z., A.R.A., S.Z.

Transparency document

Transparency document.

Acknowledgments

This study was supported by grants from the Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) (grant numbers 1120512 to A.R.A. and 1110310 and 1150186 to S.Z.); Fondo de Fomento al Desarrollo Científico y Tecnológico FONDEF D10I1077 (to A.R.A. and S.Z.); Fondo Nacional de Desarrollo de Areas Prioritarias, FONDAP, Project no. 15090007, Center for Genome Regulation (CGR) to S.Z. and Projecto Basal PFB12/2007, 2013–2017 to A.R.A. M.G-Z. acknowledges support from CONICYT, VRI and

References (55)

  • R.A. Nixon

    Niemann–Pick type C disease and Alzheimer's disease: the APP-endosome connection fattens up

    Am. J. Pathol.

    (2004)
  • J. Poirier

    Apolipoprotein E and cholesterol metabolism in the pathogenesis and treatment of Alzheimer's disease

    Trends Mol. Med.

    (2003)
  • A. Klein et al.

    Perez de arce K, et al. Oxidative stress activates the c-Abl/p73 proapoptotic pathway in Niemann–Pick type C neurons

    Neurobiol. Dis.

    (2011)
  • A.R. Alvarez et al.

    Activation of the neuronal c-Abl tyrosine kinase by amyloid-beta-peptide and reactive oxygen species

    Neurobiol. Dis.

    (2004)
  • S.C. Tsai et al.

    Regulation of histone deacetylase 2 by protein kinase CK2

    J. Biol. Chem.

    (2002)
  • L.F. Yevenes et al.

    Lysosomal vitamin E accumulation in Niemann–Pick type C disease

    Biochim. Biophys. Acta

    (2012)
  • J. Penney et al.

    Histone deacetylases in memory and cognition

    Sci. Signal.

    (2014)
  • A. Fischer et al.

    Recovery of learning and memory is associated with chromatin remodelling

    Nature

    (2007)
  • J. Graff et al.

    An epigenetic blockade of cognitive functions in the neurodegenerating brain

    Nature

    (2012)
  • J.S. Guan et al.

    HDAC2 negatively regulates memory formation and synaptic plasticity

    Nature

    (2009)
  • M. Haberland et al.

    The many roles of histone deacetylases in development and physiology: implications for disease and therapy

    Nat. Rev. Genet.

    (2009)
  • R. Brunmeir et al.

    Histone deacetylase HDAC1/HDAC2-controlled embryonic development and cell differentiation

    Int. J. Dev. Biol.

    (2009)
  • R.L. Montgomery et al.

    Histone deacetylases 1 and 2 control the progression of neural precursors to neurons during brain development

    Proc. Natl. Acad. Sci. U. S. A.

    (2009)
  • M. Jawerka et al.

    The specific role of histone deacetylase 2 in adult neurogenesis

    Neuron Glia Biol.

    (2010)
  • J.L. MacDonald et al.

    Histone deacetylases 1 and 2 are expressed at distinct stages of neuro-glial development

    Dev. Dyn.

    (2008)
  • E.D. Carstea et al.

    Niemann–Pick C1 disease gene: homology to mediators of cholesterol homeostasis

    Science

    (1997)
  • S. Naureckiene et al.

    Identification of HE1 as the second gene of Niemann–Pick C disease

    Science

    (2000)
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