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Altered Nup153 Expression Impairs the Function of Cultured Hippocampal Neural Stem Cells Isolated from a Mouse Model of Alzheimer’s Disease

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Abstract

Impairment of adult hippocampal neurogenesis is an early event in Alzheimer’s disease (AD), playing a crucial role in cognitive dysfunction associated with this pathology. However, the mechanisms underlying defective neurogenesis in AD are still unclear. Recently, the nucleoporin Nup153 has been described as a new epigenetic determinant of adult neural stem cell (NSC) maintenance and fate. Here we investigated whether Nup153 dysfunction could affect the plasticity of NSCs in AD. Nup153 expression was strongly reduced in AD-NSCs, as well as its interaction with the transcription factor Sox2, a master regulator of NSC stemness and their neuronal differentiation. Similar Nup153 reduction was also observed in WT-NSCs treated with amyloid-β (Aβ) or stimulated with a nitric oxide donor. Accordingly, AD-NSCs treated with either a γ-secretase inhibitor or antioxidant compounds showed higher Nup153 levels suggesting that both nitrosative stress and Aβ accumulation affect Nup153 expression. Of note, restoration of Nup153 levels in AD-NSCs promoted their proliferation, as assessed by BrdU incorporation, neurosphere assay, and stemness gene expression analysis. Nup153 overexpression also recovered AD-NSC response to differentiation, increasing the expression of pro-neuronal genes, the percentage of cells positive for neuronal markers, and the acquisition of a more mature neuronal phenotype. Electrophysiological recordings revealed that neurons differentiated from Nup153-transfected AD-NSCs displayed higher Na+ current density, comparable to those deriving from WT-NSCs. Our data uncover a novel role for Nup153 in NSCs from animal model of AD and point to Nup153 as potential target to restore physiological NSC behavior and fate in neurodegenerative diseases.

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All data generated or analyzed during this study are included in this published article and in the additional files.

Abbreviations

AD :

Alzheimer’s disease

Nup153 :

nucleoporin153

NSCs :

neural stem cells

NO :

nitric oxide

NAC :

N-acetylcysteine

AA :

ascorbic acid

LNAME :

L-NG-nitro-arginine methyl ester

GSI :

γ-secretase inhibitor

MFI :

mean fluorescence intensity

TLX/NR2E1 :

nuclear receptor subfamily 2 group E member 1

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Acknowledgements

We thank Professor D. Puzzo, from University of Catania, who kindly provided tissues from APP knock-out mice (B6.129S7-Apptm1Dbo/J; 4 months old) that were used as negative control in Western blot experiments.

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Author notes

  1. Lucia Leone and Claudia Colussi contributed equally to this work.

Authors and Affiliations

  1. Institute of Human Physiology, Università Cattolica del Sacro Cuore, Roma, Italia

    Lucia Leone, Katia Gironi, Valentina Longo, Salvatore Fusco, Domenica Donatella Li Puma, Marcello D’Ascenzo & Claudio Grassi

  2. Fondazione Policlinico Universitario A. Gemelli IRCCS, Roma, Italia

    Lucia Leone, Salvatore Fusco, Domenica Donatella Li Puma, Marcello D’Ascenzo & Claudio Grassi

  3. Institute of Cell Biology and Neurobiology, National Research Council, Largo F. Vito 1, 00168, Rome, Italy

    Claudia Colussi

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  1. Lucia Leone
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Contributions

Electrophysiology: MD, VL; RT-qPCR: KG, LL, SF; ChIP: SF; NSC experiments and analysis: LL, KG, CC; confocal analysis: CC; WB and IP: CC, DDLP; conceptualization: LL, CC, CG; writing: LL, CC, CG. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Claudia Colussi.

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Mice were used in agreement with the guidelines of the European Parliament (Directive 2010/63/EU for the protection of laboratory animals) and with the guidelines of the Italian National Institute of Health and were approved by the Institutional Animal Care of Università Cattolica (approval number: 553/2016PR, Rome, Italy).

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Electronic Supplementary Material

Supplementary Fig. 1

Evaluation of Nup153 levels in GFAP positive radial and non-radial NSCs in the hippocampus from WT and AD mice. a) Representative images showing the merged signals for Nup153, GFAP and DAPI immunostaining in the DG of the hippocampus from WT and AD mice (scale bar 50 μm). b) Representative images showing Nup153, Nup153/GFAP or merged signals with DAPI of DG from WT and AD mice at higher magnification (scale bar 10 μm). c) Mean fluorescence intensity (MFI) for Nup153 levels in GFAP positive cells showing radial or non-radial morphology (ANOVA, Bonferroni test, n=4). Data plotted as mean ± SEM, ***p<0.001. (PDF 10710 kb)

Supplementary Fig. 2

Aβ levels in AD-NSCs. a) Analysis of Aβ levels by dot blot in WT- and AD-NSCs treated with vehicle or GSI (1μM) for 72 h. The lower panel shows the densitometry of Aβ signal normalized to red ponceau staining (ANOVA, Bonferroni test, n=6). b) Representative WB showing the presence of small oligomers (trimers and tetramers are indicated by arrowheads) in hippocampal tissue from APP-KO (negative control) or 3×Tg (positive control) mice and in AD-NSCs compared with synthetic Aβ preparation (200 nM). RP: red ponceau staining. Data plotted as mean ± SEM, ***p<0.001. (PDF 595 kb)

Supplementary Fig. 3

Effect of LNAME treatment on nitro-tyrosine levels in AD-NSCs. a) Analysis of nitro-tyrosine levels by dot blot in AD-NSCs treated with LNAME (LN) for 72h and compared to vehicle treated-AD-NSCs. RP: red ponceau staining. b) Densitometry of nitro-tyrosine signal normalized to red ponceau staining (RP). (Student’s t test, n=4). Data plotted as mean ± SEM, ***p<0.001. (PDF 1189 kb)

Supplementary Fig. 4

Nup153 modulation regulates neurosphere formation in WT-NSCs. a) Confocal analysis showing Nup153 expression in scramble- (sc) or Nup153 silenced-neurospheres (siNup153) and GFP signal in GFP- or GFP-Nup153 transfected neurospheres (scale bar 50 μm). Nuclei were counterstained with DAPI. b) Neurosphere assay in WT-NSCs treated with: scramble- or siNup153 oligos, GFP- or GFP-Nup153 vectors (mean±SEM, ***p<0.001, ANOVA, Bonferroni test, n=4). (PDF 2264 kb)

Supplementary Fig. 5

Evaluation of transfection efficiency in AD-NSCs. a) Representative images showing the level of GFP in neurospheres transfected either with GFP or GFP-Nup153 vectors. Nuclei were counterstained with DAPI, scale bar 50 μm. The graph shows the percentage of GFP+ cells in both conditions. Data plotted as mean ± SEM, n=4). (PDF 2344 kb)

Supplementary Fig. 6

Nup153 overexpression increases histone H3 acetylation level in AD-NSCs. Representative images showing the level of acetylation on lysine 9 and 14 of histone H3 (H3K9-14ac) in neurospheres isolated from AD mice overexpressing Nup153 compared to GFP-controls. Nuclei were counterstained with DAPI, scale bar 50 μm. (PDF 2447 kb)

Supplementary Fig. 7

Nup153 silencing modulates differentiation of WT-NSCs. a) Confocal analysis of Nup153 and DCX expression in proliferating WT-NSCs and after two days of differentiation (D2) (scale bar 20 μm). b) RT-qPCR showing the expression levels of Mef2c, Mash1 and NeuroD1 in scramble or siNup153-treated WT-NSCs. (Student’s t test, n=3). c) Evaluation of β–III tubulin and MAP2 expression at D6 in scramble- or siNup153-treated WT-NSCs. Nuclei were counterstained with DAPI (scale bar 50 μm). d) Neurite length in scramble- or siNup153-treated WT-NSCs at D10 (Student’s t test, n=5) e) Representative traces of voltage-gated Na+ currents evoked by a series of depolarizing steps from -90 to +40 mV in scramble- or siNup153 treated WT-NSCs. Inset: voltage protocols. The right panel shows current-voltage relationship of peak Na+ current densities for NSCs in the above conditions (black circles, scramble; red circles, siNup153; Student’s t test, scramble n=25, siNup n=18). Data plotted as mean ± SEM, *p<0.05, **p<0.01. (PDF 6675 kb)

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Leone, L., Colussi, C., Gironi, K. et al. Altered Nup153 Expression Impairs the Function of Cultured Hippocampal Neural Stem Cells Isolated from a Mouse Model of Alzheimer’s Disease. Mol Neurobiol 56, 5934–5949 (2019). https://doi.org/10.1007/s12035-018-1466-1

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