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Loss of Tau protein affects the structure, transcription and repair of neuronal pericentromeric heterochromatin

View Article: PubMed Central - PubMed

ABSTRACT

Pericentromeric heterochromatin (PCH) gives rise to highly dense chromatin sub-structures rich in the epigenetic mark corresponding to the trimethylated form of lysine 9 of histone H3 (H3K9me3) and in heterochromatin protein 1α (HP1α), which regulate genome expression and stability. We demonstrate that Tau, a protein involved in a number of neurodegenerative diseases including Alzheimer’s disease (AD), binds to and localizes within or next to neuronal PCH in primary neuronal cultures from wild-type mice. Concomitantly, we show that the clustered distribution of H3K9me3 and HP1α, two hallmarks of PCH, is disrupted in neurons from Tau-deficient mice (KOTau). Such altered distribution of H3K9me3 that could be rescued by overexpressing nuclear Tau protein was also observed in neurons from AD brains. Moreover, the expression of PCH non-coding RNAs, involved in PCH organization, was disrupted in KOTau neurons that displayed an abnormal accumulation of stress-induced PCH DNA breaks. Altogether, our results demonstrate a new physiological function of Tau in directly regulating neuronal PCH integrity that appears disrupted in AD neurons.

No MeSH data available.


Related in: MedlinePlus

Tau deficiency is associated with an altered distribution of γH2AX and an accumulation of PCH DNA breaks in adult mouse hippocampus under stress conditions.(a) Single confocal sections of nuclei from primary culture of WT or KOTau (KO) neurons visualized with anti-NeuN antibodies or GFP fluorescence and labelled with Hoechst and anti-γH2AX antibodies indicate that the nuclear distribution of γH2AX at the periphery of chromocenters is affected in KOTau neurons. (b) The Comet assays show a tendency towards the accumulation of DNA damage in KOTau neurons compared with WT neurons. Each OTM (Olive tail moment) value is the median value of 150–200 cells from n = 7 independent WT and KOTau cultures. (c) Western blot analysis of lysates from n = 7 WT and n = 7 KOTau independent primary cultures. The level of γH2AX in each sample was estimated densitometrically with respect to the level of actin protein in the same sample. (d) Single confocal sagittal sections of hippocampus from WT or KOTau mice subjected to HS and labelled with DAPI and either TUNEL (green) or anti-γH2AX antibodies (red) show the accumulation of PCH DNA breaks within chromocenters of neurons from KOTau mice after HS treatment, which is correlated with the persistent co-localization of γH2AX within chromocenters of these neurons. (e) Quantification of the fluorescence signals for DAPI and TUNEL or DAPI and γH2AX along the indicated line scans drawn across a confocal section of nuclei from KOTau (KO) or WT neurons indicate that TUNEL and γH2AX labelling co-localize with chromocenters throughout the entire length of chromocenters of KOTau nuclei compared with chromocenters of WT nuclei, which remain mostly devoid of γH2AX. (f) Single confocal sections of hippocampus from adult WT or KOTau (KO) neurons indicate that under HS conditions the presence of H3K9me3 within chromocenters observed in WT adult neurons is diminished in nuclei of KOTau neurons as confirmed by (g) the quantification of the fluorescence signals for DAPI and H3K9me3 along the indicated lin scans drawn across chromocenters in a confocal section of nuclei from WT or KOTau (KO) neurons. Data are means ± s.e.m. (b) ± s.d. (c). (b) Mann-Whitney test. (c) Student’s t-test, NS (non-significant). Bars = 10 μm.
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f6: Tau deficiency is associated with an altered distribution of γH2AX and an accumulation of PCH DNA breaks in adult mouse hippocampus under stress conditions.(a) Single confocal sections of nuclei from primary culture of WT or KOTau (KO) neurons visualized with anti-NeuN antibodies or GFP fluorescence and labelled with Hoechst and anti-γH2AX antibodies indicate that the nuclear distribution of γH2AX at the periphery of chromocenters is affected in KOTau neurons. (b) The Comet assays show a tendency towards the accumulation of DNA damage in KOTau neurons compared with WT neurons. Each OTM (Olive tail moment) value is the median value of 150–200 cells from n = 7 independent WT and KOTau cultures. (c) Western blot analysis of lysates from n = 7 WT and n = 7 KOTau independent primary cultures. The level of γH2AX in each sample was estimated densitometrically with respect to the level of actin protein in the same sample. (d) Single confocal sagittal sections of hippocampus from WT or KOTau mice subjected to HS and labelled with DAPI and either TUNEL (green) or anti-γH2AX antibodies (red) show the accumulation of PCH DNA breaks within chromocenters of neurons from KOTau mice after HS treatment, which is correlated with the persistent co-localization of γH2AX within chromocenters of these neurons. (e) Quantification of the fluorescence signals for DAPI and TUNEL or DAPI and γH2AX along the indicated line scans drawn across a confocal section of nuclei from KOTau (KO) or WT neurons indicate that TUNEL and γH2AX labelling co-localize with chromocenters throughout the entire length of chromocenters of KOTau nuclei compared with chromocenters of WT nuclei, which remain mostly devoid of γH2AX. (f) Single confocal sections of hippocampus from adult WT or KOTau (KO) neurons indicate that under HS conditions the presence of H3K9me3 within chromocenters observed in WT adult neurons is diminished in nuclei of KOTau neurons as confirmed by (g) the quantification of the fluorescence signals for DAPI and H3K9me3 along the indicated lin scans drawn across chromocenters in a confocal section of nuclei from WT or KOTau (KO) neurons. Data are means ± s.e.m. (b) ± s.d. (c). (b) Mann-Whitney test. (c) Student’s t-test, NS (non-significant). Bars = 10 μm.

Mentions: In Drosophila and mammalian cells, repair of PCH DSBs occurs through a two-step response27. First, the early DNA damage detection step that leads to the recruitment of DNA damage response proteins, such as the phosphorylated form of the histone variant H2AX (γH2AX) within the core of PCH structures, is followed by a second repair step that requires the relocation of the damaged sequences from the inside to the periphery of PCH, leading to the formation of long-lasting γH2AX foci positioned at the periphery of chromocenters27282930. Whereas foci of γH2AX positioned at the periphery of chromocenters could be detected in nuclei of WT neurons, they were absent from nuclei of KOTau neurons where dispersed γH2AX labelling was detected, concentrated in the cytoplasm and scattered as small foci in the nucleus (Fig. 6a). The monitoring of DNA damage by COMET assay (Fig. 6b) revealed a global increase in DNA damage in KOTau neurons compared with that observed in WT neurons, which did not reach statistical significance because of intrinsic variations between independent cultures from one KOTau mouse to another. Similarly to DNA damage, a global increase in γH2AX in KOTau neurons compared with that observed in WT neurons was detected (Fig. 6c), which again did not reach statistical significance because of intrinsic variations between independent cultures from different animals. Therefore, the absence of γH2AX foci at the periphery of chromocenters in KOTau neurons could not be considered a consequence of a decrease in these neurons of either the degree of DNA damage or of the level of γH2AX but rather of an abnormal localization of γH2AX reminiscent of what we have observed here for H3K9me3 in KOTau neurons.


Loss of Tau protein affects the structure, transcription and repair of neuronal pericentromeric heterochromatin
Tau deficiency is associated with an altered distribution of γH2AX and an accumulation of PCH DNA breaks in adult mouse hippocampus under stress conditions.(a) Single confocal sections of nuclei from primary culture of WT or KOTau (KO) neurons visualized with anti-NeuN antibodies or GFP fluorescence and labelled with Hoechst and anti-γH2AX antibodies indicate that the nuclear distribution of γH2AX at the periphery of chromocenters is affected in KOTau neurons. (b) The Comet assays show a tendency towards the accumulation of DNA damage in KOTau neurons compared with WT neurons. Each OTM (Olive tail moment) value is the median value of 150–200 cells from n = 7 independent WT and KOTau cultures. (c) Western blot analysis of lysates from n = 7 WT and n = 7 KOTau independent primary cultures. The level of γH2AX in each sample was estimated densitometrically with respect to the level of actin protein in the same sample. (d) Single confocal sagittal sections of hippocampus from WT or KOTau mice subjected to HS and labelled with DAPI and either TUNEL (green) or anti-γH2AX antibodies (red) show the accumulation of PCH DNA breaks within chromocenters of neurons from KOTau mice after HS treatment, which is correlated with the persistent co-localization of γH2AX within chromocenters of these neurons. (e) Quantification of the fluorescence signals for DAPI and TUNEL or DAPI and γH2AX along the indicated line scans drawn across a confocal section of nuclei from KOTau (KO) or WT neurons indicate that TUNEL and γH2AX labelling co-localize with chromocenters throughout the entire length of chromocenters of KOTau nuclei compared with chromocenters of WT nuclei, which remain mostly devoid of γH2AX. (f) Single confocal sections of hippocampus from adult WT or KOTau (KO) neurons indicate that under HS conditions the presence of H3K9me3 within chromocenters observed in WT adult neurons is diminished in nuclei of KOTau neurons as confirmed by (g) the quantification of the fluorescence signals for DAPI and H3K9me3 along the indicated lin scans drawn across chromocenters in a confocal section of nuclei from WT or KOTau (KO) neurons. Data are means ± s.e.m. (b) ± s.d. (c). (b) Mann-Whitney test. (c) Student’s t-test, NS (non-significant). Bars = 10 μm.
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f6: Tau deficiency is associated with an altered distribution of γH2AX and an accumulation of PCH DNA breaks in adult mouse hippocampus under stress conditions.(a) Single confocal sections of nuclei from primary culture of WT or KOTau (KO) neurons visualized with anti-NeuN antibodies or GFP fluorescence and labelled with Hoechst and anti-γH2AX antibodies indicate that the nuclear distribution of γH2AX at the periphery of chromocenters is affected in KOTau neurons. (b) The Comet assays show a tendency towards the accumulation of DNA damage in KOTau neurons compared with WT neurons. Each OTM (Olive tail moment) value is the median value of 150–200 cells from n = 7 independent WT and KOTau cultures. (c) Western blot analysis of lysates from n = 7 WT and n = 7 KOTau independent primary cultures. The level of γH2AX in each sample was estimated densitometrically with respect to the level of actin protein in the same sample. (d) Single confocal sagittal sections of hippocampus from WT or KOTau mice subjected to HS and labelled with DAPI and either TUNEL (green) or anti-γH2AX antibodies (red) show the accumulation of PCH DNA breaks within chromocenters of neurons from KOTau mice after HS treatment, which is correlated with the persistent co-localization of γH2AX within chromocenters of these neurons. (e) Quantification of the fluorescence signals for DAPI and TUNEL or DAPI and γH2AX along the indicated line scans drawn across a confocal section of nuclei from KOTau (KO) or WT neurons indicate that TUNEL and γH2AX labelling co-localize with chromocenters throughout the entire length of chromocenters of KOTau nuclei compared with chromocenters of WT nuclei, which remain mostly devoid of γH2AX. (f) Single confocal sections of hippocampus from adult WT or KOTau (KO) neurons indicate that under HS conditions the presence of H3K9me3 within chromocenters observed in WT adult neurons is diminished in nuclei of KOTau neurons as confirmed by (g) the quantification of the fluorescence signals for DAPI and H3K9me3 along the indicated lin scans drawn across chromocenters in a confocal section of nuclei from WT or KOTau (KO) neurons. Data are means ± s.e.m. (b) ± s.d. (c). (b) Mann-Whitney test. (c) Student’s t-test, NS (non-significant). Bars = 10 μm.
Mentions: In Drosophila and mammalian cells, repair of PCH DSBs occurs through a two-step response27. First, the early DNA damage detection step that leads to the recruitment of DNA damage response proteins, such as the phosphorylated form of the histone variant H2AX (γH2AX) within the core of PCH structures, is followed by a second repair step that requires the relocation of the damaged sequences from the inside to the periphery of PCH, leading to the formation of long-lasting γH2AX foci positioned at the periphery of chromocenters27282930. Whereas foci of γH2AX positioned at the periphery of chromocenters could be detected in nuclei of WT neurons, they were absent from nuclei of KOTau neurons where dispersed γH2AX labelling was detected, concentrated in the cytoplasm and scattered as small foci in the nucleus (Fig. 6a). The monitoring of DNA damage by COMET assay (Fig. 6b) revealed a global increase in DNA damage in KOTau neurons compared with that observed in WT neurons, which did not reach statistical significance because of intrinsic variations between independent cultures from one KOTau mouse to another. Similarly to DNA damage, a global increase in γH2AX in KOTau neurons compared with that observed in WT neurons was detected (Fig. 6c), which again did not reach statistical significance because of intrinsic variations between independent cultures from different animals. Therefore, the absence of γH2AX foci at the periphery of chromocenters in KOTau neurons could not be considered a consequence of a decrease in these neurons of either the degree of DNA damage or of the level of γH2AX but rather of an abnormal localization of γH2AX reminiscent of what we have observed here for H3K9me3 in KOTau neurons.

View Article: PubMed Central - PubMed

ABSTRACT

Pericentromeric heterochromatin (PCH) gives rise to highly dense chromatin sub-structures rich in the epigenetic mark corresponding to the trimethylated form of lysine 9 of histone H3 (H3K9me3) and in heterochromatin protein 1α (HP1α), which regulate genome expression and stability. We demonstrate that Tau, a protein involved in a number of neurodegenerative diseases including Alzheimer’s disease (AD), binds to and localizes within or next to neuronal PCH in primary neuronal cultures from wild-type mice. Concomitantly, we show that the clustered distribution of H3K9me3 and HP1α, two hallmarks of PCH, is disrupted in neurons from Tau-deficient mice (KOTau). Such altered distribution of H3K9me3 that could be rescued by overexpressing nuclear Tau protein was also observed in neurons from AD brains. Moreover, the expression of PCH non-coding RNAs, involved in PCH organization, was disrupted in KOTau neurons that displayed an abnormal accumulation of stress-induced PCH DNA breaks. Altogether, our results demonstrate a new physiological function of Tau in directly regulating neuronal PCH integrity that appears disrupted in AD neurons.

No MeSH data available.


Related in: MedlinePlus