Calcium dysregulation and Cdk5-ATM pathway involved in a mouse model of fragile X-associated tremor/ataxia syndrome - PubMed (original) (raw)
Calcium dysregulation and Cdk5-ATM pathway involved in a mouse model of fragile X-associated tremor/ataxia syndrome
Gaëlle Robin et al. Hum Mol Genet. 2017.
Abstract
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurological disorder that affects premutation carriers with 55-200 CGG-expansion repeats (preCGG) in FMR1, presenting with early alterations in neuronal network formation and function that precede neurodegeneration. Whether intranuclear inclusions containing DNA damage response (DDR) proteins are causally linked to abnormal synaptic function, neuronal growth and survival are unknown. In a mouse that harbors a premutation CGG expansion (preCGG), cortical and hippocampal FMRP expression is moderately reduced from birth through adulthood, with greater FMRP reductions in the soma than in the neurite, despite several-fold elevation of Fmr1 mRNA levels. Resting cytoplasmic calcium concentration ([Ca2+]i) in cultured preCGG hippocampal neurons is chronically elevated, 3-fold compared to Wt; elevated ROS and abnormal glutamatergic responses are detected at 14 DIV. Elevated µ-calpain activity and a higher p25/p35 ratio in the cortex of preCGG young adult mice indicate abnormal Cdk5 regulation. In support, the Cdk5 substrate, ATM, is upregulated by 1.5- to 2-fold at P0 and 6 months in preCGG brain, as is p-Ser1981-ATM. Bax:Bcl-2 is 30% higher in preCGG brain, indicating a greater vulnerability to apoptotic activation. Elevated [Ca2+]i, ROS, and DDR signals are normalized with dantrolene. Chronic [Ca2+]i dysregulation amplifies Cdk5-ATM signaling, possibly linking impaired glutamatergic signaling and DDR to neurodegeneration in preCGG brain.
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Figures
Figure 1
Fragile X mental retardation protein (FMRP) is decreased in preCGG mice across postnatal stages. (A) Quantification of FMRP expression level relative to β-actin in cortical and hippocampal tissues from preCGG (Cortex: P0, n = 7; 6 week, n = 8; 6 months, n = 7. Hippocampus: P0, n = 4; 6 week, n = 7; and 6 months, n = 8) and age-matched Wt mice (Cortex: P0&6 week, n = 8; 6 months, n = 7, Hippocampus: P0, n = 3; 6 weeks and 6 months, n = 8), normalized to Wt FMRP expression. (B) Representative western blot of FMRP (70kDa) and β-actin (45kDa) from cortical and hippocampal tissues from indicated genotype and age. (C) Quantification of FMRP expression levels relative to β-actin in paired Wt (n = 3) and preCGG (n = 4) male (M) and Wt (n = 3) and preCGG (n = 4) female (F) cultures of hippocampal neurons at 7 DIV. (D) Representative western blot in paired culture of Wt and preCGG hippocampal neurons. Significance was determined using t-test *P < 0.05; **P < 0.01; ***P < 0.001. Error bars indicate mean ± SEM.
Figure 2
Reduced FMRP is redistributed throughout the neurites in cultured preCGG hippocampal neurons. (A) 7 DIV Wt and preCGG cultured hippocampal neurons stained for FMRP (red) and MAP2b (green). (B) FMRP fluorescence intensity was quantified in the entire neuron (total), in the soma, and in the neurites of Wt and preCGG male (M) and female (F) hippocampal neurons at 7 DIV and 14 DIV. At 7DIV: 51 Wt M, 11 Wt F, 60 preCGG M and 11 preCGG F hippocampal neurons were measured from 3 differente cultures; For 14DIV: 10 Wt M, 14 Wt F, 8 preCGG M and 15 preCGG F hippocampal neurons were measured from 3 different cultures. Significance was determined using an ANOVA followed by Tukey’s multiple comparison test *P < 0.05; **P < 0.01; ***P < 0.001. Error bars indicate mean ± SEM.
Figure 3
Altered glutamatergic responses in preCGG hippocampal neurons at 14 DIV. (A) Representative traces of the Ca2+ transient recording in the soma of the neurons in response to the addition of 3 µM glutamate in Wt and preCGG M hippocampal neurons. (B) Dot plots with mean and SEM of the maximal amplitude, (C) Area under the curve, and (D) full width half maximum (FWHM) normalized to Wt M (% of Wt M) measured on the glutamate-induced Ca2+ transients of Wt M (black, n = 65), Wt F (grey, n = 34), preCGG M (blue, n = 70), and preCGG F (pink, n = 20) hippocampal neurons. (E) Non-linear fit of the percentage of frequency distribution of SCO amplitudes during baseline recording (solid line) and after 5 min of incubation with 50 µM DHPG (dotted line) in Wt M (n = 17), Wt F (n = 10), preCGG M (n = 17), and preCGG F (n = 10). Significance was determined using an ANOVA followed by Tukey’s multiple comparison test for the glutamate-induced Ca2+ transient parameters and a Kolmogorov-Smirnov test to compare distribution. ***P < 0.001. Error bars indicate mean ± SEM.
Figure 4
Cytosolic resting calcium concentration ([Ca2+]i) is elevated in preCGG hippocampal neurons. (A) Representatives Ca2+ potential recording (VCa) from Wt male (Wt M), Wt female (Wt F), preCGG male (preCGG M) and preCGG female (preCGG F) hippocampal neurons. The initial portion of each trace was obtained prior to impalement of the cells. Penetration and removal of microelectrode was accompanied by immediate downward and upward deflection, respectively. (B) Dot plot with mean and SEM of [Ca2+]i values from all Wt M and F (respectively for 7, 14 and 21 DIV: n = 23, 13, 9 Wt M and n = 14, 12, 9 Wt F) and preCGG M and F (respectively for 7, 14 and 21 DIV n = 16, 17, 9 preCGG M and n = 16, 16, 9 preCGG F) neurons in Lockes’ solution or in presence of dantrolene 10µM (respectively for 7, 14 and 21 DIV: n = 13, 9, 5 Wt M and n = 16, 10, 5 Wt F, n = 15, 9, 5 preCGG M and n = 14, 9, 5 preCGG F). (C) Dot plot with mean and SEM of [Ca2+]i measurement in paired F and M neuronal cultures from preCGG and Wt pups in control (CT) or in the presence of 1 µM TTX at 7 DIV (Wt M, n = 7, Wt F, n = 8, preGG M, n = 10, preCGG F, n = 8). (D) Dot plot of membrane potential (Vm) values of paired F and M neuronal cultures from preCGG and Wt pups in Lockes’ buffer. Significance was determined using an ANOVA followed by Tukey’s multiple comparison test. ***P < 0.001; ##P < 0.01, compared to Wt values without dantrolene treatment. Error bars indicate mean ± SEM.
Figure 5
Elevated µ-calpain activity in preCGG cortical and hippocampal tissue lysates. Quantification of (A) µ-calpain and (B) α II spectrin breakdown products (or SBDP150) expression level relative to GAPDH in cortical and hippocampal tissues of preCGG and age-matched Wt mice, normalized to Wt. Representative western blots from indicated genotype, brain region, and age are shown below. (C) Dot plot of µ-calpain activity using a fluorometric assay in 6-month-old Wt (n = 8) and preCGG male (n = 8) cortical and hippocampal tissue lysates. Cortical lysates incubated with the calpain inhibitor Z-LLY-FMK are showing in the middle. Controls (calpain) with 0.5 µl of calpain are represented at the right of the graphic with or without calpain inhibitor Z-LLY-FMK. The numbers of animals tested is at least, for preCGG cortex: P0, n = 7, 6 weeks, n = 5, 6 months, n = 7; preCGG hippocampus: P0, n = 4, 6 weeks and 6 months, n = 5) and for Wt cortex: P0, 6 weeks and 6 months, n = 7; Hippocampus: P0, n = 3, 6 weeks, n = 7 and 6 months, n = 6). Significance was determined using t-test, *P < 0.05; **P < 0.01. Error bars indicate mean ± SEM.
Figure 6
Elevated p25/p35 ratio in preCGG cortical and hippocampal tissue lysates. Quantification of (A) Cdk-5 to β-actin and (B) the ratio p25/p35 level in cortex and hippocampus of preCGG and age-matched Wt mice, normalized to Wt expression. Representative western blots from indicated genotype, brain region, and age are shown below. Gels separated by a grey line were run on the same gel but were noncontiguous. The numbers of animals tested is at least, for preCGG cortex: P0, n = 7, 6 weeks, n = 5, 6 months, n = 7; preCGG Hippocampus: P0, n = 4, 6 weeks and 6 months, n = 5) and for Wt cortex: P0, 6 weeks and 6 months n = 7; Hippocampus: P0, n = 3, 6 weeks, n = 7 and 6 months, n = 6). Significance was determined using t-test, *P < 0.05; Error bars indicate mean ± SEM.
Figure 7
ATM and p-Ser1981-ATM upregulation and redistribution in preCGG neurons. Quantification of ATM (A) and p-Ser1981-ATM (B) expression level relative to β-actin in cortical and hippocampal lysates of preCGG (Cortex: P0, n = 8, 6 weeks and 6 months, n = 7; Hippocampus: P0, n = 6, 6 weeks, n = 7 and 6 months, n = 5) and age-matched Wt mice (Cortex: P0, n = 6, 6 weeks and 6 months, n = 5; Hippocampus: P0, n = 6, 6 weeks, n = 8 and 6 months, n = 5), normalized to Wt. (C) Quantification of the ratio of p-Ser1981-ATM to ATM in cortical and hippocampal lysates of preCGG (Cortex: P0, n = 8, 6 weeks and 6 months, n = 7; Hippocampus: P0, n = 6, 6 weeks, n = 7 and 6 months, n = 5) and age-matched Wt mice (Cortex: P0, n = 6, 6 weeks and 6 months, n = 5; Hippocampus: P0, n = 6, 6 weeks, n = 8 and 6 months, n = 5) normalized to Wt. (D) Representative ATM (upper row), p-Ser1981-ATM (middle row), and β-actin (lower row) western blots from indicated genotype, brain region, and age. Gels separated by a grey line were run on the same gel but were noncontiguous. (E) Immunolocalization of p-Ser1981-ATM (red) forming foci (indicated by white arrows) in the nucleus (DAPI, blue) in hippocampal neurons labeled with MAP2b (green) in Wt (upper panel) and preCGG (lower panel) hippocampal neurons at 7 DIV. Bar = 6 µm. (F) Dot plot with mean and SEM of the average intensity of p-Ser1981-ATM in the cytoplasm relative to the intensity in the nucleus (left side of the figure) in Wt (n = 12) and preCGG (n = 13) hippocampal neurons; number of p-Ser1981-ATM foci per nucleus in each cell (right side of the figure) in Wt (n = 12) and preCGG (n = 13) hippocampal neurons. Significance was determined using t-test *P < 0.05; **P < 0.01. Error bars indicate mean ± SEM.
Figure 8
Ratio of Bax to Bcl-2 is elevated at 6 months in preCGG cortices and hippocampi. (A) Quantification of the ratio Bax/Bcl-2 in cortical and hippocampal lysates of preCGG (Cortex: P0, n = 4, 6 weeks, n = 3, 6 months, n = 4; Hippocampus: P0, 6 weeks and 6 months, n = 4) and age-matched Wt mice (Cortex: P0, n = 4, 6 weeks and 6 months, n = 3; Hippocampus: P0, 6 weeks and 6 months, n = 4), normalized to Wt. (B) Representative Bax (upper row), Bcl-2 (middle row), and β-actin (lower row) western blots from indicated genotype, brain region, and age. Significance was determined using t-test *P < 0.05. Error bars indicate mean ± SEM.
Figure 9
Elevated ROS level in preCGG hippocampal neurons. (A) Representative pictures of Wt and preCGG hippocampal neurons (HN) after 30 min CellROX® Green (green staining) incubation under control conditions. Nuclei are labeled in blue. Bar =3µM. (B) Dot plot of the basal ROS level using CellROX® Green fluorescence measurement in paired cultures of M and F Wt and preCGG HN under control conditions (plain bar) and in the presence of 10 µM dantrolene in the media (striped bar). 50 µM 1,4-naphthoquinone was used as a positive control (CT +) and 100 µM N-acetylcysteine as a negative control (CT -). N = 3 separated cultures with 3 or 4 wells for each experimental group. (C) Representative traces of the live measurement of CellROX® green fluorescence in Wt and preCGG HN at 7 DIV. Addition of 100 µM 1,4-naphthoquinone induced a faster increase in fluorescence in preCGG HN compared to Wt. (D) Quantification of the rate of increase (slope) in the first 60 s following the addition of several 1,4-naphthoquinone concentrations to Wt and preCGG male and female HN. Vehicle was also added as a control (2% DMSO) without any changes in fluorescence level (data not shown). N = 3 separated cultures with 3 wells for each experimental group. Significance was determined using an ANOVA followed by Tukey’s multiple comparison test *P < 0.05; **P < 0.01; ***P < 0.001. Error bars indicate mean ± SEM.
Figure 10
Working model for early events occurring in premutation neurons. Premutation neurons exhibit an early chronic elevated [Ca2+]i associated with Ca2+ signaling dysregulation. This early Ca2+ overload leads to mitochondrial dysfunction yielding to an excessive ROS production that can activate ATM. Around 6 weeks of age, elevated [Ca2+]i increases the activity of the µ-calpain, inducing a degradation of spectrins at the inner membrane and a activation of Cdk5 through the cleavage of p35 in p25. Cdk5 migrates then in the nucleus to phosphorylate substrates and among them, ATM. P-ATM redistributes in the cytosol where it can act on channels amplifying the Ca2+. Later on, P-ATM activates p53 which induces the recruitment and increase in Bax and ultimately induces the downregulation of Bcl-2, causing apoptosis via mitochondrial-mediated mechanisms. Not shown in this figure are late stages of the neurodegenerative process that include the formation of intranuclear inclusions, and the possible participation of RAN-mediated events.
Figure 11
Chronology of Fmr1 premutation neuropathology in vivo and in vitro using the Dutch preCGG KI mouse, including the literature and our findings (bold). The left panel summarizes the chronological events occurring in vivo in preCGG KI mouse model reported in the literature and in our study (bold). The right panel summarizes the chronological events occurring in vitro in cultured neurons from preCGG KI mouse reported in the literature and in our study (bold). Additional citations, not mentioned in the text have been added: (88–97).
References
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