GFAP hyperpalmitoylation exacerbates astrogliosis and neurodegenerative pathology in PPT1-deficient mice

Abstract

The homeostasis of protein palmitoylation and depalmitoylation is essential for proper physiological functions in various tissues, in particular the central nervous system (CNS). The dysfunction of PPT1 (PPT1-KI, infantile neuronal ceroid lipofuscinosis [INCL] mouse model), which catalyze the depalmitoylation process, results in serious neurodegeneration accompanied by severe astrogliosis in the brain. Endeavoring to determine critical factors that might account for the pathogenesis in CNS by palm-proteomics, glial fibrillary acidic protein (GFAP) was spotted, indicating that GFAP is probably palmitoylated. Questions concerning if GFAP is indeed palmitoylated in vivo and how palmitoylation of GFAP might participate in neural pathology remain unexplored and are waiting to be investigated. Here we show that GFAP is readily palmitoylated in vitro and in vivo; specifically, cysteine-291 is the unique palmitoylated residue in GFAP. Interestingly, it was found that palmitoylated GFAP promotes astrocyte proliferation in vitro. Furthermore, we showed that PPT1 depalmitoylates GFAP, and the level of palmitoylated GFAP is overwhelmingly up-regulated in PPT1-knockin mice, which lead us to speculate that the elevated level of palmitoylated GFAP might accelerate astrocyte proliferation in vivo and ultimately led to astrogliosis in INCL. Indeed, blocking palmitoylation by mutating cysteine-291 into alanine in GFAP attenuate astrogliosis, and remarkably, the concurrent neurodegenerative pathology in PPT1-knockin mice. Together, these findings demonstrate that hyperpalmitoylated GFAP plays critical roles in regulating the pathogenesis of astrogliosis and neurodegeneration in the CNS, and most importantly, pinpointing that cysteine-291 in GFAP might be a valuable pharmaceutical target for treating INCL and other potential neurodegenerative diseases.

Keywords: GFAP; PPT1; astrogliosis; neurodegeneration; protein palmitoylation.

Fig. 1.

GFAP is palmitoylated at C291. (A) GFAP expressed in HEK-293T cells or from mice brain was analyzed for protein palmitoylation by Acyl-RAC assay. HA⁺, with HA, HA⁻, without HA. (B) Astroglioma cell line U251 was incubated with 2-BP for different period of time, and then were subjected for evaluating the level of GFAP palmitoylation by Acyl-RAC assay. (C) Protein sequence of GFAP from various species were aligned together for analyzing cysteine conservation. (D) Purified GFAP was probed by mass-spectrometry, a mass-shift of 238 Da linked to cysteine is a hallmark for palmitoylation. (E) WT or point-mutated (C291A, cysteine to alanine mutation) GFAP were expressed in HEK-293T cells and subjected for protein palmitoylation by Acyl-RAC assay.

Fig. 2.

Palmitoylated GFAP is involved in controlling astrocyte proliferation. (A) Constructed GFAP-KO cell was examined by Western blot to confirm its loss of GFAP in U251. (B) The removal of GFAP does not cause apparent morphological alteration in U251 cell. (C) The CCK8 was used to quantify cell proliferation in U251 cells for different genotypes (WT and GFAP-KO). (D and E) CCK8 was used to quantify cell proliferation in U251 (D) or U87 cells (E) expressing either empty vector (Flag), GFAP-Flag or GFAP-C291A-Flag construct. (F and G) CCK8 assay was conducted to quantify cell proliferation in U251 (F) or U87 (G) as treated with either DMSO, 2-BP, or none (WT). Data are mean ± SEM; P values calculated using paired two-sided t test. n = 3. **P ≤ 0.01, ***P ≤ 0.001.

Fig. 3.

GFAP hyperpalmitoylation stimulates astrocyte proliferation in PPT1-KI mice. (A and B) All known thioesterases (APT1/2, PPT1/2, and ABHD17a) were coexpressed with GFAP in HEK-293T cells for evaluating their ability to depalmitoylate GFAP in vitro (A), and the ratio of palmitoylated-GFAP (Upper)/total GFAP (Lower) was calculated and quantified (B). P values calculated using paired two-sided t test. n = 3. (C–E) Mice brains of WT and PPT1-KI were used to examine the levels of GFAP and palmitoylated GFAP at different timepoints (1 mo, 3 mo, and 6 mo, C); accordingly, the relative level of total GFAP (D) and ratio of palmitoylated-GFAP (Upper)/total GFAP (Lower) was calculated (E) and quantified. P values calculated using unpaired two-sided t test. n = 3. (F and G) Astrocytes isolated from WT and PPT1-KI mice brains were tested for the levels of GFAP (F) and GFAP palmitoylation (G). (H) Isolated astrocytes from WT and PPT1-KI mice brains were cultured and assayed for the rate of cell proliferation by CCK8. P value calculated using paired two-sided t test. n = 3. Data are mean ± SEM; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, otherwise not significant (n.s.).

Fig. 4.

Blocking GFAP palmitoylation down-regulate astrocyte proliferation and alleviate astrogliosis in PPT1-KI mice. (A–D) Mice brains were collected from different genotypes (WT, GFAP-C291A, PPT1-KI, and PPT1-KI/GFAP-C291A) at various timepoints (1 mo, 3 mo, and 6 mo) and subjected for the evaluation of GFAP (A) and GFAP palmitoylation by Acyl-RAC (C); correspondingly, the relative level of GFAP expression (B) and the ratio of palmitoylated-GFAP (Upper)/total GFAP (Lower) was calculated (D) and quantified. P values calculated using paired two-sided t test. n = 3. (E and F) Mice brains of different genotypes were paraffin-sectioned and immune-stained with GFAP (E) for the quantification of astrocytes in vivo (F). Biological replicates are indicated by scattered dots on the bars. P values calculated using a one-way ANOVA followed by Dunnett’s test. (G) Isolated astrocytes from various genotypes were examined for the levels of GFAP palmitoylation. (H) Isolated astrocytes from all genotypes were cultured and assayed for the rate of cell proliferation by CCK8. P values calculated using paired two-sided t test. n = 3. Data are mean ± SEM *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, otherwise not significant (n.s.).

Fig. 5.

Blocking GFAP palmitoylation partially rescue the neurodegenerative pathology in PPT1-KI mice. (A–D) Mice brains of all genotypes were processed for transparency with X-clarity and stained for GFAP and NeuN for visualizing astrocytes (A) and neurons (C) in vivo; the number of astrocytes (B) and neurons (D) was quantified manually after imaging. (E and F) Brain lysate from different genotypes were subjected for Western blot (E) to evaluate the protein levels of Tau-1 (axonal marker), NeuN (neuronal marker), GADD153 (ER stress marker), cleaved-PARP1 (apoptosis marker), all of which was quantified accordingly (F). n = 3. (G) Rotarod test was carried out in 6-mo-old mice of different genotypes to examine the motor coordination. n = 10. (H) Tail suspension test was performed for checking seizure behavior in 6-mo-old mice of different genotypes. (I) Kaplan–Meier plot comparing lifespans between genotypes. n = 18, 14, 20, 16 for wild-type, GFAP-C291A, PPT1-KI, and PPT1-KI/GFAP-C291A, respectively, P values calculated using log-rank test. Biological replicates are indicated by scattered dots on the bars. P values calculated using a one-way ANOVA followed by Dunnett’s test, except where indicated. Data are mean ± SEM; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, otherwise not significant (n.s.).

Fig. 6.

Schematic representation of the pathological mechanism in INCL mice model and a proposed therapeutic strategy. The homeostasis of palmitoylation and depalmitoylation is critical for proper physiological functions in the CNS, which is literally disrupted by the dysfunction of PPT1 in INCL. Specifically, GFAP, as a potential substrate of PPT1, its depalmitoylation process is inhibited by the loss of PPT1 and thus palmitoylated-GFAP is exaggerated in INCL. Importantly, hyperpalmitoylated GFAP promotes astrocyte proliferation and eventually urges the formation of astrogliosis. Blocking GFAP palmitoylation by introducing C291A in GFAP in PPT1-KI mice diminish the pathological burden of astrogliosis and neurodegeneration simultaneously, and therefore rescue seizure and extend longevity in PPT1-KI/GFAP-C291A mice.