Acid sphingomyelinase deficiency protects mitochondria and improves function recovery after brain injury

Abstract

Traumatic brain injury (TBI) is one of the leading causes of disability worldwide and a prominent risk factor for neurodegenerative diseases. The expansion of nervous tissue damage after the initial trauma involves a multifactorial cascade of events, including excitotoxicity, oxidative stress, inflammation, and deregulation of sphingolipid metabolism that further mitochondrial dysfunction and secondary brain damage. Here, we show that a posttranscriptional activation of an acid sphingomyelinase (ASM), a key enzyme of the sphingolipid recycling pathway, resulted in a selective increase of sphingosine in mitochondria during the first week post-TBI that was accompanied by reduced activity of mitochondrial cytochrome oxidase and activation of the Nod-like receptor protein 3 inflammasome. TBI-induced mitochondrial abnormalities were rescued in the brains of ASM KO mice, which demonstrated improved behavioral deficit recovery compared with WT mice. Furthermore, an elevated autophagy in an ASM-deficient brain at the baseline and during the development of secondary brain injury seems to foster the preservation of mitochondria and brain function after TBI. Of note, ASM deficiency attenuated the early stages of reactive astrogliosis progression in an injured brain. These findings highlight the crucial role of ASM in governing mitochondrial dysfunction and brain-function impairment, emphasizing the importance of sphingolipids in the neuroinflammatory response to TBI.

Keywords: astrogliosis; inflammasome; neuroinflammation; sphingolipids.

Fig. 1.

TBI triggered an activation of ASM via posttranscriptional mechanisms. Brain tissue samples were prepared from the WT mouse brain after the injury imposed by a CCI device. Sham-injured animal brain was used as a control. A: Time course of specific ASM and NSM activity changes was determined. The enzyme activity is expressed as pmol of C15-SM/min per mg of protein. Data are means ± SE. * P < 0.05 (n = 10). B: Time course of ASM protein expression changes was assessed following brain trauma. Brain lysates (30 μg/lane) were analyzed by Western blotting using anti-ASM (Santa Cruz) antibody. To confirm equal loading of samples, the membranes were stripped and probed with anti-β-actin antibody. Data are representative of four independent experiments.

Fig. 2.

ASM is required for TBI-induced elevation of sphingosine in mitochondria. Brain tissue and mitochondria samples were prepared from the injured brain of WT and ASM KO mice. Sham-injured animal brain (sham) was used as a control. Sphingosine (A) and ceramide (B) content changes were determined in the brain tissue during the first week post-TBI. Data are means ± SE. * P < 0.05 (n = 16). Sphingosine (C) and ceramide (D) content changes were determined in cerebral mitochondria during the first week post-TBI. Data are means ± SE. * P < 0.05 (n = 16). Each sample was normalized to its respective total protein levels.

Fig. 3.

TBI-induced mitochondrial dysfunction and protein oxidative damage is rescued in the ASM-deficient brain. Mitochondria were purified from the injured brain of WT and ASM KO mice at various time points post-TBI. Sham-injured animal brain (sham) was used as a control. A: Time course of mitochondrial respiration changes was assessed by recording oxygen consumption in the presence of complex I substrate (5 mM glutamate supplemented with 5 mM malate) (glutamate), complex II substrate (10 mM succinate) (succinate), or complex IV (COX) substrate (2 mM ascorbate plus 250 μM TMPD) (ascorbate) and 50 μM 2,4-DNP (State 3u). Data are means ± SE. * P < 0.05 (n = 16). nAO, nano atoms oxygen. B: Mitochondrial COX activity was measured by recording oxygen consumption in the presence of COX substrate (2 mM ascorbate plus 250 μM TMPD), 1 μg/ml antimycin, and 50 μM 2,4-DNP. Data are means ± SE. * P < 0.05 (n = 16). C: Time course of protein carbonyls changes in mitochondria was determined. Data are means ± SE. * P < 0.05 (n = 16).

Fig. 4.

Elevated autophagy in the ASM-deficient brain is augmented after TBI. Brain tissue samples were prepared from the injured brain of WT and ASM KO mice at 168 h (7 days) post-TBI. Sham-injured animal brain (sham) was used as a control. A: Equal amount of sample (30 μg) was loaded into the lane. The expression of autophagy protein markers was characterized by Western blotting using anti-LC3, anti-Beclin-1, anti-PINK-1, and anti-p62 specific antibodies. To confirm equal loading of samples, the membranes were stripped and probed with anti-β-actin antibody. Representative data are from six independent experiments. Quantification of the LC3-II/LC3-I protein expression ratio (B) and the protein expression of p62 and Beclin-1 (C) using ImageJ software. Data are means ± SE. * P < 0.05 (compared with sham); ^#P < 0.05 (WT versus ASM KO, n = 6).

Fig. 5.

Hindering ASM improved sensorimotor deficits recovery after brain trauma. Sensorimotor deficits were assessed using a standard rotarod test. Each day for 3 days prior to injury, animals were trained on the rotarod at a speed of 18 rpm in the acceleration mode (0–18 rpm/90 s). Animals were tested with the rotarod apparatus using three trials in session, with a minimum of 5 min resting between trials. Time course of latency changes was determined in WT and ASM KO mice after CCI (A) or in WT mice treated with Reclast (Reclast) compared with vehicle-treated WT mice (control) (B). Data are means ± SE. * P < 0.05 (compared with sham or control); ^# P < 0.05 (WT versus ASM KO, n = 16).

Fig. 6.

ASM deficiency improved spatial learning and memory after TBI. Cognitive deficits were assessed using the MWM test in WT and ASM KO mice. A: Spatial learning was assessed in training trials starting at day 14 post-TBI. The mice were exposed to four MWM trials/day, with the hidden-platform location unchanged. Data are means ± SE. * P < 0.05 (WT-TBI versus ASM KO-TBI); ^# P < 0.05 (WT-TBI or ASM KO-TBI compared with sham, n = 16). B: On day 18 post-TBI, the platform was removed, and the mice were placed back in the MWM for 90 s. The percent of total time spent in the correct quadrant was recorded as an indicator of spatial memory. Data are means ± SE. * P < 0.05 (WT-TBI versus ASM KO-TBI); ^# P < 0.05 (WT-TBI or ASM KO-TBI compared with sham, n = 16).

Fig. 7.

TBI-triggered NLRP3 inflammasome activation is attenuated in the ASM-deficient brain. Brain tissue samples were prepared from the injured brain of WT, ASM KO, and WT mice treated with Reclast (2 mg/kg, ip). Sham-injured animal brain (sham) was used as a control. A: Equal amount of sample (30 μg) was loaded into the lane. The expression of protein markers of the NLRP3 inflammasome were characterized by Western blotting. To confirm equal loading of samples, the membranes were stripped and probed with anti-β-actin antibody. Representative data are from six independent experiments. Quantification of the normalized NLRP3 protein expression (B) and the normalized protein expression of caspase-1 (C) using ImageJ software. Data are means ± SE. * P < 0.05 (n = 6 compared with sham); ^#P < 0.05 (n = 6, WT versus WT+ Reclast or ASM KO).

Fig. 8.

ASM deficiency impacted the early reactive astrogliosis after brain trauma. Brain tissue samples were prepared from the injured brain cortex of WT, ASM KO, and WT mice treated with Reclast (2 mg/kg, ip). Sham-injured animal brain (sham) was used as a control. A: Equal amount of sample (30 μg) was loaded into the lane. The expression of panastrocyte marker Aldh1l1 and reactive astrocyte marker GFAP were determined by Western blotting. To confirm equal loading of samples, the membranes were stripped and probed with anti-β-actin antibody. Representative data are from six independent experiments. B: Quantification of the normalized GFAP protein expression using ImageJ software. Data are means ± SE. * P < 0.05 (n = 6 compared with sham); ^# P < 0.05 (n = 6, WT versus ASM KO).