Parkin deficiency accentuates chronic alcohol intake-induced tissue injury and autophagy defects in brain, liver and skeletal muscle

Abstract

Alcoholism leads to organ injury including mitochondrial defect and apoptosis with evidence favoring a role for autophagy dysregulation in alcoholic damage. Parkin represents an autosomal recessive inherited gene for Parkinson’s disease and an important member of selective autophagy for mitochondria. The association between Parkinson’s disease and alcoholic injury remains elusive. This study aimed to examine the effect of parkin deficiency on chronic alcohol intake-induced organ injury in brain, liver and skeletal muscle (rectus femoris muscle). Adult parkin-knockout (PRK^−/−) and wild-type mice were placed on Liber-De Carli alcohol liquid diet (4%) for 12 weeks prior to assessment of liver enzymes, intraperitoneal glucose tolerance, protein carbonyl content, apoptosis, hematoxylin and eosin morphological staining, and mitochondrial respiration (cytochrome c oxidase, NADH:cytochrome c reductase and succinate:cytochrome c reductase). Autophagy protein markers were monitored by western blot analysis. Our data revealed that chronic alcohol intake imposed liver injury as evidenced by elevated aspartate aminotransferase and alanine transaminase, glucose intolerance, elevated protein carbonyl formation, apoptosis, focal inflammation, necrosis, microvesiculation, autophagy/mitophagy failure and dampened mitochondrial respiration (complex IV, complexes I and III, and complexes II and III) in the brain, liver and rectus femoris skeletal muscle. Although parkin ablation itself did not generate any notable effects on liver enzymes, insulin sensitivity, tissue carbonyl damage, apoptosis, tissue morphology, autophagy or mitochondrial respiration, it accentuated alcohol intake-induced tissue damage, apoptosis, morphological change, autophagy/mitophagy failure and mitochondrial injury without affecting insulin sensitivity. These data suggest that parkin plays an integral role in the preservation against alcohol-induced organ injury, apoptosis and mitochondrial damage.

parkin, chronic alcohol intake, tissue damage, mitochondria, autophagy

Introduction

Alcohol is the most frequently abused substance, accounting for a high morbidity and mortality with nearly 3.3 million alcohol-associated deaths worldwide annually [1–3]. Chronic and heavy alcohol intake is associated with a rapid rise in a wide array of chronic diseases such as Parkinson’s disease, dementia, cancer, muscle atrophy, acute respiratory distress syndrome, diabetes mellitus, liver cirrhosis, and cardiovascular diseases [4–13]. Not surprisingly, alcoholism imposes a rather heavy financial burden on public health and economy with an estimated $200 billion attributed to healthcare expense, productivity loss, and other unfavorable socioeconomic impacts [2,14]. Up-to-date, a number of scenarios have been postulated for alcohol-induced organ damage including ethanol/acetaldehyde toxicity, oxidative stress, apoptosis, impaired protein synthesis, protein catabolism, altered fatty acid extraction and deposition, as well as epigenetic modification of vital proteins [4,5,15]. In addition, presence of neurodegenerative diseases such as Parkinson’s disease is also suggested to impact alcoholic organ damage, although inconsistent findings have been reported [16–18].

Although liver is considered to be the predominant site for alcohol metabolism afflicted with liver cirrhosis and alcoholic fatty liver diseases with alcoholism [19], other organs including brain, lungs, intestines and skeletal muscle may also be negatively affected with chronic alcohol exposure [19,20]. For example, chronic alcoholism has been demonstrated to elicit pronounced brain damage encompassing atrophy, neuronal loss, as well as stimulant and compulsive behavior changes [21]. Meanwhile, nearly half of chronic alcohol consumers develop alcoholic skeletal myopathy involving lipid peroxidation, muscle fiber atrophy and loss of muscle strength [22]. To this end, understanding the mechanisms of action underscoring the debilitating effects of chronic alcoholism on organs should be pertinent to organ-specific therapeutic interventions. Recent evidence has depicted a role for dysregulated autophagy, a conservative recycling process for engulfment and encapsulation of cytoplasmic proteins or organelles [6,23], in the onset and development of alcoholic organ damage [24–28]. Dysfunction of autophagy in response to chronic and heavy alcohol intake leads to compromised protein quality control in various organ system and therefore perturbs protein and mitochondrial homeostasis in organs following chronic alcoholism [29]. Nonetheless, the precise nature of autophagy dysregulation in response to alcohol challenge remains elusive. This study was designed to examine the role of parkin, an essential component of mitochondria-selective autophagy (mitophagy) and, more importantly, an autosomal recessive gene for Parkinson’s disease, in chronic alcoholism-induced organ damage in the brain, liver and rectus femoris skeletal muscle. Parkin, an E3 ubiquitin ligase ubiquitously expressed in cytosol, is translocated to mitochondrial outer membrane to initiate ubiquitination of mitochondrial substrates, resulting in degradation of long-lived or damaged mitochondria by mitophagy [30]. Parkin is a sentinel molecule to regulate mitophagy, mitochondrial dynamics and thus mitochondrial homeostasis [30]. The PINK1/parkin-regulated mitophagy is considered as a gatekeeper for mitochondrial quality control [23,31–33]. Dysregulation of parkin-related mitophagy has been demonstrated in a number of pathological settings including cancers, aging, atherosclerosis, metabolic diseases, and cardiovascular diseases [30,34,35].

In this study, we planned to examine the effect of parkin deletion on chronic alcohol intake-induced changes in protein carbonyl formation, apoptosis, inflammation, necrosis, microvesiculation, autophagy, mitophagy and mitochondrial respiration in the brain, skeletal muscle and liver.

Materials and Methods

Experimental animals and chronic alcohol intake

All animal experimental procedures carried out here were in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996) and approved by the Animal Use and Care Committee at the Zhongshan Hospital Fudan University (Shanghai, China). In brief, 5-month-old adult male parkin-knockout (PRK^−/−) mice were purchased from the Jackson Laboratory (Bar Harbor, Farmington, USA; B6.129S4-Parktm1Shn/J, stock number 006582) and their wild-type (WT) littermates were used as WT controls. All mice were housed in a temperature-controlled room under a 12 h/12 h-light/dark and placed on a standard Lieber-De Carli alcohol liquid (with alcohol providing 36% of total energy; Shake & Pour Bio-Serv Inc., Frenchtown, USA) or control diet for an acclimation period of 1 week. The choice of a liquid diet is originated from the fact that ethanol self-administration is less stressful while providing adequate nutrition compared with intravenous administration, aerosolized inhalation, or forced-feeding regimens. Following acclimation, mice were kept on regular (alcohol-free) or isocaloric 4% (v/v) alcohol liquid diet for 12 weeks. A pair-feeding method was utilized to avoid possible nutritional deficits where non-ethanol group received equal volume of diet the alcohol group consumed the day before.

Serum aspartate aminotransferase, alanine aminotransferase, blood ethanol and blood acetaldehyde assays

To evaluate the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), as well as ethanol and acetaldehyde (the ethanol metabolite), blood was collected. Serum was extracted using centrifugation and was stored at −80°C. AST/ALT, ethanol and acetaldehyde levels were determined using the VetAce Analyzer (Alfa Wassermann, Inc., West Caldwell, USA), the ethanol assay kit (Abcam, Cambridge, USA), and the Megazyme acetaldehyde kit (Megazyme, Wicklow, Ireland), respectively.

Intraperitoneal glucose tolerance test

Intraperitoneal glucose tolerance test (IPGTT) was conducted to evaluate global metabolism. Following the 12-week feeding, mice fasted for 12 h were given an i.p. injection of glucose (2 g/kg body weight). Blood glucose levels were measured using an Accu-Chek III glucose analyzer (Roche, Indianapolis, USA) immediately before the challenge, or at 15, 30, 60 and 120 min thereafter [36].

Table 1

General biometric and plasma parameters in adult male WT and parkin-knockout (PRK^−/−) mice fed with liquid alcohol diet (4%) for 12 weeks

Mouse group	WT	WT-ETOH	PRK−/−	PRK−/−-ETOH
Body weight (g)	25.5 ± 0.8	26.0 ± 0.8	25.5 ± 0.8	26.1 ± 0.9
Brain weight (mg)	429 ± 10	401 ± 9*	431 ± 11	372 ± 11*,#
BrW/BW (mg/g)	16.9 ± 0.4	15.5 ± 0.3*	17.0 ± 0.7	14.3 ± 0.4*,#
Liver weight (g)	1.44 ± 0.05	1.61 ± 0.03*	1.45 ± 0.04	1.81 ± 0.08*,#
LW/BW (mg/g)	56.5 ± 1.8	62.1 ± 1.7*	57.0 ± 1.5	69.3 ± 1.5*,#
Skeletal muscle (mg)	210 ± 5	180 ± 4*	215 ± 6	162 ± 5*,#
SkW/BW (mg/g)	8.26 ± 0.26	6.95 ± 0.16*	8.50 ± 0.44	6.22 ± 0.13*,#
Serum AST (U/L)	86.5 ± 6.9	166.1 ± 9.9*	87.5 ± 5.7	197.0 ± 13.1*,#
Serum ALT (U/L)	54.8 ± 4.8	78.6 ± 3.5*	57.4 ± 4.2	94.4 ± 5.9*,#
Serum AST/ALT	1.61 ± 0.11	2.12 ± 0.10*	1.57 ± 0.13	2.10 ± 0.10*
Blood ethanol (mg/dl)	0.156 ± 0.149	50.0.63 ± 5.817*	0.300 ± 0.196	51.375 ± 5.732*
Blood acetaldehyde (μM)	1.14 ± 0.48	61.25 ± 6.25*	1.00 ± 0.51	63.80 ± 6.28*

BrW, brain weight; LW, liver weight; SkW, skeletal muscle weight; BW, body weight; AST, aspartate aminotransferase; ALT, alanine aminotransferase. Data were expressed as the mean ± SEM, n = 8.

Data were expressed as the mean ± SEM, n = 8.

^*P < 0.05 vs WT or corresponding PRK^−/− group.

^#P < 0.05 vs WT-ETOH group.

Figure 1

Effect of chronic alcohol intake (4%, 12 weeks) on IPGTT and protein carbonyl formation in WT and parkin-knockout(PRK^−/−) mice (A) Validation of parkin deletion in various organs by western blot analysis. (B) Intraperitoneal glucose tolerance test (IPGTT, 2 g/kg) in WT and PRK^−/− mice fasted for 12 h. (C) Area underneath the IPGTT curve plotted in panel B. (D) Brain protein carbonyl formation. (E) Liver protein carbonyl formation. (F) Rectus femoris skeletal muscle protein carbonyl formation. Data were expressed as the mean ± SEM, n = 8 (B,C) or 4 (D–F) mice per group. ^*P< 0.05 vs WT or corresponding PRK^−/− group; ^#P< 0.05 vs WT-ETOH.

Figure 2

Effect of chronic alcohol intake (4%, 12 weeks) on mitochondrial respiration in various organs in WT and parkin-knockout (PRK^−/−) mice (A) Brain CCO activity/complex IV. (B) Liver CCO activity/complex IV. (C) Skeletal muscle CCO activity/complex IV. (D) Brain complexes I and III. (E) Liver complexes I and III. (F) Skeletal muscle complexes I and III. (G) Brain complexes II and III. (H) Liver complexes II and III. (I) Skeletal muscle complexes II and III. Data were expressed as the mean ± SEM, n = 7 mice per group. ^*P < 0.05 vs WT or corresponding PRK^−/− group; ^#P< 0.05 vs WT-ETOH.

Protein carbonyl assay

To assess tissue oxidative damage, protein carbonyl content was measured. Proteins were extracted and minced to reduce proteolytic degradation. Protein was extracted using 20% trichloric acid and samples were resuspended in 10 mM 2,4-dinitrophenylhydrazine (2,4-DNPH) solution. Following centrifugation (11,000 g for 10 min), supernatants were removed, pellets were rinsed in ethanol:ethyl acetate (1:1, v:v) and precipitated in 6 M guanidine solution. Absorbance was measured using a microplate reader (BioTek, Winnooski, USA) at 360–390 nm and carbonyl content was determined using the equation of carbonyl content = absorption at 360 nm × 45.45 nmol/ml per protein content (mg) with a molar absorption coefficient of 22,000 M⁻¹ cm⁻¹ [37].

Mitochondrial electron transport chain enzymatic activity analysis

To measure the mitochondrial electron transport chain enzymatic activity, including cytochrome c oxidase (CCO), succinate:cytochrome c reductase, and reduced nicotinamide adenine dinucleotide (NADH):succinate cytochrome c reductase, mitochondria were isolated from respective tissues. CCO activity was measured through oxidation of reduced cytochrome c absorbance at 550 nm (∆A₅₅₀). To terminate the CCO reaction, potassium cyanide (0.17 mM) and succinate were added to measure succinate:cytochrome c reductase activity through ∆A₅₅₀. Then malonate was added to stop succinate:cytochrome c reductase. NADH (2.8 nM) was provided prior to assessment of NADH:cytochrome c reductase activity through ∆A₅₅₀ with a microplate reader (BioTek). The rate of cytochrome c oxidation or reduction (nmole per minute) was determined using a molar extinction coefficient of 19,600 for cytochrome c [38].

Tissue morphological assessment

Organs of interest were harvested, rinsed with PBS and were embedded in the optimal cutting temperature compound prior to snap frozen in liquid nitrogen. Frozen blocks were cut into 7-μm slides under −20°C using a cryostat microtome. Tissue slides were fixed with ice-cold 4% paraformaldehyde for 10 min, embedded in paraffin, cut into 5-μm sections and stained with hematoxylin and eosin (H&E) for tissue morphology examination [39].

Assessment of autophagic flux

To assess the role of autophagy flux in alcohol intake- and parkin knockout-induced organ damage, mice (5–6 mice per group) were treated with the lysosomal inhibitor bafilomycin A1 (BafA1, 3 μmol/kg, i.p.) daily for 7 days in the last week of the 12-week alcohol liquid diet feeding regimen. Brain, liver and skeletal muscle tissues were then collected prior to assessment of LC3BII-to-LC3BI ratio and p62 level by western blot analysis [40].

Western blot analysis

Tissue was homogenized in 1× RIPA lysis buffer and were centrifuged at 10,000 g for 15 min at 4°C. Bicinchoninic acid protein assay was utilized to measure the protein concentrations in the supernatant. A total of 50 μg of protein sample was loaded and separated on 10% or 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. After being blocked, the membranes were incubated with the following primary antibodies (Cell Signaling Technology, Danvers, USA) against parkin, Bcl-2, Bcl-XL, Bax, Atg5-Atg12 conjugate, Atg7, Beclin1, LC3B, TOM20 and GAPDH. The membranes were then incubated with the horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology) for 1 h at 37°C. After immunoblotting, films were detected using a Bio-Rad calibrated densitometer (BioRad, Hercules, USA) and the intensity of immunoblot bands was normalized with corresponding band intensity of GAPDH [41].

Figure 3

Effect of chronic alcohol intake (4%, 12 weeks) on brain morphology and autophagy protein markers in WT and parkin-knockout(PRK^−/−) mice (A) Representative brain H&E staining images (×200). (B) Brain LC3B ratio from WT and PRK^−/− mice treated with or without the lysosomal inhibitor Bafilomycin A1 (3 μmol/kg, i.p.) for 7 days during the last week of alcohol feeding regimen; Inset: Representative gel blot of LC3B and GAPDH (loading control). (C) Representative western blots of Atg5-Atg12 conjugate, Atg7, Beclin1, p62 and TOM20 using specific antibodies (GAPDH as loading control). (D) Atg5-Atg12 conjugate. (E) Atg7. (F) Beclin1. (G) p62. (H) TOM20. Data were expressed as the mean ± SEM, n = 6–9 mice per group. ^*P< 0.05 vs WT or corresponding PRK^−/− group; ^#P< 0.05 vs WT-ETOH.

Figure 4

Effect of chronic alcohol intake (4%, 12 weeks) on liver morphology and autophagy protein markers in WT and parkin-knockout(PRK^−/−) mice (A) Representative liver H&E staining images (× 400). (B) Liver LC3B ratio from WT and PRK^−/− mice treated with or without the lysosomal inhibitor Bafilomycin A1 (3 μmol/kg, i.p.) for 7 days during the last week of alcohol feeding regimen. Inset: representative gel blot of LC3B and GAPDH (loading control). (C) Representative western blots of Atg5-Atg12 conjugate, Atg7, Beclin1, p62 and TOM20 using specific antibodies (GAPDH as loading control). (D) Atg5-Atg12 conjugate. (E) Atg7. (F) Beclin1. (G) p62. (H) TOM20. Data were expressed as the mean ± SEM, n = 6–9 mice per group. ^*P < 0.05 vs WT or corresponding PRK^−/− group; ^#P < 0.05 vs WT-ETOH.

Figure 5

Effect of chronic alcohol intake (4%, 12 weeks) on rectus femoris skeletal muscle morphology and autophagy protein markers in WT and parkin-knockout (PRK^−/−)mice (A) Representative skeletal muscle H&E staining images (× 200). (B) Skeletal muscle LC3B ratio from WT and PRK^−/− mice treated with or without the lysosomal inhibitor Bafilomycin A1 (3 μmol/kg, i.p.) for 7 days during the last week of alcohol feeding regimen. Inset: representative western blots of LC3B and GAPDH (loading control). (C) Representative western blots of Atg5-Atg12 conjugate, Atg7, Beclin1, p62 and TOM20 using specific antibodies (GAPDH as loading control). (D) Atg5-Atg12 conjugate. (E) Atg7. (F) Beclin1. (G) p62. (H) TOM20. Data were expressed as the mean ± SEM, n = 6–9 mice per group. ^*P< 0.05 vs WT or corresponding PRK^−/− group; ^#P< 0.05 vs WT-ETOH.

Caspase-3 activity assay

Tissues were homogenized and centrifuged, and pellets were lysed in 100 μl of ice-cold lysis buffer containing 50 mM HEPES, 0.1% CHAPS, 1 mM dithiothreitol, 0.1 mM EDTA, and 0.1% NP40. Following cell lysis, reaction buffer (70 μl) was added to each well of 96-well plates, followed by addition of caspase-3 colorimetric substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA, 20 μl/well; Sigma-Aldrich, St Louis, USA). After 1 h of incubation at 37°C, the OD values were measured with a microplate reader (BioTek) at 405 nm. Caspase-3 cleaved the chromophore pNA from its substrate molecule, and caspase-3 activity was expressed as picomoles of pNA released per microgram of protein per minute [41].

Statistical analysis

Data are expressed as the mean ± SEM. All statistical analyses were performed using a one-way analysis of variance (2-way for IPGTT). Statistical significance was set at P < 0.05.

Results

Impact of parkin ablation on chronic alcohol intake-induced biometrics, IPGTT, liver function and protein carbonyl formation changes

The 12-week chronic alcohol intake did not affect body weight, although it significantly decreased brain and rectus femoris weights (or size), while promoting hepatomegaly, with a more pronounced effect in parkin-knockout mice. Parkin deletion itself did not overtly affect body and organ weights. Levels of liver enzymes including AST, ALT and AST/ALT ratio were overtly elevated following chronic alcohol intake. Although parkin ablation itself failed to affect the levels of AST and ALT, it accentuated chronic alcoholism-induced liver injury (without affecting AST/ALT ratio). Blood ethanol and the ethanol metabolite acetaldehyde levels were elevated in a comparable manner in both WT and PRK^−/− mice following chronic alcohol intake (Table 1). Parkin deletion was confirmed in brain, liver and rectus femoris skeletal muscle, the effect of which was not affected by chronic alcohol intake (Fig. 1A). Following intraperitoneal glucose challenge, serum glucose level began to drop after peaking at 15 min and returned toward near baseline after 120 min. Chronic alcohol intake provoked a subtle although significantly higher blood glucose levels between 30 and 120 min following glucose challenge with a peak at 30 min, in a comparable manner in both WT and PRK^−/− groups (Fig. 1B). This is further supported by a greater area underneath the IPGTT curve (AUC) in alcohol-fed WT or PRK^−/− mice (Fig. 1C), indicating overt and comparable glucose intolerance following chronic alcohol intake in both WT and PRK^−/− mice. Evaluation of tissue damage using protein carbonyl formation revealed higher levels of carbonyl content in brain, liver and skeletal muscle following chronic alcohol intake, the effect of which was exacerbated by parkin deletion with little effect from parkin knockout itself (Fig. 1D–F). These data indicated a role of intrinsic parkin in the preservation against alcohol-induced protein carbonyl damage.

Impact of parkin ablation on chronic alcohol intake-induced mitochondrial injury

CCO, the terminal respiratory complex (complex IV) of the mitochondrial respiratory chain, has been shown to be compromised with chronic alcohol intake [7,42], which prompts oxidative stress in alcoholism. Data from our study revealed reduced CCO levels in brain, liver and skeletal muscle following chronic alcohol intake, the effect of which was augmented by parkin deletion with little effect from parkin deletion itself (Fig. 2A–C). To assess the contribution of the CCO-independent mitochondrial respiration in chronic alcoholism, NADH:cytochrome c reductase, which denotes joint function of respiratory complexes I and III, and succinate:cytochrome c reductase, which represents joint activity of respiratory complexes II and III, were evaluated. Consistent with the effects on CCO activity, chronic alcohol intake significantly dampened NADH:cytochrome c reductase or succinate:cytochrome c reductase in brain, liver and skeletal muscle, the effects of which were accentuated by parkin ablation; in addition, parkin deletion itself did not seem to affect these electron transport chain function (Fig. 2D–I). These findings supported the presence of compromised electron transport chain in chronic alcoholism, with a more pronounced response with the combination of parkin ablation.

Effect of parkin ablation on chronic alcohol intake-induced tissue morphology and autophagy or mitophagy protein markers

The effect of chronic alcohol intake and parkin knockout on tissue morphology in brain, liver and skeletal muscle was further assessed by H&E staining. It was found that chronic alcohol intake elicited focal inflammation (e.g. neutrophil infiltration), necrosis and microvesiculation in WT and PRK^−/− mouse brain (Fig. 3A), liver (Fig. 4A) and skeletal muscle (Fig. 5A). Deletion of the mitophagy protein parkin itself did not affect tissue morphology in the absence of alcohol challenge. To examine the possible role of autophagy in tissue injury in chronic alcohol intake- and parkin deletion-induced responses, autophagy protein markers including Atg5-Atg12 conjugate, Atg7, Beclin1, LC3B and p62 were analyzed by western blot analysis. Our data showed that chronic alcohol intake significantly downregulated protein levels of Atg5-Atg12 conjugate, Atg7, Beclin1 and LC3BII-to-LC3BI ratio, but upregulated the protein levels of p62 and mitophagy protein marker TOM20 (indicative of decreased mitophagy) in brain (Fig. 3), liver (Fig. 4) and skeletal muscle (Fig. 5), the effect of which was accentuated by parkin deletion with little effect from parkin ablation itself with the exception of brain Beclin1. Brain Beclin1 level was downregulated by parkin deletion in the absence of alcohol challenge (Fig. 3F). To further evaluate the possible contribution of lysosomal degradation (autophagy flux) to ethanol- and parkin deletion-induced autophagy response, the lysosomal inhibitor Bafilomycin A1 was given to experimental animals during the last week of the 12-week feeding regimen prior to assessment of LC3BII-to-LC3BI ratio. Our data revealed that Bafilomycin A1 treatment did not overtly affect the patterns of ethanol- and parkin deletion-elicited responses on LC3BII-to-LC3BI ratio in brain (Fig. 3B), liver (Fig. 4B) and skeletal muscle (Fig. 5B). These data suggested that the early autophagosome formation might be decreased in ethanol-induced autophagy response, the effect of which may be further dampened by parkin deletion.

Effects of parkin ablation on chronic alcohol intake-induced apoptosis markers

To investigate the possible contribution of apoptosis in chronic alcohol intake- and parkin ablation-induced tissue damage, the protein markers of apoptosis including Bax and Bcl-2/Bcl-XL as well as Caspase-3 activity were evaluated. Western blot analysis results indicated that level of Bax was overtly elevated in brain, liver and rectus femoris muscle following chronic alcohol intake, the effect of which was accentuated by parkin ablation (Fig. 6A–C). Chronic alcohol intake downregulated Bcl-2 in brain tissues but upregulated Bcl-2/Bcl-XL in liver and skeletal muscle; the effect of which was reversed by parkin ablation (Fig. 6D–F), while parkin knockout itself did not affect the levels of these apoptotic protein markers. Assessment of Caspase-3 activity suggested that chronic alcohol intake overtly increased Caspase-3 activity in brain, liver and skeletal muscle, the effect of which was overtly exacerbated by parkin deletion with little effect from parkin deletion itself (Fig. 6G-I). These data indicated that parkin insufficiency exacerbated chronic alcohol intake-induced cell death in brain, liver and skeletal muscle.

Figure 6

Effect of chronic alcohol intake (4%, 12 weeks) on apoptosis markers in various organs in WT and parkin-knockout(PRK^−/−) mice (A) Brain Bax level. (B) Liver Bax level. (C) Skeletal muscle Bax level. (D) Brain Bcl-2 level. (E) Liver Bcl-XL level. (F) Skeletal muscle Bcl-2 level. (G) Brain Caspase-3 activity. (H) Liver Caspase-3 activity. (I) Skeletal muscle Caspase-3 activity. Insets: representative western blots of Bax, Bcl-2/Bcl-XL using specific antibodies (GAPDH as loading controls). Data were expressed as the mean ± SEM, n = 6–9 (A–F) and 5 (G–I) mice per group. ^*P< 0.05 vs WT or corresponding PRK^−/− group; ^#P< 0.05 vs WT-ETOH.

Discussion

The salient findings from our study suggested that parkin deletion exacerbates chronic alcohol intake-induced protein carbonyl damage, mitochondrial injury, apoptosis, and autophagy failure in brain, liver, and rectus femoris skeletal muscle. Morphometric examination suggested the presence of atrophy in brain and skeletal muscle along with hepatomegaly following chronic alcohol intake. Previous reports have demonstrated a rather important role for derangement of autophagy and particularly parkin-mediated mitophagy in the organ and tissue homeostasis in alcoholic diseases [25,27,29,43–45]. While effective clinical management of alcoholic organ injury remains challenging, our findings favored the role of the mitophagy protein parkin as a potential therapeutic target in the management of organ damage in the face of chronic alcohol intake.

In our hands, parkin deletion by itself did not generate any notable phenotype or effect on protein carbonyl formation, mitochondrial respiration, apoptosis or autophagy (except for brain Beclin1), suggesting a lesser role of parkin in cellular and tissue homeostasis in the physiological settings. This is supported by the comparable morphology in various tissues between WT and PRK^−/− mice in the absence of alcohol intake. Nonetheless, parkin deficiency accentuated chronic alcohol intake-induced apoptosis, protein damage, mitochondrial injury and autophagy failure. Morphological data revealed subtle but identifiable focal inflammation (neutrophil infiltration), necrosis and microvesiculation in brain, liver and skeletal muscle tissues from alcohol-fed mice. Chronic alcohol intake overtly elevated blood ethanol and its metabolite acetaldehyde levels, compromised liver function (serum AST and ALT), glucose handling capacity (glucose tolerance), protein carbonyl formation, mitochondrial electron transport (complex IV, complexes I and III, complexes II and III), autophagy failure along with apoptosis (elevated caspase-3 activity, Bax and Bcl-2/Bcl-XL, with the exception of a drop in brain Bcl-2). The comparable blood acetaldehyde levels suggest little effect of parkin deletion on ethanol metabolism. These findings from whole body metabolism, brain, liver and rectus femoris skeletal muscle are consistent with the earlier notion of overt body and organ damage in response to chronic alcohol intake [1,5,19]. It should be noted that post ethanol feeding body weights were similar among all four experimental groups, consistent with previous studies [27,46]. This is mostly courtesy of the pair-feeding protocol using the nutritionally adequate Liber De Carli ethanol liquid diet (36% from ethanol), despite reduced body weight reported elsewhere [47] possibly due to fluid balance, sex and animal strain.

Perhaps the most intriguing finding from this study is the augmented tissue damage in parkin-knockout mice following alcohol intake. Our data suggested that ethanol consumption may be more devastating in individuals with parkin deficiency. Mitophagy denotes the selective removal of long-lived and damaged mitochondria using evolutionarily conserved autophagy process in mitochondria [33]. Mitophagy helps to preserve tissue homeostasis under both physiological and pathological conditions [23,30]. Earlier evidence has convincingly depicted a protective role of mitophagy induction against certain diseases such as cancer, inflammatory diseases, aging, diabetes, and insulin resistance [34,48]. Proper mitophagy levels are required to maintain organ and tissue homeostasis, whereas dysregulated autophagy was reported to impair muscle health [49]. At this time, direct evidence is still limited for mitophagy change in response to chronic alcohol challenge. Earlier evidence revealed that parkin loss exacerbated ethanol-induced dopaminergic neurodegeneration through p38-dependent inhibition of autophagy and mitochondrial function [17], in line with data from our current study where brain atrophy, protein damage, apoptosis, mitochondrial respiration injury and autophagy failure (likely in early autophagosome formation as evidenced by the Bafilomycin A1 data) were noted. Although it was suggested that autophagy may serve as a protective measure against neurotoxicity of ethanol [50], prolonged exposure to ethanol simply depletes autophagy pool, leading to autophagy failure. To date, no information is available for mitophagy response in skeletal muscle under alcoholism. It was reported that binge alcohol intake compromised mitochondrial-related genes (e.g. PGC-1α) in skeletal muscle, although change of mitophagy remained uncertain [51]. Data from our current study revealed exacerbated muscle loss, protein damage, mitochondrial respiration injury, apoptosis and autophagy failure (likely in early autophagosome formation as evidenced by the Bafilomycin A1 data) in rectus femoris from alcohol-fed PRK^−/− mice. In contrary to atrophy in brain and skeletal muscle, chronic alcohol intake led to hepatomegaly as seen in our current study, consistent with previous reports [19,46,52]. Despite hepatomegaly, alcohol intake elicited hepatic responses in carbonyl damage, mitochondrial respiration injury, apoptosis and autophagy failure (likely in early autophagosome formation) reminiscent of those in brain and skeletal muscle. Bcl-2/Bcl-XL is an anti-apoptotic protein and upregulated levels in liver and skeletal muscle in the face of chronic alcoholism (more pronounced in PRK^−/− mice) may suggest compensatory responses. Our data revealed downregulated autophagy initiating genes including Beclin1, a core complex of class III PI3K, Atg5-Atg12 conjugate and Atg7 following chronic alcohol intake, the effect of which was accentuated by parkin deficiency. These autophagy proteins are essential in the early autophagosome formation through conversion of cytosolic LC3I to the autophagosome-bound LC3II [53,54]. It is noteworthy that parkin deletion does not affect macroautophagy levels with the exception of brain Beclin1, suggesting a higher sensitivity for brain Beclin1 to parkin (albeit little morphological deficit was noted in PRK^−/− mouse brain). Earlier evidence supported that alcohol (using Gao-Binge model) provoked greater mitochondrial damage (swollen and damaged mitochondria lacking cristae, mitochondrial respiration defect), loss in mitophagy, β-oxidation, and cytochrome c oxidase function along with oxidative stress in livers from parkin-knockout mice compared with those from WT mice [29], in line with our present study.

Results from our study revealed that chronic alcohol intake dampened tissue autophagy in association with greater levels of protein carbonyl damage, mitochondrial injury and apoptosis, the effects of which were accentuated by parkin ablation. Ample evidence has consolidated a close association between autophagy and apoptosis. On one hand, the apoptotic protein Bcl-2/Bcl-XL suppresses autophagy by way of binding and separating Beclin1 from class III PI3K complex, thus suffocating Beclin1-dependent autophagy [54]. On the other hand, Beclin1 was reported to promote apoptosis by way of Caspase-9 [55]. These findings favor a reciprocal regulation between autophagy and apoptosis in the face of chronic alcohol intake and parkin deficiency. Last but not the least, parkin deletion did not alter chronic alcohol intake-induced glucose intolerance, suggesting minimal role of parkin in global insulin and glucose metabolism. The unaltered AST/ALT ratio (both greater than 2.0) in alcohol-fed WT and PRK^−/− groups indicates the presence of alcoholic hepatitis following chronic alcohol intake [52].

In summary, findings from our study provided convincing evidence that deficiency in the mitophagy adaptor parkin accentuates chronic alcohol intake-induced organ injury (protein carbonyl formation, apoptosis, mitochondrial respiration and autophagy failure) in brain, liver and skeletal muscle. These outcomes should shed some light toward a better understanding of the role of parkin in alcoholic organ damage. Given the apparent controversy of adaptive or maladaptive autophagy and mitophagy in alcoholic models, the use of pharmacological inducers of parkin or transgenic overexpression of the mitophagy protein merits further scrutiny to better define the precise role of parkin and parkin-mediated mitophagy as well as the mechanism of action involved in alcoholic organ injury.

Funding

The work was supported in part by the grants from the National Natural Science Foundation of China (Nos. 81570225 and 81671938).

Author notes

Hu Peng and Xing Qin These authors contributed equally to this work.