Dihydrolipoic Acid Inhibits Lysosomal Rupture and NLRP3 Through Lysosome-Associated Membrane Protein-1/Calcium/Calmodulin-Dependent Protein Kinase II/TAK1 Pathways After Subarachnoid Hemorrhage in Rat
Background and Purpose—The NLRP3 (nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3) inflammasome is a crucial component of the inflammatory response in early brain injury after subarachnoid hemorrhage (SAH). In this study, we investigated a role of dihydrolipoic acid (DHLA) in lysosomal rupture, NLRP3 activation, and determined the underlying pathway.
Methods—SAH was induced by endovascular perforation in male Sprague–Dawley rats. DHLA was administered intraperitoneally 1 hour after SAH. Small interfering RNA for lysosome-associated membrane protein-1 and CaMKIIα (calcium/calmodulin-dependent protein kinase II α) was administered through intracerebroventricular 48 hours before SAH induction. SAH grade evaluation, short- and long-term neurological function testing, Western blot, and immunofluorescence staining experiments were performed.
Results—DHLA treatment increased the expression of lysosome-associated membrane protein-1 and decreased phosphorylated CaMKIIα and NLRP3 inflammasome, thereby alleviating neurological deficits after SAH. Lysosome-associated membrane protein-1 small interfering RNA abolished the neuroprotective effects of DHLA and increased the level of phosphorylated CaMKIIα, p-TAK1 (phosphorylated transforming growth factor-β-activated kinase), p-JNK (phosphorylated c-Jun-N-terminal kinase), and NLRP3 inflammasome. CaMKIIα small interfering RNA downregulated the expression of p-TAK1, p-JNK, and NLRP3 and improved the neurobehavior after SAH.
Conclusions—DHLA treatment improved neurofunction and alleviated inflammation through the lysosome-associated membrane protein-1/CaMKII/TAK1 pathway in early brain injury after SAH. DHLA may provide a promising treatment to alleviate early brain injury after SAH.
- calcium-calmodulin-dependent protein kinase type 2
- dihydrolipoic acid
- lysosomal-associated membrane protein 1
- subarachnoid hemorrhage
Early brain injury (EBI), which occurs within 72 hours after subarachnoid hemorrhage (SAH), has recently been considered a major cause of the poor outcome of patients with SAH.1 The underlying mechanisms include a reduction in cerebral blood flow, increased intracranial pressure, oxidative stress, apoptosis, blood–brain barrier disruption, and inflammation.2 Recently, increasing evidence has indicated the role of the NLRP3 (nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3) inflammasome as a key component of post-SAH inflammatory response.3,4 The disruption of the lysosomal membrane and lysosomal rupture leads to the release of cathepsin B/D, which has been shown to induce the activation of the NLRP3 inflammasome.5 In the SAH rat model, it has also been shown that the lysosomal membrane may be damaged after SAH, which leads to the release of cathepsin B/D and induces apoptosis.6,7 Previous studies have shown that lysosomal rupture regulates NLRP3 inflammasome activation through the TAK1/JNK pathway. In addition, Ca2+ ions from lysosomal rupture are an important factor to activate this pathway through CaMKII (calcium/calmodulin-dependent protein kinase II) in vitro.8 However, the mechanisms between lysosomal rupture and NLRP3 activation after SAH are still unclear.
LAMP1 (lysosomal-associated membrane protein-1) is the most abundant lysosomal membrane protein and is regarded as a marker to evaluate the stability of lysosomes.9 In addition, studies have shown that the LAMP1 protein not only maintains the structural integrity of the lysosomal membranes but also closely associated with cell autophagy and apoptosis.10,11 However, the role of LAMP1 contributing to inflammation in EBI after SAH has not been studied.
Dihydrolipoic acid (DHLA), an active form of lipoic acid (LA), is a powerful electron donor, induced by lipoamide dehydrogenase in the cell.12 It can stabilize the lysosomal membrane, decrease oxidative stress, and exert beneficial effects in rat models of various diseases.6,13 To date, no study has investigated the anti-inflammation role of DHLA in SAH models. In the present study, we hypothesized that DHLA treatment could inhibit lysosomal rupture and attenuate NLRP3 activation through the LAMP1/CaMKII/TAK1 pathway in EBI after SAH.
Materials and Methods
The authors declare that all supporting data are available in the article and in the online-only Data Supplement.
Animals and SAH Model
Adult male Sprague–Dawley rats (n=184; 290–330 g) were housed in a room with constant temperature (25°C), humidity control, a 12/12 hour light/dark cycle, and free access to food and water. All the experimental procedures were approved by the Institutional Animal Care and Use Committee at Loma Linda University and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
The SAH model was conducted by the modified endovascular perforation method.14 Briefly, rats were anesthetized and kept on a ventilator during surgery with 3% isoflurane in 65/35% medical air/oxygen. The left external and internal carotid arteries were exposed, and then a 4.0-monofilament nylon suture was inserted into the left internal carotid artery through the external carotid artery stump until resistance was felt. Then, the suture was advanced 3 mm to perforate the bifurcation of the anterior and middle cerebral artery. Sham rats underwent the same procedures except the perforation. The incision was then closed, and rats were housed individually in heated cages after recovery from anesthesia.
Thirty-eight rats were divided into 6 groups (sham, and SAH after 3, 6, 12, 24, and 72 hours, n=6). The additional 2 rats in the SAH (24 hours) group were used for immunofluorescence staining. The temporal expression of LAMP1 and phosphorylated CaMKIIα (p-CaMKIIα) was detected by Western blot. Immunofluorescence staining was performed to test the localization of LAMP1 and p-CaMKIIα in the neurons, astrocytes, and microglia (Figure I in the online-only Data Supplement).
A total of 72 rats were divided into 5 groups: the sham (n=20 including 6 rats for neurological tests and Western blot, 4 rats for immunofluorescence staining, and 10 rats for long-term study), SAH+vehicle (n=20), SAH+DHLA (10 mg/kg; n=6), SAH+DHLA (30 mg/kg; n=20), and SAH+DHLA (90 mg/kg, n=6). Based on neurological tests, 30 mg/kg of DHLA-treated group of SAH was chosen for Western blot, immunofluorescence, and long-term neurobehavior experiments.
Thirty rats were randomly divided into 5 groups: sham (n=6), SAH+vehicle (n=6), SAH+DHLA (best dosage; n=6), SAH+DHLA+scramble small interfering ribonucleic acid (siRNA; n=6), and SAH+DHLA+LAMP1 siRNA (n=6). Another 18 rats were randomly divided into 3 groups: sham (n=6), SAH+scramble siRNA (n=6), and SAH+CaMKIIα siRNA (n=6). The relevance factors were tested by Western blot.
DHLA was purchased from Sigma. Before injection, it was diluted in dimethyl sulfoxide and PBS. DHLA was administered in the SAH+DHLA group 1 hour after surgery by intraperitoneal injection. The SAH+vehicle group received an equal volume of dimethyl sulfoxide and PBS.
LAMP1 siRNA (SR508640), CaMKIIα siRNA (SR500835), and scramble siRNA were purchased from Origene Technologies, Inc (MD). A total of 500 pmol in 5 μL was injected intracerebroventricularly at 48 hours pre-surgery as previously described.15
Measurement of SAH Grade
The SAH grading score was used to estimate the degree of SAH as previously described.14 The grading of SAH was performed by a partner who was blind to the experiment. Rats with the SAH grade <9 were excluded from this study.
Assessment of Short-Term Neurological Function
The neurological status of all rats was evaluated at 24 hours after SAH induction using the previously described modified Garcia scoring system and beam balance test.14 The assessment of neurological score was performed by a partner who was blind to the experiment.
Assessment of Long-Term Neurobehavior
The rotarod test was performed at the first-, second-, and third-week post-SAH to assess sensorimotor coordination and balance as previously described.16 Water maze test was performed at days 21 to 25 post-SAH as previous study showed.17
Western Blot Analysis
Western blot was performed as previously described.18 Briefly, proteins of the left hemisphere were extracted by homogenizing in radio immunoprecipitation assay (RIPA) buffer (Santa Cruz Biotechnology, CA). The primary antibodies were from Abcam (MA) used with following dilution: LAMP1 (1:1000, ab24170), CaMKII (1:5000, ab52476), p-CaMKII (1:2000, ab32678), TAK1 (1:1000, ab109536), p-TAK1 (1:1000, ab109404), JNK (1:2000, ab179461), p-JNK (1:2000, ab131499), NLRP3 (1:500, ab214185), and interleukin (IL)-1β (1:500, ab9787). Caspase-1 was from NOVUS (CO, 1:500, NBP1-45433).
Rats were euthanized at 24 hours after SAH induction. A series of 10-μm slices were prepared. Double immunofluorescence staining was performed as previously described.19 The primary antibodies were LAMP1 (1:200, ab24170), p-CaMKII (1:250, ab32678), and ionized calcium binding adaptor molecule-1 (1:200, ab5076).
Data were presented as mean±SEM. One-way ANOVA was used to compare means of different groups followed by a Tukey multiple comparisons test. Statistical significance was defined as P<0.05.
Mortality and SAH Severity Scores
A total of 184 rats were used, 38 rats were sham and 146 rats underwent SAH induction. The mortality of SAH rats was 13.0% (19 of 146), and no rats dying in the sham group. Seven rats were excluded because of low-grade SAH. Blood clots were mainly observed around the circle of Willis and ventral brain stem after SAH induction. The average SAH grade score had no statistical difference among all SAH groups (Figure II in the online-only Data Supplement).
Temporal Patterns of LAMP1, p-CaMKII Were Detected in Left Hemisphere After SAH
Western blot was performed to determine the protein expression of LAMP1, p-CaMKIIα at 3, 6, 12, 24, and 72 hours in the left hemisphere of the rat brain after SAH. Results showed that LAMP1 level decreased as early as 3 hours after SAH and reached its lowest level ≈24 hours (P<0.05; Figure 1A). In addition, the expression of p-CaMKIIα increased after SAH and peaked at 24 hours, and there was notable difference compared with sham animals (P<0.05; Figure 1B). Double immunofluorescence staining was performed to detect localization of the LAMP1 and p-CaMKIIα in the neurons (neuronal nuclear), astrocytes (GFAP [glial fibrillary acidic protein]), or microglia (ionized calcium binding adaptor molecule-1). We found that LAMP1 was expressed on all type of cells and was mainly colocalized with neuron and microglia in the cortex at 24 hours after SAH (Figure 1C). We also found that p-CaMKIIα was mainly colocalized with neuron and microglia (Figure 1D).
DHLA Improved Short- and Long-Term Neurobehavior
The modified Garcia and beam balance scores were significantly lower in the SAH+vehicle group than those in the sham group (P<0.01; Figure 2A), and the administration of DHLA improved the neurological scores in SAH+DHLA group. The administration of middle dosage (30 mg/kg) significantly improved the neurological scores compared with the SAH+vehicle group (P<0.05; Figure 2A) and seemed to be the most effective dosage. Therefore, we chose this dosage for the long-term and mechanism studies.
In the Rotarod test, the SAH+vehicle group had a significantly shorter latency to fall compared with the sham group both in the 5 revolution per minute (RPM) and 10RPM accelerating velocity tests (P<0.01; Figure 2B). One week after SAH, DHLA treatment improved performance significantly in the 5RPM test (P<0.05; Figure 2B, left). Finally, 2 weeks after SAH, DHLA improved the performance in both the 5RPM and 10RPM tests significantly (P<0.05 in 5RPM, P<0.01 in 10RPM; Figure 2B).
In the water maze test, all the groups performed equally in velocity (Figure 2C, left). In the spatial maze test, the SAH+vehicle group traveled a longer distance to find the platform and had a longer escape latency than sham group, and it was significantly improved by DHLA treatment on the performance of day 2 to 4 and block 2 to 4 in SAH+DHLA group (P<0.05; Figure 2D). In the probe trials, the SAH+vehicle group spent less time in the target quadrant when the platform was removed compared with the sham group. DHLA treatment notably improved the duration spent in the probe quadrant for the SAH+DHLA group (P<0.05; Figure 2C, right).
DHLA Increased the Expression of LAMP1 and Inhibited the Expression of p-CaMKIIα, NLRP3, Cleaved Caspase-1, and IL-1β
At 24 hours after SAH, the expression of LAMP1 was remarkably decreased, whereas p-CaMKIIα, NLRP3, cleaved caspase-1, and IL-1β were dramatically increased in SAH+vehicle group compared with the sham group (P<0.01; Figure 3). However, DHLA (30 mg/kg) treatment enhanced the level of LAMP1 and inhibited the expression of p-CaMKIIα, NLRP3, cleaved caspase-1, and IL-1β in SAH+DHLA group when compared with SAH+vehicle group but not in the sham+DHLA group (P<0.05; Figure 3; Figure III in the online-only Data Supplement).
DHLA Inhibited Lysosomal Rupture in Microglia
The results from double immunofluorescence staining of LAMP1, p-CaMKIIα, and ionized calcium binding adaptor molecule-1 indicated that LAMP1 expression in microglia was decreased (Figure 4A), but p-CaMKIIα expression was increased (Figure 4B) after SAH. These alterations were reversed by DHLA treatment in SAH+DHLA group (Figure 4).
Knockdown LAMP1 Abolished the Anti-Inflammation Effect of DHLA After SAH
LAMP1 knockdown markedly reversed the neurological improvements of SAH+DHLA (30 mg/kg) rats on modified Gracia and beam balance test at 24-hour post-SAH. In addition, there was a significant difference compared with SAH+DHLA+scramble siRNA group (P<0.05; Figure 5A and 5B). Moreover, LAMP1 siRNA intervention significantly inhibited the expression of LAMP1 while increasing p-TAK1, p-JNK, p-CaMKIIα, and NLRP3 expression when compared with SAH+DHLA+scramble siRNA group (P<0.05; Figure 5C).
CaMKIIα siRNA Decreased the Protein Level of p-CaMKIIα, p-TAK1, p-JNK, and NLRP3
To study the relationship between CaMKII and TAK1/JNK in regulation of NLRP3 after SAH, CaMKIIα siRNA was administered intracerebroventricularly at 48 hours before SAH induction. The Western blot results showed that CaMKIIα knockdown markedly increased the neurological score on modified Garcia and beam balance test in SAH+CaMKIIα siRNA group compared with SAH+scramble siRNA group (P<0.01; Figure 6A and 6B). The expressions of p-TAK1, p-JNK, and NLRP3 were significantly decreased in SAH+CaMKIIα siRNA group compared with SAH+scramble siRNA group (P<0.05; Figure 6C).
In the present study, we first found that the expression of LAMP1 decreased, the expression of p-CaMKIIα increased in the brain 24 hours after SAH, and that they were mainly expressed in neurons and microglia. In addition, the DHLA treatment improved both short- and long-term neurofunction after SAH, which were accompanied by an increase in LAMP1 expression and a decrease in p-CaMKIIα and NLRP3 inflammasome expression. Furthermore, knockdown of LAMP1 abolished the neuroprotective effects of DHLA, which were associated with the increased expression of p-CaMKIIα, p-TAK1, p-JNK, and NLRP3 inflammasome at 24 hours after SAH. Moreover, the knockdown of CaMKIIα downregulated the expression of p-TAK1, p-JNK, and NLRP3 and improved neurobehavior at 24 hours after SAH. These findings suggested that the administration of DHLA could inhibit lysosomal rupture, attenuate NLRP3 inflammasome activation, and improve neurofunction after SAH at least, in part, through the LAMP1/CaMKII/TAK1 signaling pathway.
The role of NLRP3 inflammasome in the pathophysiology of EBI after SAH has been well established.20 Once activated, the NLRP3 inflammasome causes transformation of procaspase-1 into cleaved caspase-1 and maturation of IL-1β and IL-18, subsequently contributes to inflammation after SAH.4 There are 3 key mechanisms regulating the activation of the NLRP3 inflammasome: the generation of reactive oxygen species, the efflux of potassium, and the rupture of the lysosome.21 A recent study found that lysosomal rupture triggered cathepsin-dependent protein degradation and activated the NLRP3 inflammasome.22 Another study demonstrated that the CaMKII/TAK1/JNK pathway was activated through lysosomal rupture and caused the activation of the NLRP3 inflammasome.8 However, the mechanisms between lysosomal rupture and NLRP3 activation after SAH have not been elucidated. In the present study, we found that lysosomal rupture can activate the NLRP3 inflammasome through the LAMP1/CaMKII/TAK1 signaling pathway.
LAMP1 is a major component of the lysosomal membrane. LAMP1 was originally thought to protect the lysosomal membrane and was regarded as a lysosomal marker to evaluate the stability of the lysosome state.9 However, increasing evidence has suggested that LAMP1 has functions beyond the initially suggested roles in maintaining the integrity of the lysosomal membrane.10 It has been shown that the appearance of LAMP1 accompanied apoptosis. LAMP1 has a crucial role in contributing to the formation of autophagosomes and leads to the progress of autophagy.11 In our study, we found that the LAMP1 expression decreased and reached the lowest level at 24 hours after SAH and was mainly expressed on neurons and microglia, indicating an increase of lysosomal rupture after SAH induction. It also revealed some relationship between lysosomal rupture and inflammation after SAH because the activation of microglia is associated with neuroinflammation.23
LA and its reduced form, DHLA, have been regarded as effective antioxidant molecules. LA is reduced by lipoamide dehydrogenase to the corresponding DHLA, which is more effective in performing antioxidant functions.24 The antioxidant properties of LA and DHLA has been shown in many diseases associated with redox status imbalance, such as diabetes mellitus and cardiovascular diseases.25 Moreover, the neuroprotective effects of LA and DHLA have been shown in many models of central nervous system disease, such as ischemic stroke, traumatic brain injury, and Alzheimer disease.26–28 And proper dose of LA has been proved to alleviate oxidative stress and reduce formation of reactive oxygen species in SAH model.13 In our study, we found the DHLA treatment significantly improved the short- and long-term neurobehavior after SAH. In addition, the middle dosage of DHLA (30 mg/kg) significantly increased the expression of LAMP1 and decreased the expression of the NLRP3 inflammasome after SAH, indicating that DHLA inhibited lysosomal rupture and the activation of the NLRP3 inflammasome. It is consistent with a recent reporting that DHLA can stabilize the lysosomal membrane and improve neurofunction.6 To study whether the anti-inflammation and neuroprotective effects of DHLA are associated with LAMP1 expression, we used LAMP1 siRNA intracerebroventricular injection 48 hours before SAH induction. The results showed that knockdown of LAMP1 abolished the neuroprotective effects of DHLA and reversed the expression of downstream proteins, indicating an essential role of lysosomal rupture and LAMP1 in the downstream pathway.
A study in vitro showed that lysosomal rupture regulated NLRP3 inflammasome activation through the CaMKII/TAK1/JNK pathway.8 CaMKII is a sensitive Ca2+ receptor. The Ca2+ released from lysosomal rupture is sufficient to evoke CaMKII and activate the downstream factors. CaMKIIα is one of the major forms of CaMKII, and it can sustain the activation of CaMKII. CaMKIIα has also been proved to contribute to inflammation in microglia.29 So in our study, we mainly focus on the CaMKIIα, and we found the expression of p-CaMKIIα increased after SAH and peaked at 24 hours after SAH induction, which is consistent with the degree of lysosomal rupture. In addition, DHLA, the lysosomal membrane stabilizer, significantly inhibited the expression of p-CaMKIIα. Meanwhile, the LAMP1 siRNA reversed the decreasing level of p-CaMKIIα by DHLA. These results indicate that CaMKII maybe a critical downstream effector of lysosomal rupture and LAMP1. And DHLA treatment may function through LAMP1/CaMKII pathway. However, the Western blot and immunohistochemistry staining results of CaMKIIα may have cross-reaction with CaMKIIβ, which has similar structural domain and close relationship with CaMKIIα. Because the α subunit is the predominant form in forebrain and the β subunit is the dominant form in the cerebellum,30 we used whole left hemisphere of the brain in the experiments and think that CaMKIIα should be responsible for the major function although there may be partly mixed with some CaMKIIβ. Moreover, the silencing of CaMKIIα significantly improved neurofunction. This result is consistent with a recent study showing that CaMKII inhibitor prevented impaired sensorimotor function after SAH.31 In addition, several studies have demonstrated that CaMKII regulates the activity of TAK1/JNK, which is a central molecule in multiple signaling pathways.32–34 However, little is known about the exact downstream signaling cascade initiated by CaMKII/TAK1/JNK after SAH. In the current study, results showed that after administration of DHLA, the level of p-TAK1 and p-JNK markedly decreased, which concurrently decreased p-CaMKIIα expression. In addition, with the CaMKIIα siRNA intracerebroventricular injection, the level of p-TAK1, p-JNK, and NLRP3 dramatically decreased. These results suggest that lysosomal rupture and the activation of LAMP1/CaMKII/TAK1 signaling pathway underlie the anti-inflammatory effect of DHLA after SAH.
There are some limitations in the present study. The pathophysiology of lysosomal rupture is complicated. Other pathways may exist in the activation of the NLRP3 inflammasome. Meanwhile, the mechanisms of DHLA-induced stability of the lysosomal membrane and inhibition lysosomal rupture remain to be studied further.
In conclusion, our study showed that DHLA treatment can improve neurofunction and alleviate inflammation through the LAMP1/CaMKII/TAK1 pathway in EBI after SAH. It may provide an optical method in the treatment of EBI after SAH.
Sources of Funding
This study is supported partially by grants from National Institutes of Health (NS081740 and NS082184) to Dr Zhang and a grant from National Natural Science Foundation of China (81500992, 81371433).
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.117.018593/-/DC1.
- Received June 29, 2017.
- Revision received October 28, 2017.
- Accepted November 3, 2017.
- © 2017 American Heart Association, Inc.
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