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Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FloridaDepartment of Neurological Surgery, University of Miami Miller School of Medicine, Miami, Florida
Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FloridaDepartment of Neurological Surgery, University of Miami Miller School of Medicine, Miami, Florida
Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FloridaDepartment of Neurological Surgery, University of Miami Miller School of Medicine, Miami, FloridaBruce W. Carter Department of Veterans Affairs Medical Center, Miami, Florida
Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FloridaDepartment of Physiology and Biophysics, University of Miami Miller School of Medicine, Miami, Florida
Reprint requests: W. Dalton Dietrich, Professor of Neurological Surgery, Neurology, Biomedical Engineering and Cell Biology, Scientific Director, The Miami Project to Cure Paralysis, University of Miami Leonard M. Miller School of Medicine, 1095 NW 14th Terrace, Suite 2-30, Miami, FL 33136-1060.
Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FloridaDepartment of Neurological Surgery, University of Miami Miller School of Medicine, Miami, Florida
Traumatic Brain Injury (TBI) is a major cause of death and disability in the US and a recognized risk factor for the development of Alzheimer's disease (AD). The relationship between these conditions is not completely understood, but the conditions may share additive or synergistic pathological hallmarks that may serve as novel therapeutic targets. Heightened inflammasome signaling plays a critical role in the pathogenesis of central nervous system injury (CNS) and the release of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) speck from neurons and activated microglia contribute significantly to TBI and AD pathology. This study investigated whether inflammasome signaling after TBI was augmented in AD and whether this signaling pathway impacted biochemical and neuropathological outcomes and overall cognitive function. Five-month-old, 3xTg mice and respective wild type controls were randomized and underwent moderate controlled cortical impact (CCI) injury or served as sham/uninjured controls. Animals were sacrificed at 1 hour, 1 day, or 1 week after TBI to assess acute pathology or at 12 weeks after assessing cognitive function. The ipsilateral cerebral cortex was processed for inflammasome protein expression by immunoblotting. Mice were evaluated for behavior by open field (3 days), novel object recognition (2 weeks), and Morris water maze (6 weeks) testing after TBI. There was a statistically significant increase in the expression of inflammasome signaling proteins Caspase-1, Caspase-8, ASC, and interleukin (IL)-1β after TBI in both wild type and 3xTg animals. At 1-day post injury, significant increases in ASC and IL-1β protein expression were measured in AD TBI mice compared to WT TBI. Behavioral testing showed that injured AD mice had altered cognitive function when compared to injured WT mice. Elevated Aβ was seen in the ipsilateral cortex and hippocampus of sham and injured AD when compared to respective groups at 12 weeks post injury. Moreover, treatment of injured AD mice with IC100, an anti-ASC monoclonal antibody, inhibited the inflammasome, as evidenced by IL-1β reduction in the injured cortex at 1-week post injury. These findings show that the inflammasome response is heightened in mice genetically predisposed to AD and suggests that AD may exacerbate TBI pathology. Thus, dampening inflammasome signaling may offer a novel approach for the treatment of AD and TBI.
TBI is a known risk factor for the development of AD. Inflammasome activation is a common pathological mechanism seen in both TBI and AD pathologies.
Translational Significance
The inflammasome protein adaptor protein, ASC interacts with AD pathology-associated proteins, such as Aβ, and increased inflammasome activity is associated with worsened pathological outcomes. We show that TBI induces heightened inflammasome activation in AD mice and causes worsened cognitive outcomes and increased pathological progression. Moreover, IC100, a monoclonal antibody against ASC inhibited inflammasome activation in AD mice after TBI. Thus, the inflammasome signaling pathway is a promising target for therapeutic intervention for AD and TBI.
INTRODUCTION
Traumatic Brain Injury (TBI) represents a major public health burden in the US and throughout the world. An average of 1.7 million Americans suffer a TBI each year and it is a significant source of death and disability.
Approximately 30% of people who suffer a moderate TBI present worsening symptoms over a 5-year period, with deficits in learning and memory as one of the major disabling results.
Prevention CfDCa Surveillance Report of Traumatic Brain Injury-related Hospitalizations and Deaths by Age Group, Sex, and Mechanism of Injury—United States, 2016 and 2017.
U.S. Department of Health and Human Services,
Atlanta, Georgia2021
These deficits stem from the initial acute traumatic injury as well as changes in central nervous system (CNS) homeostasis and increased chronic neuroinflammation.
AD onset is governed by a combination of genetic predispositions and environmental triggers, whereas TBI pathology is primarily caused by environmental factors.
Two main pathological hallmarks of AD are the formation and the accumulation of amyloid beta (Aβ) plaques and hyperphosphorylated tau (pTau) neurofibrillary tangles that contribute to chronic inflammation and neuronal loss.
AD is of growing concern in public health and it appears that TBI and AD synergize to worsen outcomes in this patient population. The US Centers for Disease Control and Prevention (CDC) has reported that individuals with a history of moderate TBI have a 2.3 times greater risk of developing AD.
Prevention CfDCa Surveillance Report of Traumatic Brain Injury-related Hospitalizations and Deaths by Age Group, Sex, and Mechanism of Injury—United States, 2016 and 2017.
U.S. Department of Health and Human Services,
Atlanta, Georgia2021
Individuals who sustain a TBI within 10 years of AD onset are at a greater risk for the development of AD even if they sustained an earlier TBI, and TBI may induce AD pathology as early as 4–10 years before the age of onset.
Unintentional falls are the leading cause of TBI-induced hospitalizations, and according to the CDC, falls are increasing in individuals 55 and older. Historically, TBI and AD have been considered distinct pathologies. However, heightened neuroinflammation is characteristic in both conditions, and therapeutic interventions that block the increased neuroinflammatory response may prove beneficial as a treatment strategy.
The inflammasome is a multi-protein complex that regulates the activation of caspase-1, and subsequent release of inflammatory cytokines interleukin-1β (IL-1β) and IL-18, resulting in gasdermin-D (GSDMD)-induced pyroptosis.
Nod-like receptor protein 3 inflammasome (NLRP3) has been the most intensely studied inflammasome, but other inflammasome complexes have been implicated to play a role in neurodegenerative diseases.
Several investigations have observed increased inflammasome expression after TBI and importantly, a chronic increase in apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and IL-1β.
Additionally, recent studies have shown that formation and accumulation of tau is NLRP3-linked, further implicating the role of microglia-induced inflammatory activity.
Inflammasome signaling in TBI and AD may act synergistically to worsen cognitive decline through the exacerbation of neuroinflammation. Thus, the inflammasome may represent a promising therapeutic target for reducing the effects of TBI and AD pathologies on CNS homeostasis and cognitive decline. In this study, we investigated whether a genetic predisposition to AD alters the CNS inflammatory response to TBI. We hypothesized that AD predisposition alters the CNS inflammatory response and that TBI induces increased inflammasome activity in the 3XTg mouse model of AD.
METHODS
Animal model
In this study, we utilized the 3XTg (B6; 129-Tg(APPSwe,tauP301L)1Lfa Psen1tm1Mpm) AD mouse model, along with their respective wild type (B6129SF2) controls. All mice were 5 months of age and between 20 and 40 g at the time of injury including a 1:1 mixed male and female cohort in these initial studies. This AD mouse model is characterized as a triple transgenic model including humanized genes for mutation of the amyloid precursor protein and presenilin 1 resulting in the formation of pathological Aβ plaques, as well as tau mutation encouraging the formation of hyperphosphorylated tau tangles.
Intracellular Aβ can be detected immunologically as early as 3–4 months within the cortex and at 6 months within the hippocampus with the onset of extracellular Aβ plaque accumulation first evident at 6 months of age and tau at 12 months.
Controlled cortical impact traumatic brain injury in 3xTg-AD mice causes acute intra-axonal amyloid-beta accumulation and independently accelerates the development of tau abnormalities.
Aβ plaque deposits and tau neurofibrillary tangles, are major pathological hallmarks of AD and, documented interactors with the inflammasome have been discussed with this genetic AD model.
Animals were first weighed and then administered anesthesia via intraperitoneal injections (IP) of ketamine (100 mg/kg) and xylazine (10 mg/kg). Animals then had their eyes covered with petroleum jelly to protect their eyes during the procedure. Hair was then removed from the animal's skull utilizing Nair. Animals were then set into a stereotaxic frame and secured by ear bars and a bite plate. Once secured, a small incision was made using surgical scissors to expose the skull. A craniotomy was performed utilizing a surgical drill to produce a bore hole no deeper than the dura matter, bone fragments were cleared away using cotton tipped applicators. The craniotomy was located between lambda and bregma with a 4 mm diameter, 0.5 mm away from midline. Once the craniotomy was complete, the animal was placed under the piston of the CCI device. The piston was calibrated, and test fired prior to animal placement. Once placed, the animal was administered a moderate CCI utilizing a 4 m/s velocity and 0.8 mm depth for 150 m seconds duration.
At the conclusion of the injury, animals were removed from the stereotaxic device and excess blood was wiped away using cotton tipped applicators. Animals then had their incision closed with surgical staples and placed on a heating pad for recovery. Animals were administered buprenorphine 0.1 mg/kg SC post operatively for pain management. Animals were observed during recovery and were returned to animal quarters upon awakening. Shams received all surgical interventions and respective drugs but did not receive the craniotomy or TBI insult.
Tissue collection and preparation
Prior to tissue collection, mice were anesthetized with either 4% isoflurane or IP injections of ketamine and xylazine. Tissue samples collected for immunoblot experiments were dissected into left and right cerebral cortex and hippocampus and snap frozen in liquid nitrogen prior to storage at –80°C. Prior to immunoblot use, tissue was homogenized and lysed in RIPA buffer with the cellular debris removed via centrifugation. Aliquots were mixed with laemmli sample buffer with β-mercaptoethanol and stored at –80°C until use. Tissue collected for immunohistochemistry was first perfused via transcardial perfusion. Mice brains were perfused with 4% paraformaldehyde (PFA) (4°C, at pressure 2–3 mm Hg for 15 minutes) with the brain removed upon completion. Brains were placed in 4% PFA overnight at 4°C and then transferred into 20% sucrose solution and stored at 4°C until sectioning. Brains were sectioned at 40 μm thickness using a Leica freezing microtome with the sections stored in antifreeze solution at –20°C until use.
Immunoblot
Western blotting was performed as previously described.
Briefly, tissue samples were boiled then separated on 4%–20% Tris-TGX Stain free Citron gels (Bio-Rad). Gels were then transferred onto polyvinylidene difluoride (PVDF) membranes utilizing Turbo Transfer mixed molecular weight protocol (Bio-Rad). Membranes were blocked in I-block (Tropix) or blocking buffer (Biorad). Membranes were incubated in antibodies (1:1000 dilution in blocking buffer) to inflammatory associated proteins: caspase-1 (Novus), caspase-8 (Santa Cruz), IL-1β (Novus), NLRP3 (Novus), and ASC (Santa Cruz). Membranes were washed in TBS-T prior to exposure to secondary antibodies and prior to exposure to imaging reagents. After primary antibody exposure, membranes were incubated in secondary antibodies containing horseradish peroxidase: IgG antirabbit (Cell Signaling) and IgG antimouse (Cell Signaling). Membranes were imaged using a Chemidoc touch imaging system (Bio-Rad) with results analyzed utilizing Image-Lab (Bio-Rad) software. The amount of total density of the proteins of interest was determined by measuring specific band intensity and dividing it by the respective band intensity of a beta actin (Thermo) loading control or by total lane protein density of stain free gel prior to transfer.
Pyroptosome isolation and western blotting
Partial isolation of ASC pyroptosome was conducted as previously described.
Briefly, homogenized tissue was first filtered through a 5 μm low-binding PVDF membrane (Millipore). Samples were then centrifuged at 2700 × g for 8 minutes with the resulting pellet resuspended in 40 μL of dimethylammonio]-propanesulfonic acid (CHAPS) buffer (20 mmol/L HEPES-KOH, pH 7.5, 5 mmol/L MgCl2, 0.5 mmol/L EGTA, 0.1 mmol/L phenylmethylsulfonyl fluoride, protease inhibitor cocktail, and 0.1% CHAPS). Samples were next spun at 2700 × g for 8 minutes with the resulting pellet resuspended in 27.8 μL of CHAPS buffer with 2.2 μL of disuccinimidyl substrate. Samples were allowed to incubate for 30 minutes at room temperature for ASC oligomerization. After incubation, equal volumes (2X) of Laemmli buffer were added to the samples and stored at –80°C until use. Samples were probed for ASC utilizing methods outlined in the immunoblot section above.
Immunohistochemistry
Immunohistochemistry was performed on cortical tissues utilizing 40 um free floating brain tissue sections as previously described.
Sections were washed in PBS+ (0.4% triton X100) and antigen retrieval (citrate buffer, 20 minutes in steamer) prior to blocking. Tissue sections were then blocked with 3% normal animal serum in PBS+ for 1 week at 4°C and then incubated in both antibodies for ASC (Santa Cruz), MOAB (Abcam) and Iba1 (Abcam), 1:500, overnight at 4°C. Tissue sections were washed in PBS+ and incubated in respective fluorescent secondary antibodies (Abcam) for 2 hours at room temperature. Sections were then washed in PBS, mounted on slides, and cover slipped with DAPI Vector shield (Vector Labs). Slides were stored in darkness at 4°C until imaging using a confocal microscope with a 60x objective and analyzed utilizing Leica or Olympus software.
Behavioral experiments
All animals were randomized and assigned unique nonspecific identifiers to increase scientific rigor and reproducibility. Investigators were blinded to which mice were in what group, and unique identifiers were not decoded until behavioral experiments were completed and results fully analyzed. For the behavioral studies, Open field assessment was conducted at 3 days post-CCI surgery, novel object recognition was conducted at 14 days postsurgery, and Morris water maze testing was initiated 6 weeks postsurgery. All recordings were made with an Ultra 720+ Resolution DSP, True Day/Night Color camera (Everfocus).
Open field assessment
Open field assessment was carried out to assess locomotor and anxiety-like behaviors as previously described.
Briefly, mice were allowed to habituate for 30 minutes in the behavioral room prior to assessment. Mice were prehandled and placed into the open field box (43.2 cm width, 43.2 cm length, 30.5 cm height with white plexiglass walls and floor) and allowed to explore for 10 minutes while being recorded. Videos and open field activity heat maps were analyzed utilizing Ethovision (Noldus) software. Time in center, time in borders, velocity, and total distance traveled were assessed.
Novel object recognition
Novel object recognition was carried out as previously described.
Briefly, mice were placed in the behavior room to habituate for 30 minutes prior to assessment on each day. Mice were first allowed to habituate in the box for 10 minutes on day 1 with no objects present. On day 2 mice were exposed to 2 identical objects, placed equidistant, and allowed to explore the box for 10 minutes. On day 3 one object was replaced with a novel object and mice were allowed to explore the box for 10 minutes. Mouse behavior was recorded, and videos were analyzed utilizing Ethovision (Noldus) software. Time spent with the novel object was compared to the time spent with the old object. Results were calculated using the following equation: (Novel – Old)/(Novel + Old), if the result was positive then this represents novel object recognition, if negative this represented no novel object recognition, if 0 then there was equal recognition. The preferential exploration of a novel object measured as the discrimination index, in which –0.5 index indicates a preference for one object relative to the other, is a measurement of recognition memory sensitivity.
Briefly, animals were placed into a pool containing a hidden platform and distinct symbols denoting the cardinal directions and were allowed 65 seconds to find the platform. Upon finding the platform, animals were allowed to sit 10 seconds prior to being retrieved by the investigator. If the platform was not found in the allotted time, animals were guided and held on the platform for 20 seconds on day 1 or 10 seconds the following days. Prior to testing each day, animals were placed in the room for 30 minutes for habituation. Animals were tested 4 times each day for 4 days with alternating entry points into the pool and an inter trial interval of 25 minutes. On day 5 the hidden platform was removed, and animals were allowed to swim in the pool for a 65 second probe trial. The pool water was paint stained and kept at 25°C and the platform was located 1 cm below the water level. Mouse behavior was recorded, and videos were analyzed utilizing Ethovision (Noldus) software.
AD protein V-Plex analysis
Ipsilateral cortex and hippocampus tissue was dissected from mice after the completion of behavior as outlined above. Tissue was homogenized and loaded into 96 well V-Plex (MSD) plate according to manufacturer's instructions and as described in.
Cohort study on the differential expression of inflammatory and angiogenic factors in thrombi, cerebral and peripheral plasma following acute large vessel occlusion stroke.
Briefly, plate was prepared by first adding 150 μL of diluent to each well and allowing the plate to incubate at room temperature for 1 hour. Plate was then washed with 150 μL of washing buffer. Five μL of sample with 20 μL of diluent or 25 μL of standard were then added along with 25 μL of detection antibody solution to each well. Plate was then incubated at room temperature for 2 hours. Following incubation and washing, 150 μL reading buffer were added, and the assay was run in the MESO QuickPlex SQ 120 (MSD) and data analyzed utilizing Discovery Workbench software (MSD). Total Aβ load was calculated for the ipsilateral cortex and hippocampus, along with total Aβ-38, Aβ-40, Aβ-42, and Aβ42/40 ratio. We additionally measured tau pathology in these mice utilizing a V-Plex kit for measuring total tau and P-tau (Thr 231, Tau231). Levels of total tau and ptau were calculated for the ipsilateral cortex and hippocampus.
Inflammasome inhibition with IC100
We have previously shown that IC100 treatment improves the clinical score in an animal model of multiple sclerosis
Mechanism of action of IC 100, a humanized IgG4 monoclonal antibody targeting apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC).
In this study 3XTg AD mice underwent CCI or sham surgery as described above. Mice were administered 30 mg/kg IP of either IC100 (Zyversa) for drug treatment, or 30 mg/kg IP human IgG at 30 minutes post injury and again at 3 days post injury. Mice were sacrificed at 1 week post injury and the ipsilateral cortex was collected as described above. Tissues were then analyzed for IL-1β expression utilizing V-Plex mouse proinflammatory panel for IL-1β (MSD) using an Electrochemiluminescent immunoassay (ECLIA) as described in.
IL-1β concentration was calculated for the ipsilateral cortex in sham, controls, and drug treated animals.
Statistical analyses
Statistical analysis was conducted utilizing GraphPad Prism 8. Outliers were removed prior to further analysis by the robust regression and outlier removal (ROUT) method as described in.
Normality was first tested by the Shapiro-Wilk normality test, and nonparametric data was transformed using a logarithmic transformation to approximately conform the data to normality. Thus, immunoblot and V-Plex data were normalized through log transformation prior to data analysis. Data analysis included 1-way ANOVAs with post hoc Dunnett's correction or Brown-Forsythe one-way ANOVA with post hoc Dunnett's correction if standard deviations were significantly different. Behavioral data were not transformed and were analyzed utilizing 1-way ANOVAs with post hoc Bonferroni correction, Brown-Forsythe one-way ANOVA with post hoc Dunnett's correction (if standard deviations were significantly different), or Kruskal-Wallis nonparametric testing with post hoc Dunn's correction (if data were lognormal) for open field and novel object, and 2-way repeated measures ANOVA with post hoc Newman-Keuls correction for Morris water maze.
RESULTS
Caspase-1, Caspase-8, and IL-1β are increased in AD after TBI
To determine the intensity and composition of TBI-induced neuroinflammation in mice, we performed western blot analysis of ipsilateral cerebral cortex tissue from individual mouse cohorts sacrificed at 1 hour, 1 day, and 1 week after moderate CCI or sham surgery controls (Fig 1A). Western blot analysis of cortical tissue showed significantly increased activate (cleaved) caspase-1, caspase-8, and IL-1β after CCI in AD mice when compared to shams at 1 hour post injury and maintained at 1 day and at 1 week post injury (Fig 1C–E). These findings provide evidence for CCI injury resulting in increased neuroinflammatory activity which was maintained over the course of 1 week after injury. However, there was no significant increase in NLRP3 sensor protein expression after CCI (Fig 1B). Additionally, to verify the severity of our injury,
we performed western blot analysis of ipsilateral hippocampal tissue from the same group of AD mice and found that hippocampal tissue did not show significant increases in caspase-1, caspase-8, ASC, or IL-1β at 1 hour, 1 day, or 1 week post-CCI or sham surgery (Supplementary Fig. 1). Overall, these results show that moderate CCI surgery was able to activate the inflammasome in the injured cortex of AD mice as early as 1 hour, and it was sustained as long as 1 week after injury.
Fig 1Increased Inflammasome Expression in the Cerebral Cortex of AD mice after CCI. A) Representative immunoblot images and densitometric analysis of NLRP3, Caspase-1, Caspase-8, and IL-1β within the ipsilateral cerebral cortex after CCI in AD mice at 1 hour, 1 day, and 1 week post CCI surgery. B) There was no significant increase in NLRP3 sensor protein expression after injury in AD mice. C) Caspase-1 was significantly increased at 1 hour and 1 day compared to Sham. D) Caspase-8 levels were significantly increased at 1 hour, 1 day and 1 week compared to Sham and E) IL-1β protein expression after injury was also significantly increased at 1 hour, 1 day and 1 week compared to sham. β-actin was used as an internal standard and control for protein loading. Log transformation was performed followed by one-way ANOVA and Dunnett's post hoc test. Data shown as mean+/-SEM. N = 5 per group with each group a separate cohort with significant P values shown.
To determine whether AD predisposition impacts the neuroinflammatory response to TBI, we performed western blot of ipsilateral cortical tissue from WT and AD mice at 1 day post-CCI or sham surgery (Fig 2A). Western blot of cortical tissue showed increased IL-1β after CCI injury in AD mice when compared to CCI WT mice at 1 day post injury (Fig 2E). Additionally, CCI resulted in significantly increased levels of activated caspase-1, caspase-8, and IL-1β in WT and AD mice after CCI compared to sham controls (Fig 2C–D). These results show that inflammasome activation was significantly greater in injured AD mice when compared to WT mice.
Fig 2Increased Inflammasome Expression in AD and WT Mice at 24 hours after CCI. A) Representative immunoblot data and densitometric analysis showing expression of NLRP3 sensor protein, Caspase-1, Caspase-8, and IL-1β within the ipsilateral cerebral cortex of injured WT and AD mice 24 hours post-CCI injury. B) There was no significant increase in the expression of NLRP3 after injury. C) Caspase-1 levels were significantly increased in sham AD, CCI WT and CCI AD mice compared to WT sham mice. Caspase-1 protein levels CCI AD mice were also significantly higher than WT sham mice. D) Caspase-8 levels were significantly increased after CCI in AD mice compared to Sham AD and Sham WT. TBI WT Caspase-8 levels were also significantly increased compared to Sham WT. E) There was a significant increase in IL-1β levels after injury in AD mice compared to all groups. IL-1 β levels in WT CCI mice were significantly higher compared to WT sham mice. β-actin was used as an internal standard and control for protein loading. Log transformation was performed followed by one-way ANOVA and Dunnett's post hoc test. Data shown as mean+/-SEM. N = 5–8 per group with each group a separate cohort with significant P values shown.
ASC expression in Iba1+ Microglia/Macrophages after TBI in AD and WT mice
To determine if ASC expression was elevated after injury and evaluate microscopic locations of ASC speck formation, we conducted a combination of ASC western blot, ASC isolation, and immunohistochemical staining of cortical brain tissue sections (Fig 3). Studies have shown that microglia activation occurs minutes after TBI and can remain chronically activated for weeks to years after injury.
We observed increased ASC expression in injured AD mice that was maintained over 1 week (Fig 3B). Moreover, ASC levels were also increased in uninjured AD mice when compared to sham WT and TBI-WT mice (Fig 3B). After injury, both WT and AD mice presented significant increases in ASC expression when compared to sham uninjured controls, and that TBI-AD mice had significantly increased ASC expression compared to TBI-WT mice (Fig 3B). In addition, pyroptosome isolation showed increased ASC oligomerization after injury in both genotypes, suggesting the formation of ASC specks (Fig 3C). Finally, IHC staining of the perilesional cortex showed that ASC specks were associated with Iba1 positive microglia/macrophages in WT and AD mice after injury appearing within the cytosol including perinuclear locations (Fig 3D). These results show that ASC expression and oligomerization is elevated within the injured cortex of both WT and AD mice after CCI, and that ASC expression is significantly higher in AD animals compared to WT animals after injury.
Fig 3Increased ASC Expression and Activity After CCI in WT and AD mice. A) Representative immunoblot data and densitometric analysis showing the temporal profile of total ASC expression after CCI in AD mice at 1 hour, 1 day, and 1 week. B) ASC levels were significantly increased after CCI in AD mice compared to Sham at all time points. There was a significant increase in ASC in CCI AD mice compared to all groups. Additionally, there was a significant increase in ASC levels in WT TBI and AD sham compared to WT Sham. Log transformation was performed followed by one-way ANOVA and Dunnett's post hoc test. Data shown as mean+/-SEM. N = 5–8 per group with each group a separate cohort with significant p values shown. C) Representative immunoblot data and densitometric analysis of immunoblots showing isolated ASC oligomers by weight and level of oligomerization in WT and AD mice at 24 hours post injury. D) ASC speck localization in Iba1+ microglia/macrophages within the pericontusional cerebral cortex at 1 week post injury in WT and AD mice. DAPI (blue), Iba1 (green), and ASC (red). Scale bars: 5 μm.
Altered cognitive behavior in AD mice acutely and chronically after TBI
To assess alterations in cognitive function after CCI in AD and WT mice, we tested mice in open field at 3 days postsurgery, novel object recognition at 14 days postsurgery and Morris water maze at 6 weeks postsurgery (Fig 4). In open field testing, there was a significant decrease in overall velocity and total distance traveled in AD TBI compared to AD sham. (Fig 4A–B). Significant differences were seen in that injured AD mice spent less time in the border with a trend to spend more time in the center when compared to injured WT mice (Fig 4C–D). One explanation for this difference was that injured AD mice had a reduced anxiety behavior compared to injured WT mice.
For the novel object recognition test, there was no significant difference in recognition of the novel object between groups (Fig 4F). In Morris water maze testing, there was a significant difference in cognitive function across the 4 day time span, as well as between injury and genotypic groups (Fig 4H). Additionally, there was a significant decrease in cognitive function in AD TBI, WT TBI, and AD sham when compared to WT sham respectively (Fig 4H). There was no significant difference between groups for the probe trial (Fig 4I). These results show that AD mice had decreased anxiety-associated behavior compared to WT mice after injury; whereas spatial learning and memory functionality was significantly reduced in all genotype and injury groups when compared to uninjured WT mice. However, mice showed no significant difference in their ability to recognize the novel object regardless of genotype or treatment group.
Fig 4Decreased Anxiety and Reduced Learning and Memory Functionality in AD Mice. Open field data and illustrative heatmaps showing differences between WT and AD mice after CCI. A) There is no significant difference between WT and AD TBI mice in overall distance traveled. B) WT and TBI mice were not significantly different in overall velocity. C) The AD CCI mice spent significantly more time in the center zone compared to WT CCI mice. D) In contrast, the AD CCI mice spent significantly less time in the border zone. E) Illustrative heatmaps and template of open field behavior between WT and AD TBI mice. F) There was no significant difference between WT and AD mice on the novel object recognition task. G) Illustrative heatmaps and template of novel object recognition between WT and AD TBI mice. H) There were significant differences over time (<0.001) and between groups (0.003) in total time to find the platform during Morris water maze testing. There were significant differences between AD sham (P = 0.009), WT TBI (P = 0.035), and AD TBI (P = 0.003) when compared to WT sham animals. I) There was no significant difference between groups during probe trial. Behavioral data was analyzed using one-way ANOVAs with post hoc Bonferroni correction, Brown-Forsythe one-way ANOVA with post hoc Dunnett's correction (if standard deviations were significantly different), or Kruskal-Wallis non parametric testing with post hoc Dunn's correction (if data was lognormal).or 2-way repeated measures ANOVA with post hoc Newman-Keuls correction. Data shown as mean+/–SEM. N = 9–11 per group with significant P values shown.
To assess chronic changes in Aβ load after injury, we analyzed protein lysates from ipsilateral cortex and hippocampal tissue at 12 weeks postsurgery (Fig 5). In injured cortex tissue we observed that there was no change in Aβ38 levels between groups (Fig 5A). However, we did observe that Aβ40 and Aβ42 were significantly elevated in AD sham compared to WT sham and in AD TBI mice compared to WT TBI (Fig 5B–C). In addition, this was consistent with increased total Aβ load (Fig 5D). A decrease in the ratio between Aβ42/40 has been reported as a predictor of AD pathology.
When analyzing the ratio between Aβ42 and Aβ40, although we observed a visual trend wherein the ratio decreased when comparing shams and injury groups, we did not observe any significant changes (Fig 5E). In the ipsilateral hippocampus, we saw comparable results in that Aβ38 was not changed between groups (Fig 5F) but there were significant increases in Aβ40 and Aβ42 load in AD sham and AD TBI when compared to respective WT groups (Fig 5G–H). This was reflected in a significant elevation in total Aβ levels in the AD sham and TBI groups compared to their respective WT (Fig 5I) and a significant decrease in the Aβ ratio when comparing AD sham to WT sham (Fig 5J). After injury, Aβ was seen colocalized (arrows) in the injured cortex with ASC in WT and AD mice at 7 days post injury (Fig 5K). Additionally, (Supplementary Fig 2) total tau was only elevated in injured AD mice when compared to injured WT mice, but that there was no difference when compared to AD shams (Supplementary Fig 2C). Moreover, Tau231 was not elevated in any of the groups we analyzed (Supplementary Figs 2B and D). Together, these results indicate increased Aβ expression in AD mice compared to WT mice regardless of injury group.
Fig 5Aβ is Elevated in AD mice compared to WT mice regardless of injury group. A–C) V-Plex assay of ipsilateral cortex tissue at 12 weeks post injury showed significantly increased Aβ40 and Aβ42 between genotypes, not injury groups. D) Total Aβ load in the ipsilateral cortex showing significant differences between genotypes. E) There was no significant difference in the ratio of Aβ42/40 in the cortex. F–H) Assay of ipsilateral hippocampal tissue showed significant increases in Aβ40 and Aβ42 between genotypes. I) Total Aβ load within the hippocampus was significantly elevated between genotypes. J) There was a significant decrease in the Aβ ratio when comparing AD sham to WT sham. K) ASC was observed colocalized (arrows) with Aβ in the injured cortex of WT and AD mice at 7 days post injury. Scale bars represent 5 um.
Inflammasome inhibition with IC100 after TBI in AD mice
We have previously shown that IC100 is effective in inhibiting inflammasome activity, microglia activation, and pyroptotic pathology in multiple injury and disease models through blocking of ASC speck formation and caspase-1 cleavage (Fig 6B).
Mechanism of action of IC 100, a humanized IgG4 monoclonal antibody targeting apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC).
To assess the effectiveness of IC100 treatment in reducing inflammasome activity after injury in AD, we administered 30 mg/kg IP of either IC100 or the isotype control human IgG at 30 minutes and 3 days post injury. We then sacrificed and collected injured cortex tissue at 7 days post injury and measured IL-1β cytokine expression by ECLIA. When comparing control mice to IC100 treated mice, we saw a significant decrease in IL-1β concentration (Fig 6A). Hence, these results show a decrease in inflammasome activity by neutralization of ASC in AD mice.
Fig 6Mechanism and Results of anti-ASC Administration in AD mice after TBI. A) Administration of anti-ASC significantly reduced the levels of inflammatory cytokine IL-1β after injury in AD mice when compared to injured controls. Data was analyzed using one-way ANOVAs with post hoc Bonferroni correction. Data shown as mean+/–SEM. N = 5–10 per group with significant P values shown. B) The NLRP3 inflammasome is canonically activated through the detection of PAMPs and DAMPs by TLRs and macrophages. This results first in the transcriptional upregulation of inflammasome proteins (dashed lines), followed by formation of the inflammasome complex via ASC speck formation and caspase-1 activation. This results in the cleavage of inflammatory cytokines, the formation of the GSDMD pore, and subsequent pyroptotic cell death. Anti-ASC interrupts this process by inhibiting (green lines) ASC speck formation and binding with caspase-1, resulting in the reduction of inflammatory cytokine expression.
Prevention CfDCa Surveillance Report of Traumatic Brain Injury-related Hospitalizations and Deaths by Age Group, Sex, and Mechanism of Injury—United States, 2016 and 2017.
U.S. Department of Health and Human Services,
Atlanta, Georgia2021
One pathology of particular interest which is itself a growing public health concern is AD. Considering that AD etiology has been associated with genetic and environmental triggers, and that TBI is a known risk factor for AD development, it is important to better understand shared pathological aspects of these 2 pathologies. Given that inflammasome activation is seen in both TBI and AD individually, abnormal inflammasome activation represents a potential pathological mechanism of crosstalk between the two. In this study, we examined whether 3XTg mice containing humanized AD genes showed altered activation of the inflammasome after TBI and if this resulted in a heightened inflammatory response and worsened cognitive decline. Accordingly, AD mice and WT controls were subjected to CCI injury. We then performed immunoblotting and immunohistochemical staining of brain tissue to observe inflammasome activity and innate immune cell activity. Next, we conducted behavioral assessments utilizing open field, novel object recognition, and Morris water maze to assess cognitive functional after injury. Finally, we investigated if TBI impacted chronic Aβ expression and total Aβ load.
Previous studies have reported that the inflammasome is an active participant in the pathologies of both TBI and AD, with inflammasome and innate immune cell activation increased after TBI and the neurodegenerative processes associated with AD.
Our results provide evidence that the inflammasome response to TBI is exacerbated in AD prior to the accumulation of extracellular Aβ plaques. Furthermore, we showed that the heightened inflammasome response after TBI is associated with a worsened loss of cognitive function. This result suggests that AD predisposition not only exacerbates inflammation after TBI, but also contributes to the cognitive decline traditionally seen after TBI. Interestingly, we found a heightened inflammasome response after TBI, but this increased inflammasome activity did not result in a chronic change in Aβ expression. This finding suggests that in our model that although TBI and AD together results in worsened cognitive decline, they do not increase AD pathological severity, but rather exacerbate the inflammatory response to TBI in animals predisposed to AD development. Therefore, the increased inflammasome activation appears to be a shared pathological feature of TBI and AD, and may be a potential target for therapeutic intervention.
In TBI, the release of danger-associated molecular patterns (DAMPs) by injured and dying cells combined with innate immune activation and changes to neuronal ionic homeostasis results in the priming and activation of the inflammasome as indicated by increased activated IL-1β.
of patients with TBI. Biomarker studies of TBI patients have shown that not only is the inflammasome activated after injury, but that measuring the levels of inflammasome proteins, such as caspase-1 and ASC, are effective tools for measuring injury severity and for determining probable prognosis.
In our study, we observed elevated caspase-1, ASC, and IL-1β as early as 1 hour and as late as 1 week post-TBI in the injured cortex of AD mice, and that both ASC and IL-1β levels were significantly increased when compared to injured WT controls. This not only shows that the inflammatory response to TBI in AD mice is exacerbated but suggests that AD mice have a worsened probable prognosis after injury compared to injured controls. Considering that monomeric Aβ can already be seen at this time point in this AD mouse model, the exacerbated inflammatory response can be most likely attributed to priming of the inflammasome by Aβ. Accordingly, inflammasome proteins have also been found to be elevated in the early and advanced stages of AD in the serum of patients,
emphasizing the importance of abnormal inflammasome activation in the pathology of AD and TBI.
ASC is a major player in the inflammasome response in various types of neural injury and ASC levels in tissue fluids may be an effective measure of injury severity and may also contribute to pathology.
These composites hinder Aβ clearance and along with Aβ plaques can activate the inflammasome through microglia cytokine release, lysosome damage, and disruption to neuronal ionic homeostasis.
As such, we evaluated ASC activity after TBI and determined whether this response was altered in AD animals. Our results showed that ASC expression was elevated after CCI in both WT and AD mice, and that the levels in AD mice were significantly higher when compared to WT mice. These results are consistent with other studies showing that ASC is elevated after TBI.
Furthermore, we detected increased ASC expression prior to the traditional age of Aβ plaque aggregation in this AD model. This observation implies that increased ASC expression may be due to monomeric Aβ priming of the inflammasome in AD mice with subsequent injury resulting in a more robust triggering of the inflammasome. In addition to observing total ASC load, we also evaluated the level of ASC oligomerization and subsequent ASC speck formation. This finding is consistent with a previous report showing that ASC is elevated in stages of human AD.
Furthermore, pyroptosome isolation and western blotting showed that ASC oligomerization was noticeably increased after CCI in WT and AD mice, indicating TBI-induced ASC speck formation regardless of genotype.
Caspase-8 has traditionally been associated with the cell death process of apoptosis. However, caspase-8 has also been shown to form noncanonical inflammasomes in the CNS together with NLRP1 and ASC.
Thus, the increase of caspase-8 implicates increased cell death, representing another source of inflammasome activation. However, in the current study, we did not detect an increase in NLRP3 protein levels after injury, suggesting that other inflammasomes besides NLRP3 may play a role in increased ASC expression and subsequent AD pathology after CCI. Interestingly, studies in other AD mouse models have implicated the role of the NLRP3 inflammasome in that NLRP3 deficiency through knock out of NLRP3 sensor and or caspase-1 resulted in increased “M2” phenotype microglia polarization, and decreased Aβ expression.
Considering that caspase-1 activity is shared by all inflammasomes canonical and noncanonical, future studies should investigate the activation of other inflammasomes that may be targeted for the treatment of inflammasome-mediated TBI-exacerbated AD pathology.
Activated microglia, astrocytes, and invading peripheral macrophages have been observed after TBI, and chronically activated microglia are thought to play a contributing role in the resulting inflammatory pathology.
Immunofluorescent staining of tissue from AD and WT mice at 7 days after CCI showed microglia/macrophage associated Iba1+ immunoreactivity in perilesional cortical regions that colocalized with ASC. This observation is consistent with previous studies showing TBI-induced microglia activation and that ASC is associated with activated microglia. ASC specks have been shown to bind with Aβ to form insoluble Aβ/ASC plaques and the heightened ASC expression after CCI could be an underlying mechanism for TBI as a risk factor for AD development by altering the onset or severity AD pathology.
To better understand the extent of injury and investigate the onset of AD pathology-induced inflammasome activation, we also performed western blot analysis of the hippocampal lysates over the same time course. We observed that the expression of inflammasome signaling proteins was not significantly changed following CCI in hippocampus. This finding indicates that the injury-induced changes in inflammasome activation after moderate CCI appear to be most prominent to the injured ipsilateral cerebral cortex.
To establish whether TBI in AD mice affected cognition over time, we conducted behavioral analysis testing at 3 days, 2 weeks, and 6 weeks post injury. Behavioral assessments using quantitative open field testing demonstrated a significant decrease in the time that injured AD mice spent in the border region compared to injured WT mice which spent more time adjacent to border regions. This general response has been associated with anxiety behavior.
Our results suggest that animal genotype altered this behavioral response in that TBI-AD mice presented with a dampened anxiety response compared to WT-TBI mice. Interestingly, although we noted an increase in distance traveled and velocity in AD mice after TBI when compared to AD shams, there was no difference between WT-TBI and AD-TBI.
In the novel object recognition test, injured AD animals showed a trend in decreased novel object recognition, but there was no significant difference when compared to TBI-WT animals or to respective sham groups. This fining is in agreement with, previous studies that report no significant differences in object recognition between AD and WT animals around 6 months of age.
Additionally, since our injury model was moderate in severity, it is possible that the initial primary injury was not severe enough to acutely impact hippocampal dependent learning and memory.
Morris water maze testing indicated a significant group difference between WT Sham and all other groups. However, there was no significant difference between AD-TBI and WT-TBI groups although there was a trend for the AD-TBI mice to take longer to find the hidden platform compared to the WT-TBI group. Interestingly, there was no significant difference between injured animals and AD sham animals, suggesting that the injury was not severe enough to worsen learning and memory function. However, considering that AD sham animals had significantly worse cognitive function when compared to WT sham animals, it may imply that AD pathology had already started impacting cognition. The lack of significant differences between the TBI groups may be a consequence of the time point assessed in this study. This post injury time point may have been too early to document the full effects of TBI on cognitive function with genotypic-specific injury effects possibly being seen at more later chronic time points. It will be important in future studies to therefore evaluate more chronic effects of TBI on AD function using additional genetic models and behavioral outcomes. Additionally, due to the numerous AD mouse models, future studies will need to consider the results that specific genetic models of AD illicit, and determine if they respectively represent observations uniform to AD as a whole or specific to the genetic model itself.
Aβ is one of the major chronic pathological hallmarks of AD and is a known activator of the inflammasome.
RT Vontell, JP de Rivero Vaccari, X Sun, SH Gultekin, HM Bramlett, WD Dietrich and RW Keane, Identification of inflammasome signaling proteins in neurons and microglia in early and intermediate stages of Alzheimer’s disease, Brain Pathol, 2022:e13142. doi: 10.1111/bpa.13142. Epub ahead of print.
As such, we assessed if the chronic expression of Aβ was altered as a result of injury. Numerous studies have shown that Aβ expression is seen in multiple AD mouse models after TBI.
However, these studies have had varying results often depending on the type of injury, age at injury, and mouse model utilized. In the 3XTg mouse model, one study have observed increased Aβ as early as 24 hours post injury, only to return to sham levels by day 7,
In our study, we observed that Aβ40 and Aβ42 along with total Aβ load was increased in the injured cortex and ipsilateral hippocampus of AD mice compared to WT mice regardless of injury group. Since there were no significant differences between shams and injury groups, we concluded that Aβ elevation was a result of either age, analysis time point, injury model, or genotype. These studies suggest that increased cognitive loss is a result of increased inflammasome activation, and not as a result of altered AD pathology in our experimental model. The colocalization of ASC with Aβ we observed in the cortex of WT and AD mice after injury supports previous studies that demonstrate interactions between ASC and Aβ.
We observed similar results in tau expression in that it was only seen elevated in the hippocampus of injured AD mice compared to injured WT mice. Interestingly, although elevated tau and P-Tau231levels have been observed after injury, we did not measure significant changes as a result of injury or genotype.
However, it should be noted that our 12-week post injury time point may have been too early to measure direct injury effects that may become apparent at more chronic time points. This concern is apparent in the development of tau pathology in which this mouse strain produces tau tangles starting at much later time points. Thus, future studies are needed to determine the precise role of tau on TBI-AD pathology.
Finally, we tested if blocking inflammasome activity by inhibiting ASC reduces the elevated inflammatory response in AD mice after TBI. Administration of IC100 resulted in reduction of the inflammasome-mediate IL-1 cytokine IL-1β in the injured cortex of AD mice at 1-week post injury. Considering that the primary injury phase of TBI is traditionally seen during the first week post injury, this finding demonstrates a reduction in TBI induced neuroinflammation through a reduction in inflammasome signaling and inflammatory cytokine production.
In conclusion, the inflammasome is a key pathological component of a variety of brain diseases and conditions. Since TBI is a known risk factor for AD development, and that AD can result from genetic predisposition, it is important to understand how these 2 pathologies interact with one another. Our results suggest that genetic predisposition to AD alters the CNS response to trauma through heightened inflammasome activation, and that inhibition of the inflammasome by targeting the adaptor protein ASC reduces inflammatory cytokine expression after TBI in AD mice. Together, these results support the idea that a heightened inflammasome response could play a pivotal role in worsening inflammation after TBI in AD predisposed individuals and ultimately exacerbate the development of AD through neuroinflammation.
Supplementary Material
Supplementary Fig 1: No Change in Inflammasome Protein Expression After TBI in the Hippocampus of AD mice. A, Representative immunoblot data and densitometric analysis for Caspase-1, Caspase-8, ASC, and IL-1β within the ipsilateral hippocampus after CCI in AD mice at 1 hour, 1 day, and 1-week post injury. B–E, There was no significant difference across the temporal profile for all inflammasome proteins compared to sham. Log transformation was performed followed by 1-way ANOVA and Dunnett's post hoc test. Data shown as mean +/–SEM. N = 5 per group with significant Pvalues shown.
Supplementary Fig. 2: AD Mouse Cortical and Hippocampal tau Load. A, V-Plex Elisa data showing no significant changes in total tau load of the ipsilateral cortex. B, Data showing no significant changes to ptau (Thr 231) load in the ipsilateral cortex. C, Data showing significant increase in tau load in injured AD mice compared to injured WT mice. D, Data showing no significant changes to ptau (Thr 231) expression in the ipsilateral hippocampus. Data was analyzed using one-way ANOVAs with post hoc Bonferroni correction. Data shown as mean +/–SEM. N = 5 per group with significant Pvalues shown.
Authors’ contribution statement
The manuscript has been reviewed and approved by all the authors. All authors have read the journal's authorship agreement and policy on disclosure of potential conflicts of interest.
Acknowledgments
JPdRV, HMB, RWK, and WDD are co-founders and managing members of InflamaCORE, LLC and have licensed patents on inflammasome proteins as biomarkers of injury and disease as well as on targeting inflammasome proteins for therapeutic purposes. JPdRV, HMB, RWK, and WDD are Scientific Advisory Board Members of ZyVersa Therapeutics. NHJ and NAK declare no conflicts of interest.
This research was funded by a FDOH grant (21A13) to WDD, an RF1 grant from the NIH/NINDS/NIA (1RF1NS125578-01) to WDD and JPdRV, an R01 grant from the NIH/NINDS to RWK and JPdRV (R01NS113969-01), and a Lois Pope Life Fellowship Award to NHJ. Graphics generated using Biorender.
Data Availability
The data supporting the findings of this study are available from the corresponding author upon reasonable request.
Surveillance Report of Traumatic Brain Injury-related Hospitalizations and Deaths by Age Group, Sex, and Mechanism of Injury—United States, 2016 and 2017.
U.S. Department of Health and Human Services,
Atlanta, Georgia2021
Controlled cortical impact traumatic brain injury in 3xTg-AD mice causes acute intra-axonal amyloid-beta accumulation and independently accelerates the development of tau abnormalities.
Cohort study on the differential expression of inflammatory and angiogenic factors in thrombi, cerebral and peripheral plasma following acute large vessel occlusion stroke.
Mechanism of action of IC 100, a humanized IgG4 monoclonal antibody targeting apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC).
RT Vontell, JP de Rivero Vaccari, X Sun, SH Gultekin, HM Bramlett, WD Dietrich and RW Keane, Identification of inflammasome signaling proteins in neurons and microglia in early and intermediate stages of Alzheimer’s disease, Brain Pathol, 2022:e13142. doi: 10.1111/bpa.13142. Epub ahead of print.
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