Cannabidiol Normalizes Caspase 3, Synaptophysin, and Mitochondrial Fission Protein DNM1L Expression Levels in Rats with Brain Iron Overload: Implications for Neuroprotection
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- da Silva, V.K., de Freitas, B.S., da Silva Dornelles, A. et al. Mol Neurobiol (2014) 49: 222. doi:10.1007/s12035-013-8514-7
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We have recently shown that chronic treatment with cannabidiol (CBD) was able to recover memory deficits induced by brain iron loading in a dose-dependent manner in rats. Brain iron accumulation is implicated in the pathogenesis of neurodegenerative diseases, including Parkinson’s and Alzheimer’s, and has been related to cognitive deficits in animals and human subjects. Deficits in synaptic energy supply have been linked to neurodegenerative diseases, evidencing the key role played by mitochondria in maintaining viable neural cells and functional circuits. It has also been shown that brains of patients suffering from neurodegenerative diseases have increased expression of apoptosisrelated proteins and specific DNA fragmentation. Here, we have analyzed the expression level of brain proteins involved with mitochondrial fusion and fission mechanisms (DNM1L and OPA1), the main integral transmembrane protein of synaptic vesicles (synaptophysin), and caspase 3, an apoptosis-related protein, to gain a better understanding of the potential of CBD in restoring the damage caused by iron loading in rats. We found that CBD rescued iron-induced effects, bringing hippocampal DNM1L, caspase 3, and synaptophysin levels back to values comparable to the control group. Our results suggest that iron affects mitochondrial dynamics, possibly trigging synaptic loss and apoptotic cell death and indicate that CBD should be considered as a potential molecule with memory-rescuing and neuroprotective properties to be used in the treatment of cognitive deficits observed in neurodegenerative disorders.
KeywordsCannabidiol Iron Mitochondria Apoptosis Neurodegenerative disorders
Brain iron accumulation is implicated in the pathogenesis of neurodegenerative diseases , mainly because of the formation of free radicals that cause significant oxidative damage . Iron accumulation in neurons, astrocytes, and microglia has been reported in the basal ganglia as well as in the cortex and hippocampus, regions affected in neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), and multiple sclerosis (MS) [2, 3, 4, 5, 6, 7, 8, 9]. Studies using imaging techniques show that iron accumulation in specific brain areas correlates with poor performance in cognitive tests, both in healthy elderly individuals [10, 11, 12, 13, 14] and in patients suffering from AD and MS [9, 15, 16, 17].
Previous studies in rodents have shown that oral administration of iron during the period of rapid brain development produces iron accumulation in the basal ganglia and causes neurobehavioral deficits , including motor hypoactivity and learning, and memory impairments . The negative impact of iron on cognition may be due to increasing levels of cerebral oxidative stress, as impaired recognition memory, increased lipid peroxidation, and decreased antioxidant defenses have been observed in the hippocampus, cortex, and substantia nigra in rats presenting higher levels of iron in the brain ; for a review, see .
Through fission and fusion, mitochondria modify their morphology and length while their size and numbers also change , all dynamic processes critical for the maintenance of healthy populations of mitochondria and which can affect their function . Both mitochondrial fission and fusion are critical for the maintenance of healthy populations of mitochondria since mitochondrial fusion allows the exchange of intramitochondrial content between two or more organelles, reducing the quantity of defective mitochondria in the cells, while mitochondrial fission allows elimination of irreversibly damaged organelle content, maintaining the homogeneity and integrity of mitochondria .
Deficits in synaptic energy supply have been linked to neurodegenerative diseases such as AD and PD, evidencing the key role played by mitochondria in maintaining viable neural cells and functional circuits . Synaptic loss is a primary factor involved in the intellectual decline associated with neurodegenerative diseases such as AD . The correlation between synaptic loss and cognitive decline is observed to be stronger than that between cognitive decline and cell counts, and that synaptic loss occurs earlier and to a greater extent than neuronal death . Besides energy production, mitochondria also play a central role in starting and promoting both apoptosis as well as other types of cell death . It has also been shown that brains of patients suffering from neurodegenerative diseases have increased expression of apoptosisrelated proteins and specific DNA fragmentation .
Cannabidiol (CBD), the main nonpsychotropic constituent of Cannabis sativa, is known to have antioxidant [28, 29] and antiapoptotic [30, 31] properties. A few studies show that in newborn rats, CBD is neuroprotective against hypoxia–ischemia  and striatal lesions caused by 3-nitropropionic acid, an inhibitor of mitochondrial complex II . Additionally, we have recently shown that chronic treatment with CBD was able to recover iron-induced memory deficits in a dose-dependent manner in rats .
Lately, CBD has received attention as a promising candidate therapy in AD ; however, additional information is required to characterize the mechanisms underlying its neuroprotective properties.
To gain a better understanding of the potential of CBD in restoring the damage caused by iron loading in rats, we analyzed the expression level of proteins involved with mitochondrial fusion and fission mechanisms, namely cytosolic dynamin-related protein 1 (DNM1L) and optic atrophy protein 1 (OPA1); the main integral transmembrane protein of synaptic vesicles (synaptophysin); and caspase 3, an apoptosis-related protein. Our findings support the potential of CBD in reversing cognitive decline and its clinical use in treating neurodegenerative disorders such as PD and AD.
Material and Methods
Pregnant Wistar rats were obtained from the State Health Science Research Foundation (FEPPS-RS, Porto Alegre, Brazil).
After birth, each litter was adjusted within 48 h to eight rat pups and to contain offspring of both genders in about equal proportions. Each pup was kept with its mother in a plastic cage with sawdust bedding at a room temperature of 21 ± 1 °C and a 12/12-h light/dark cycle. At the age of 3 weeks, pups were weaned and the males were selected and raised in groups of three to five in individually ventilated cages with sawdust bedding. For postnatal treatments, animals were given standardized pellet food and tap water ad libitum.
All experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and the recommendations on animal use of the Brazilian Society of Neuroscience and Behavior (SBNec) and approved by the Institutional Ethics Committee of the Pontifical Catholic University (CEUA 11/00247). All efforts were made to minimize the number of animals used and their suffering.
Iron Neonatal Treatment
The neonatal iron treatment has been described in detail elsewhere [34, 36]. Briefly, 12-day-old rat pups received orally a single daily dose (10 mL/kg solution volume) of vehicle (5 % sorbitol in water; control group) or 30 mg/kg of body weight of Fe2+ (iron carbonyl, Sigma-Aldrich, São Paulo, Brazil) via a metallic gastric tube, over 3 days (postnatal days 12–14).
Adult (3-month-old) rats, treated neonatally with vehicle or iron were randomly distributed into two subgroups that received a daily intraperitoneal injection of vehicle (Tween 80/saline solution, 1:16 (v/v)) or CBD (approximately 99.9 % pure at 10 mg/kg; THC-Pharm, Frankfurt, Germany and STI-Pharm, Brentwood, UK) for 14 consecutive days. This experimental design resulted in four experimental groups: (1) sorbitol administered in the neonatal period and vehicle in adulthood (Sorb-Veh), (2) sorbitol administered in the neonatal period and CBD in adulthood (Sorb-CBD), (3) iron administered in the neonatal period and vehicle in adulthood (Iron-Veh), and (4) iron administered in the neonatal period and CBD in adulthood (Iron-CBD). Drug solutions were freshly prepared immediately prior to administration .
Sample Preparation for Molecular Analysis
Rats were euthanized by decapitation at 24 h after the last injection of CBD treatment. Cortex and hippocampus were quickly dissected, left hemispheres placed in a cooled RNA-later solution (Sigma-Aldrich, São Paulo, Brazil) for RT-qPCR assays, and right hemispheres placed in a cooled protease inhibitor solution (Complete Mini, Roche Applied Science, Mannheim, Germany) for Western blot assays. Samples were stored at −80 °C for subsequent molecular analyses.
Western Blot Analysis
Proteins were extracted in homogenization buffer containing 10 mM Tris–HCl (pH 8.0), 1 mM EDTA (pH 8.0), 100 mM NaCl, protease inhibitor cocktail, 0.5 % Triton X-100, and 0.1 % SDS. After 30 min in ice, samples were centrifuged at 13,500 rpm for 10 min . The supernatant was collected and the protein content was determined using Bradford assay . Aliquots were stored at −20 °C.
Twenty-five micrograms of protein was separated on a 10 % SDS polyacrylamide gel and transferred electrophoretically to a nitrocellulose membrane. Membranes were blocked with 5 % nonfat dry milk in TBS containing 0.05 % Tween 20 and were incubated overnight with one of the following antibodies: anti-β-actin (Abcam, Cambridge, UK) at 1:2,500, anti-DNM1L (Abcam, Cambridge, UK) at 1:500, anti-OPA1 (Abcam, Cambridge, UK) at 1:2,000, anti-synaptophysin (Abcam, Cambridge, UK) at 1:1,500, and anti-caspase 3 (Abcam, Cambridge, UK) at 1:1,000. Goat anti-mouse IgG and goat polyclonal anti-rabbit IgG (both from Abcam, Cambridge, UK) secondary antibodies were used and detected using ECL Western blot Substrate Kit (Abcam, Cambridge, UK). Pre-stained molecular weight protein markers (SuperSignal Molecular Weight Protein Ladder, Thermo Scientific, Rockford, USA) were used to determine the detected bands' molecular weight and confirm target specificity of antibodies. The densitometry quantification was performed using ImageJ software (http://rsb.info.nih.gov/ij/). Total blotting protein levels of samples were normalized according to each sample’s β-actin protein levels .
Quantitative Real-time PCR Analysis
Sequences of forward (F) and reverse (R) primers used in quantitative Real-time PCR analysis
The results were expressed as mean ± SEM and were analyzed using SPSS software by one-way analysis of variance (ANOVA), followed by appropriate post hoc tests when necessary. In all comparisons, p values less than 0.05 were considered to indicate statistical significance.
In the present study, we show that iron treatment significantly reduces protein levels and gene expression of the mitochondrial fission protein DNM1L in the hippocampus and mitochondrial fusion protein OPA1 levels in the cortex of rats. Additionally, we observed that iron induced a significant increase in caspase 3 protein levels both in the hippocampus and cortex while having a significant reduction of synaptophysin levels in the hippocampus. Gene expression of total caspase 3 was also increased in the cortex. CBD completely reversed iron-induced effects on the proteins here analyzed, bringing hippocampal DNM1L, caspase 3, and synaptophysin protein levels, as well as gene expression of DNM1L, back to values similar to those observed in the control group. Not all iron-induced effects on protein targets, as detected by Western blot analysis, reflected on the expression of their corresponding genes, as revealed by molecular analysis. This discrepancy was observed in the levels of OPA1 in the cortex, synaptophysin in the hippocampus, and caspase 3 in the hippocampus and suggests that a posttranscriptional modulation may have occurred in these cases. A significant reduction in OPA1 protein levels in the cortex of iron-treated rats, which was not a result of decreased OPA1 mRNA expression, could be possibly related to an increased protein ubiquitination and subsequent degradation . Alternatively, a study has demonstrated that caspase 3 stimulates OPA1 cleavage in digitonin-permeabilized rat brain mitochondria, suggesting that OPA1 is cleaved by an intermembrane space protease which is regulated by active caspases . While the present results show that mRNA expression of total caspase 3 in the hippocampus was not altered, studies show that when the apoptosis cascade is activated, caspase 3 is cleaved, generating lower molecular weight forms of caspase 3, including the active form of 17 kDa [31, 44]. Thus, the 26-kDa band quantified in our Western blot analysis is likely to represent a cleaved form of caspase 3. Additional studies analyzing the cleavage of caspase 3 substrates such as PARP, would be required to determine whether 26-kDa-cleaved caspase 3 is functional.
Little information is available on the role of mitochondrial fission and fusion proteins in neurodegenerative diseases. However, evidence indicates that an imbalance in mitochondrial dynamics, including increased mitochondrial fragmentation, is an early event associated to neurodegenerative processes. For instance, experiments using co-immunoprecipitation have shown that DNM1L interacts with hyperphosphorylated tau in post mortem AD brains and brain tissues from Amyloid precursor protein (APP), APP/presenilin-1 (PS1), and 3XTg-AD mice, possibly exacerbating mitochondrial deficiencies . In addition, APP overexpression has been shown to reduce the levels of DNM1L and OPA1 while increasing the levels of Fis1, which induces changes in mitochondrial morphology and distribution and increases reactive oxygen species (ROS) levels in human neuroblastoma M17 cells .
Mitochondria contribute to cellular iron homeostasis in which ROS related to iron deregulation acts in a feed-forward manner, as both cause and consequence of mitochondrial dysfunction . We have previously reported that iron treatment increases oxidative stress parameters and superoxide production in submitochondrial particles [20, 48] in the cortex and hippocampus of rats. We thus hypothesize that iron-induced oxidative stress may be related to mitochondrial dysfunction, resulting in alterations in the expression of fusion and fission proteins observed here, which might ultimately lead to unbalanced mitochondrial dynamics and impaired energy production in our model. A reduction in the mitochondrial fission protein DNM1L is possible which may prevent elimination of defective mitochondria and that CBD acts by antagonizing this effect.
Impaired mitochondrial function may lead to a reduction in synaptic viability, as these organelles are preferentially localized in synaptic terminals. Our results show that synaptophysin, one of the main proteins in presynaptic terminals, was significantly reduced in the hippocampus of rats treated with iron, suggesting that these rats may have synaptic abnormalities contributing to iron-induced memory impairments. These results are consistent with other studies, which show reduced synaptophysin in the posterior cerebral cortex in a mouse model of brain aging associated with cerebral atrophy and learning and memory deficits .
In AD patients, synaptophysin levels are reduced in the hippocampus and in the frontal cortex, when compared with controls, and hippocampal synaptophysin levels correlated with neuropsychological measurements, including Mini-mental State Examination scores [49, 50]. Interestingly, we show that CBD was able to reverse iron-induced reductions in synaptophysin levels and, as previously reported , to improve memory impairments associated to iron.
Although some studies have described that fission machinery promotes apoptotic death while fusion machinery protects against apoptotic stimulus [51, 52], in our study we found reduced DNM1L fission protein levels in combination with increased caspase 3 protein levels in the hippocampus. Thus, other stimuli, possibly related to the extrinsic apoptotic pathway, including a reduction in neurotrophic factors, are likely to relate to the increased levels of caspase 3 observed here. Faulty synaptic transmission can contribute to increasing and/or activating the neuronal death machinery, and may work as apoptotic stimulus . Our results indicate a combined occurrence of mitochondrial deregulation and synaptic loss with increased caspase 3 levels, corroborating the hypothesis that the mitochondrial imbalance results in energetic failure leading to an impaired synaptic function that can culminate in increased activity of the cellular death machinery. Our findings confirm a previous immunohistochemistry study in which we demonstrated that iron leads to increased levels of PAR4 and caspase 3, key apoptotic markers involved in triggering and promoting activation of apoptosis cascade in the cortex and hippocampus of adult rats, leading to a potential susceptibility to neurodegeneration . A previous in vitro study using human neuroblastoma cells exposed to ferric ammonium citrate has shown that iron overload produces increased oxidative stress and activates redox-sensitive signals, leading to apoptotic cell death, with increases in caspase 3 activity . Additionally, iron chelation with M30 decreases apoptosis in human neuroblastoma cells by reducing pro-apoptotic proteins Bad and Bax and by inhibiting the cleavage and activation of caspase 3 . The present results showing that CBD reversed iron-induced hippocampal increase of the active form of caspase 3 are consistent with previous studies indicating that CBD was able to reduce the concentration of caspase 9 in damaged tissue in an animal model of hypoxic–ischemic injury  and to enhance the levels of pro-caspase 3. Additionally, CBD reduced the levels of caspase 3 in PC12 cells treated with Aβ, preventing DNA fragmentation (a typical process of programmed cell death) induced by Aβ .
Over the years, we have focused our research on the effects of iron treatment on cognitive parameters. We have consistently demonstrated that iron, when administered in the neonatal period, induces significant and long-lasting cognitive deficits in a variety of hippocampal-based memory paradigms, including spatial , aversively motivated [19, 36], and recognition memory tasks . In this study, the most pronounced iron-induced effects were noteworthy to be observed in the hippocampus. We have recently demonstrated that CBD was able to completely abolish iron-induced deficits in hippocampus-dependent memory . The present findings show that CBD was able to rescue iron-induced alterations in synaptophysin and caspase 3 levels, as well as DNM1L expression in the hippocampus. Thus, by reducing caspase 3 levels, possibly due to CBD antiapoptotic properties, and by recovering synapthophysin levels back to control values, CBD may confer neuroprotection. These effects might explain memory-ameliorating properties of CBD. In humans, recent studies have shown that CBD protects against THC-induced reduction of hippocampal volume in chronic cannabis users  and prevents THC-induced hippocampal episodic memory impairment, which may relate to its cannabinoid receptor 1 (CB1) antagonistic effects .
Interestingly, we observed that CBD significantly increased the levels of the mitochondrial fusion protein OPA1 in the hippocampus but not in the cortex of sorbitol-treated rats, while a trend towards reduced DNML1 occurred in the hippocampus but not in the cortex in rats treated with CBD when compared with its respective control group, suggesting that CBD may have direct effects on mitochondrial dynamics, which seem to be region-specific. Corroborating our in vivo results, Ryan and colleagues  demonstrated that CBD protects against the mitochondrial uncoupler FCCP in SH cells and in cultured hippocampal neurons in vitro. To our knowledge, no other studies have investigated the effects of CBD on mitochondrial dynamics, thus, more studies are necessary to better elucidate the effects of CBD on mitochondria in the hippocampus.
Taken together, our findings support the hypothesis that CBD positively modulates hippocampal memories while it also offers neuroprotection. Given its safety and reduced side effects, CBD is a promising candidate for the treatment of neurodegenerative disorders involving memory decline, such as AD. Considering that recent studies  have implicated iron accumulation as an early pathological event in MS, CBD might also be considered as a neuroprotective agent for MS patients.
In conclusion, our study shows that rats treated with iron present altered mitochondrial dynamics, which is possibly related to iron-induced oxidative stress. Impaired mitochondrial functioning and synaptic loss may contribute to the activation of apoptotic mechanisms, increasing apoptotic markers, such as caspase 3, observed here. Impaired mitochondrial functioning and synaptic loss may play a causative role in the cognitive deficits reported in our model, as has been described in previous studies [20, 34, 36, 60, 61]. However, the lack of other molecular markers of mitochondrial dynamics, synaptic function, and apoptotic cascade limits the interpretation of the present data and warrants more studies to allow a better understanding of this complex scenario.
This is a pioneer study linking in vivo iron loading with changes in gene and protein expression that suggest alterations in mitochondrial dynamics and synaptic loss in the central nervous system. Altogether, our findings indicate that CBD should be considered as a potential molecule with memory-rescuing and neuroprotective properties to be used in the treatment of cognitive deficits observed in neurodegenerative disorders.
V.K.S. is supported by a CAPES/MEC fellowship. L.F. is supported by a FAPERGS scholarship. M.R.B, J.E.H., A.W.Z., J.A.C., and N.S. are CNPq Research fellows. This research was supported by the National Institute for Translational Medicine (INCT-TM). This manuscript was reviewed by a professional science editor and by a native English-speaking copy editor to improve readability.
Conflict of Interest
The authors declare that they have no conflict of interest.