Novel insights into mitochondrial molecular targets of iron-inducedneurodegeneration: Reversal by cannabidiol

Vanessa Kappel da Silva, Betânia Souza de Freitas, Victória Campos Dornelles,Luiza Wilges Kist, Maurício Reis Bogoc, Milena Carvalho Silvad, Emílio Luiz Streck, Jaime Eduardo Hallak, Antônio Waldo Zuardib,e, José Alexandre S. Crippab,e,Nadja Schrödera

Brain Research Bulletin, 2018, 139, 1–8

https://doi.org/10.1016/j.brainresbull.2018.01.014

 

ABSTRACT

Evidence has demonstrated iron accumulation in specific brain regions of patients suffering from neurodegen-erative disorders, and this metal has been recognized as a contributing factor for neurodegeneration. Using anexperimental model of brain iron accumulation, we have shown that iron induces severe memory deficits thatare accompanied by oxidative stress, increased apoptotic markers, and decreased synaptophysin in the hippo-campus of rats. The present study aims to characterize iron loading effects as well as to determine the molecular targets of cannabidiol (CBD), the main non psychomimetic compound of Cannabis sativa, on mitochondria. Rats received iron in the neonatal period and CBD for 14 days in adulthood. Iron induced mitochondrial DNA (mtDNA) deletions, decreased epigenetic modulation of mtDNA, mitochondrial ferritin levels, and succinate dehydrogenase activity. CBD rescued mitochondrial ferritin and epigenetic modulation of mtDNA, and restoredsuccinate dehydrogenase activity in iron-treated rats. Thesefindings provide new insights into molecular targetsof iron neurotoxicity and give support for the use of CBD as a disease modifying agent in the treatment of neurodegenerative diseases.

Keywords : Cannabidiol, Neurodegeneration, Mitochondria, mtDNA methylation, Iron, Mitochondrial ferritin

 

1. Introduction

The etiology of neurodegenerative diseases has not been completelyelucidated, but it is widely accepted that oxidative damage, linked toaccumulation of transition metals, can contribute to neurodegeneration(Salvador et al., 2010; Kim et al., 2015). Progressive iron accumulationin the brain has been described during the normal aging process. Re-markably, in neurological diseases such as Alzheimer’s (AD), Parkin-son’s (PD), and Huntington’s (HD) diseases, iron accumulates in brainareas relevant to disease-associated neurodegenerative processes(Stankiewicz and Brass, 2009; Mills et al., 2010). For instance, it hasbeen demonstrated that iron selectively accumulates in thesubstantianigra pars compactain PD patients (Dexter et al., 1991; Sofic et al.,1991), while it builds up around and within amyloid plaques and neurofibrillary tangles in brains from AD patients (Connor et al., 1992; Lovell et al., 1998).

In previous studies, aiming to examine the mechanisms of iron neurotoxicity in neurodegenerative diseases, we have established an animal model of brain iron loading, with oral administration of iron during the neonatal period, which is the period of maximal iron uptake by the brain (Taylor and Morgan, 1990). Iron neonatal treatment induces emotional memory deficits, tested in the inhibitory avoidance task (Schröder et al., 2001; Fagherazzi et al., 2012; Figueiredo et al.,2016) as well as recognition memory impairments (de Lima et al., 2005; Fagherazzi et al., 2012; Figueiredo et al., 2016). These memory deficits are accompanied by increased thiobarbituric acid reactive species (TBARS), protein carbonylation and superoxide production (Dal-Pizzolet al., 2001;de Lima et al., 2005), increased levels of apoptotic markers Par4 and caspase 3 (Miwa et al., 2011, da Silva et al., 2014), and reactive astrogliosis (Fernandez et al., 2011). In addition, recent studies have shown that iron overload leads to accumulation of ubiquitinated proteins (Figueiredo et al., 2016), decreased levels of synaptophysin, as well as alterations in the levels of DNM1L, a protein critically involved in mitochondrial fission (da Silva et al., 2014), resembling common alterations observed in neurodegenerative disorders. Kaur et al. (2007) reported that mice, treated with iron for eight days during the neonatal period with a dose four times higher than the one used in our model, developed histological and neurochemical alterations relevant to PD pathology, i.e., increased oxidative stress, decreased striatal dopamine content and nigral tyrosine hydroxylase (TH) positive cells.

Neurons are highly differentiated cells with high energy requirements, which mainly come from mitochondria, warranting neuronal survival and many essential functions, including axonal growth and
branching, generation of action potentials, and synaptic transmission and plasticity (Lin and Sheng, 2015). Therefore, it has been suggested that dysfunctions in these organelles play a role in the pathogenesis of neurological disorders (Mattson et al., 2008). Mitochondrial DNA damage has already been related to aging and neurodegenerative diseases and contribute to mitochondrial disruption, which in turn may lead to cell injury, particularly in the central nervous system (CNS) (Siddiqui et al., 2012; Mao et al., 2012; Grünewald et al., 2016). Recently, studies have shown that mtDNA is also subject of epigenetic regulation, including methylation (5mC) and hydroxymethylation (5hmC) (Manev and Dzitoyeva, 2013). Increases and decreases of DNA methylation have been observed during aging, but most of the studies are limited to nuclear DNA (Richardson, 2003). Mitochondrial epigenetics are in nascent form and should be better and extensively studied (Manev et al., 2012).

Mitochondria maintain cellular energy reserves, which are extremely important to the CNS, by keepping respiratory chain and Krebs cycle under strict control (Basha and Poojary, 2014). Creatine kinases catalyze reversible transfer of phosphoryl groups between ATP and creatine, mainly in high energetic consumption tissues, being essential to energy homeostasis (Pilla et al., 2003). Mitochondrial dysfunction compromises energetic metabolism, resulting in overproduction of reactive oxygen species (ROS) and bioenergetic failure to the cells, contributing to many neurodegenerative diseases (Arun et al., 2016).

Since mitochondria are the major sources of cellular iron utilization, these organelles play a key role in maintaining iron homeostasis (Napier et al., 2005). Iron entry across the mitochondrial inner membrane requires one of two homologous proteins of the mitochondrial solute carrier family, called mitoferrin 1 and mitoferrin 2 (Paradkar et al., 2009). Mitoferrin 1 is the main mitochondrial iron importer in haematopoietic tissues, while mitoferrin 2 contributes to iron acquisition in non-erythroid tissues (Shaw et al., 2006; Paradkar et al., 2009). In 2001, Levi and coworkers (Levi et al., 2001) identified an iron storage protein inside mitochondria, mitochondrial ferritin, with a similar structure to the cytosolic ferritin. It has been described that mitochondrial ferritin is expressed preferentially in tissues with high oxygen consumption and has a role in protecting mitochondria from oxidative damage induced by free iron rather than storing iron (Levi and Arosio, 2004; Santambrogio et al., 2007). Although it has been
proposed that both mitochondrial disruption and iron accumulation are involved in the pathophysiology of neurodegenerative disorders, there is restricted information about the regulation of mitochondrial mechanisms of iron transport and storage in these diseases.

Cannabidiol (CBD) is the main non-psychotropic constituent of Cannabis sativa, corresponding to approximately 40% of plant extract (Campos et al., 2012; Zuardi, 2008). Evidence indicates that CBD possesses antioxidant, antiapoptotic, and neuroprotective properties (Hampson et al., 1998; Iuvone et al., 2004; García-Arencibia et al., 2007; Castillo et al., 2010, Pazos et al., 2012). We have previously shown that CBD completely reverses iron-induced memory deficits (Fagherazzi et al., 2012) and normalizes hippocampal levels of caspase 3, synaptophysin, and mitochondrial fission protein DNM1L in rats with brain iron overload (da Silva et al., 2014). The aim of the present study was to characterize the effects of iron loading on mitochondrial physiology by measuring mtDNA deletions and mtDNA epigenetic modifications in the hippocampal formation, a brain region critically involved in learning and memory, known to be primarily affected in AD. We also wanted to determine if neonatal iron loading would hinder mitochondrial iron handling, by altering the expression of mitoferrin 2 and mitochondrial ferritin later in life. Relevant functional parameters of energy metabolism, succinate dehydrogenase and creatine kinase activities, were also analyzed in the hippocampus of iron-loaded rats. Considering that CBD proved to ameliorate iron-induced memory deficits, which are relevant in the context of aging and
neurodegenerative disorders, and the demand for neuroprotective treatments, we further investigated possible targets of CBD action on iron-induced mitochondrial alterations.

(…)

1-s2.0-S0361923017303714-main