Autophagy-dependent mechanism of genistein-mediated elimination of behavioral and biochemical defects in the rat model of sporadic Alzheimer's disease
Introduction
Alzheimer disease (AD) appears to be the most common neurodegenerative disease. The major symptoms include memory impairment, cognitive deficit, difficulty in decision-making, and behavioral changes (Scheltens et al., 2016). At the molecular level, this disease is characterized by appearance of senile plaques and intraneuronal neurofibrillary tangles. These pathological structures consist of β-amyloid (βA) and hyperphosphorylated tau protein (p-tau), respectively (Sanabria-Castro et al., 2017). However, one should note that the detailed mechanism of Alzheimer disease has not been elucidated yet, and some hypotheses assume that βA and p-tau might be effects rather than causes of primary pathological changes, thus, suggesting roles of other factors in the pathology (Szutowicz et al., 2017). Nevertheless, irrespective what are actual primary reasons of AD, it is generally accepted that accumulation of βA (particularly βA40 and βA42) and p-tau causes progressive degeneration of neurons, which leads to the symptoms described above (Sanabria-Castro et al., 2017).
Despite extensive studies on AD, no effective treatment is available to date, and it is estimated that even if research and development processes are very efficient, a therapy of high efficacy might be available not earlier than in 2025 (Cappa, 2018). One of novel approaches in treatment of neurodegenerative diseases is stimulation of the autophagy process. It is expected that this might lead to degradation of accumulated pathogenic aggregates of macromolecules, thus, eliminating the causes of neurodegeneration (Pierzynowska et al., 2018a). This strategy can also potentially work in treatment of AD, as suggested recently (Uddin et al., 2018). However, the most important limitation of the use of vast majority of known autophagy stimulators is that they do not meet the criteria to be employed as therapeutics for a long-term use. Particularly, such compounds should not only cross the blood-brain-barrier and stimulate autophagy, but also act sufficiently gentle to avoid cell destruction during a long treatment, and thus, to exclude a possibility of severe adverse effects (Yang et al., 2013). Unfortunately, despite effective activation of autophagy, the compounds considered to date as potential drugs revealed multiple adverse effects, which perhaps could be acceptable in a short-term therapy, but not in a putative lifelong treatment as in AD (Pierzynowska et al., 2018a). Nevertheless, recent studies indicated that genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) is a promising compound in this aspect. This molecule is able to cross the blood-brain-barrier (Tsai, 2005), and it was found to be non-toxic to animals even when used at as high concentration as 160 mg/kg/day (Malinowska et al., 2009). Clinical studies, performed with children suffering from Sanfilippo disease (one of mucopolysaccharidoses, a group of lysosomal storage diseases), demonstrated its safety in the long-term (over 1 year) use at the high (150 mg/kg/day) dose (Kim et al., 2013). Apart from stimulation of lysosomal biogenesis, due to activation of transcription factor EB (TFEB) (Moskot et al., 2014), genistein was found recently to positively regulate the autophagy induction which leads to elimination of mutated huntingtin aggregates in the cellular model of Huntington's disease (Pierzynowska et al., 2018b). Therefore, we considered reasonable to test high dose of genistein (150 mg/kg/day, i.e. the dose which has been investigated previously in Sanfilippo disease) in an animal model of AD. Since majority of AD cases are sporadic, rather than inherited (familial), we aimed to use a rat model for such a disease form.
One should note that genistein has been tested previously in cellular and animal models of AD (summarized by Devi et al., 2017). However, lower doses of this compound were employed (10 mg/kg by Bagheri et al., 2011, 200 μM in cell cultures, which is an equivalent to 54 mg/kg, by Zhou et al., 2014, 0.022 mg/kg/day by Bonet-Costa et al., 2016, about 25 mg/kg/day by Park et al., 2016, and 10, 30 and 90 mg/kg/day by Wang et al., 2016). In those studies, animal models used were either ApoE−/− mice fed with a high-fat diet or rats with intraperitoneal injection of D-galactose and/or intracerebral injection of βA, and BV-2 microglia cells were employed in in vitro tests (Bagheri et al., 2011; Zhou et al., 2014; Bonet-Costa et al., 2016; Park et al., 2016; Wang et al., 2016). Although some positive effects were reported, such models only partially mimic human AD as accumulation of some, but not all, kinds of pathogenic molecules is induced. In those studies, the action of genistein was suggested to be dependent on inhibition of apoptosis, or anti-inflammatory and antioxidative properties of this compound (Bagheri et al., 2011; Zhou et al., 2014; Bonet-Costa et al., 2016; Park et al., 2016; Wang et al., 2016).
In this study, we decided to employ the streptozotocin (STZ)-treated rat males, which have been considered as the best animal model of the sporadic AD. Intracerebroventricular infusion of STZ was shown to cause tau protein hyperphosphorylation, accumulation of βA (including βA40 and βA42 forms), as well as a loss of dendritic and synaptic plasticity (Bao et al., 2017; Rostami et al., 2017; Tong et al., 2017). Behavioral alterations in STZ-treated animals, resembling those occurring in human AD patients, were demonstrated, and have been summarized and discussed in detail (Salkovic-Petrisic and Hoyer, 2007). It is crucial that intracerebroventricular application of STZ causes development of the insulin resistant brains state, and results in progressive cholinergic deficits, glucose hypometabolism, oxidative stress and neurodegeneration, leading to memory impairment (Salkovic-Petrisic et al., 2013). We aimed to test if high dose (150 mg/kg/day) of genistein can improve biochemical and/or behavioral parameters in animals, and if yes, to investigate the mechanisms of genistein action.
Section snippets
Animals
The experiments were conducted on male Wistar rats (Tri-City Central Animal Laboratory, Research and Service Center of the Medical University of Gdansk, Poland), n = 6 for each investigated group for histological (microscopic) and biochemical (Western-blotting) analyses (which means n = 12 and n = 6 for behavioral tests at 30 and 90 days after STZ administration, respectively; see Fig. 1), weighing approximately 350 g at the beginning of the experiment. The animals were housed in polycarbonate
Genistein corrects behavior in the rat model of sporadic AD
Behavioral tests were performed 30 and 90 days after STZ/vehicle injections. Following tests were performed: Morris water maze, elevated plus maze test, open field test, and locomotor measurements in actometers. The scheme of experiments, indicating duration of particular periods and time points of specific tests, as well as number of animals used in particular tests, is presented in Fig. 1. Time points of biochemical and histological tests are also indicated in this scheme. It is important to
Discussion
Neurodegenerative diseases are defined as disorders resulting from the gradual and progressive loss of neural cells (Brown et al., 2005). Despite many years of studies, no cure is available for these diseases apart from symptomatic treatment which can only weakly alleviate major destructive effects of neurodegeneration (Chen and Pan, 2014).
In many neurodegenerative diseases, including those considered as major problems in current medicine, like AD, the pathological processes depend on formation
Conclusions
In conclusion, treatment with high dose (150 mg/kg/day) genistein can normalize behavioral and biochemical defects in the STZ-induced rat model of sporadic AD. Activation of autophagy appears to be the primary mechanism of this phenomenon. As the sporadic form of AD is undoubtedly caused by multiple factors, and there are many pathological processes ongoing in the affected organism, including accumulation of protein aggregates, enhanced apoptosis, oxidative stress, and neuroinflammation, one
Conflict of interest
The authors declare no conflict of interest.
Authors’ contributions
KP was the initiator of the project, planned all biochemical experiments and microscopic analyses and performed some of them, participated in animals care, participated in behavioral studies, analyzed all experimental results, and participated in drafting the manuscript; MP participated in animals care, conducted behavioral experiments and analyzed their results, performed statistical analyses, and prepared parts of the manuscript related to animal studies; LG performed a part of biochemical
Acknowledgments
This work was supported by National Science Center, Poland (project grant no. 2013/09/D/NZ4/01658 to IM), and Faculty of Biology of University of Gdańsk (task grant no. 530-L140-D242-17-1A).
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