Elsevier

Gene

Volume 590, Issue 2, 30 September 2016, Pages 227-233
Gene

Research paper
Age-related gene expression change of GABAergic system in visual cortex of rhesus macaque

https://doi.org/10.1016/j.gene.2016.05.010Get rights and content

Highlights

  • Expressions of 24 GABAergic function related genes were evaluated in visual cortex of rhesus macaques during ageing.

  • The mRNA of GAD65 was down-regulated in middle-aged males and the protein was down-regulated in aged male monkey.

  • 12 genes were significantly up-regulated during ageing process.

Abstract

Degradation of visual function is a common phenomenon during aging and likely mediated by change in the impaired central visual pathway. Treatment with GABA or its agonist could recover the ability of visual neurons in the primary visual cortex of senescent macaques. However, little is known about how GABAergic system change is related to the aged degradation of visual function in nonhuman primate. With the use of quantitative PCR method, we measured the expression change of 24 GABA related genes in the primary visual cortex (Brodmann's 17) of different age groups. In this study, both of mRNA and protein of glutamic acid decarboxylase (GAD65) were measured by real-time RT-PCR and Western blot, respectively. Results revealed that the level of GAD65 message was not significantly altered, but the proteins were significantly decreased in the aged monkey. As GAD65 plays an important role in GABA synthesis, the down-regulation of GAD65 protein was likely the key factor leading to the observed GABA reduction in the primary visual cortex of the aged macaques. In addition, 7 of 14 GABA receptor genes were up-regulated and one GABA receptor gene was significantly reduced during aging process even after Banjamini correction for multiple comparisons (P < 0.05). These results suggested that the dysregulation of GAD65 protein might contribute to some age-related neural visual dysfunctions and most of GABA receptor genes induce a clear indication of compensatory effect for the reduced GABA release in the healthy aged monkey cortex.

Introduction

Visual retrogression is one of the typical physiological changes during aging process in humans and nonhuman primates. Several studies suggest that visual dysfunction may be due to the retina or central visual pathways in the neural structure of a recession or loss of function (Trick et al., 1986, Spear et al., 1994, Tran et al., 1998, Porciatti et al., 1999). Considerable researches also demonstrated that the changes of neurotransmitter, such as GABAergic system, may affect the visual function in mammalian. The content of GABA, GABA synthetic enzyme (GAD) and GABA receptors are down-regulated by monocular deprivation in monkey visual cortex (Hendry and Jones, 1986, Hendry et al., 1987, Hendry and Jones, 1988, Munoz et al., 2001). Neurophysiology studies have recently demonstrated that the density of GABAergic neurons is remarkably declined with age in the primary visual cortex of cat and rat (Hua et al., 2006, Wang et al., 2006). The orientation selectivity of neurons in adult rat visual cortex could be significantly decreased by a special antagonist of GABAA receptor (bicuculline) (Eysel et al., 1998). However, the visual function can be facilitated by GABA or the agonist of GABA receptors in the primary visual cortex of the senescent primates (Leventhal et al., 2003).

More researches revealed that the inhibitory effect of GABA has indeed decreased during the aging process, such as the elderly animal cells reduce to the response for the bicuculline, the binding site of GABAA receptor agonist (agonist) reduced (Post-Munson et al., 1994). In addition, the decreased expression of GABA receptor subunits or GAD had been reported in the other cortex of rodent or primate in aged groups (Gutierrez et al., 1994, Gutierrez et al., 1996, Gutierrez et al., 1997, Caspary et al., 1999, Ling et al., 2005, Rissman et al., 2006, Caspary et al., 2013). These data suggested a reduction in GABAergic inhibition in senescence ones, which might be one of the key mechanisms explaining the decline of visual function during senescence.

GABA is the major inhibitory neurotransmitter in the adult central nervous system. Many genes are involved in GABAergic system, including synthesis, transportation, binding and reuptake. GABA is synthesized by glutamate decarboxylase (GAD) via glutamate and catabolized by two catabolic enzymes, GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). Glutaminase (GLS) is the enzyme that deaminates glutamine to glutamate, which is the precursor of GABA. GAD is present in two isoforms with molecular sizes of 65 kDa (GAD 65) and 67 kDa (GAD 67), which are encoded by two genes respectively on chromosomes 10 and 2 (Bu et al., 1992, Esclapez et al., 1993, Feldblum et al., 1993, Soghomonian and Martin, 1998). They exhibit different subcellular localizations, functions, regulatory properties and co-factor interaction (Kaufman et al., 1991). Studies indicate that GAD65 appears to be targeted to membranes and nerve endings and preferentially synthesizes GABA for vesicular release, while GAD67 is more widely distributed in the cells and synthesizes mainly non-vesicular cytoplasmic GABA (Feldblum et al., 1993, Hendrickson et al., 1994, Feldblum et al., 1995, Soghomonian and Martin, 1998).

GABA carries out its function by recognizing three major types of receptors, the ionotropic GABAA and GABAC receptors and the metabotropic GABAB receptor (Macdonald and Olsen, 1994, Lukasiewicz and Shields, 1998). GABAA, which ubiquitous in the CNS, is a GABA-gated Cl channel. The channel is the tetramer or the pentamer made of some classes of subunit (alpha, beta, gamma). To date, eight classes of GABAA receptor subunits have been cloned in mammalian brain (α1–6, β1–3, γ1–4, ρ1–3, δ, ε, π and θ) (Olsen and Tobin, 1990, Macdonald and Olsen, 1994, Rabow et al., 1995, Whiting et al., 1999, Whiting, 2003). GABAA receptors mainly mediated the inhibitory of post-synaptic neurons. The membrane is hyperpolarized when the channel opens coupled with the Cl influx in the neurons(Burt and Kamatchi, 1991). GABAB receptors appear to be coupled to Ca2 + and K+ channels of presynaptic membranes. Three subunits have been cloned and are termed GABABR1a, GABABR1b and GABABR2 (Kaupmann et al., 1997). It seems they regulate the neurotransmitters release (Chebib and Johnston, 1999). GABAC receptors are enriched in the retina, compared to other parts of the CNS (Lukasiewicz and Shields, 1998).

In CNS, four GABA transporters have been found from human brain, i.e., GAT1-4 (Nelson et al., 1990, Lam et al., 1993, Borden et al., 1994, Borden et al., 1995). GAT-1 is the most abundant and has been shown to be localized in neurons and in astrocytes (Dirkx et al., 1995). However, GABA taken up into the presynaptic GABAergic nerve terminals is reutilized as transmitter, accumulated into synaptic vesicles by a vesicular transporter VGAT (Gram et al., 1988, McIntire et al., 1997, Gasnier, 2004). Some studies demonstrate that VGAT forms a protein complex with GAD65 on the synaptic vesicles (SVs) (Jin et al., 2003) and package GABA into SV (Buddhala et al., 2009).

A considerable amount of researches are already available on these genes in the cortex of several species (Rakic et al., 1988, Drago et al., 1989, Gutierrez et al., 1994, Gutierrez et al., 1996, Caspary et al., 1999, Hwang et al., 2004, Rissman et al., 2006, Yu et al., 2006). Few studies focused on aged-related changed GABAergic system in the primate. The present study assessed the age-related expression changes of 24 GABA related genes in the primary visual cortex of young, middle-aged and aged of rhesus macaque.

Section snippets

Animal subjects

A total of sixteen rhesus macaques (Macaca mulatta) were tested in this study. Three age groups were assigned. According to previous studies, the age-related motion and cognitive decline occurs at about 20 years old (Vincent et al., 1989 ; Tigges et al., 1995). Therefore, the macaques older than 20 years were assigned to the aged group (age 23–26; n = 4; 2 male, 2 female). For the control, eight adult macaques were included in the adult group (age 8–15; n = 8; 4 male, 4 female). We also included five

Enzyme message and protein change

Five presynaptic enzyme message levels were obtained from primary visual cortex from adult, middle-aged and aged rhesus macaques. ANOVA showed that GAD65 and GAD67 have the different expression pattern in the primary visual cortex (Fig. 1). The data showed that the mRNA expression of GAD65 is decreased with age, but there had no significant difference among three groups. The level of GAD67 was significant higher in the middle-aged group relative to the adult group (P = 0.003) (Fig. 1), and the

Discussion

In the study, we systematically investigated the expression change of 24 GABAergic genes in the primary visual cortex of macaques among three different age groups, and we observed significant expression change during the aging process. The present findings supported previous studies on age-related change of GABAergic receptors in primates and rodents (Leventhal et al., 2003, Rissman et al., 2006, Caspary et al., 1990, Caspary et al., 1999, Gutierrez et al., 1994, Gutierrez et al., 1996, Vela et

Conclusion

In general, the results of this study suggested the dramatic change of gene expression in the GABAergic system of the primate cortex. The abundance of GAD65 protein were apparently remarkably decreased in the aged monkeys despite no significant changes in the mRNA expression. The increased expression of genes, especially those GABA receptors, indicated a self-regulated compensatory mechanism during aging. Taken together, these changes may affect the homeostasis of GABA in the CNS, which are

Acknowledgement

This work was supported by grants from the National 973 project of China (2011CBA00401 and 2012CBA01300), the National Natural Science Foundation of China (31130051, 31301028 and 31321002) and the Natural Science Foundation of Yunnan Province (2007C100M and 2009CD107). This study was also supported by funding from the West Light doctoral program.

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