Introduction

Amino acids (AA) are the basic units of proteins; if there is any AA deficiency, the synthesis of proteins is altered, causing a unbalance in the cellular physiology and organism. AA have different functions and are essential for the physiology of the body; for example, hormones such as insulin, growth hormone, and glucagon, are made up of AAs. It is known that some AAs regulate the in vitro and in vivo secretion of insulin in pancreatic β-cells. AAs play an important role in different processes related to gene expression, including the modulation of proteins that regulate mRNA¹.

In the central nervous system (CNS), some AA are neurotransmitters and have a key role in neuronal communication, such as glutamate and glycine. In the case of GABA, despite that chemical structure, it is not considered like AA, because it does not conform proteins. These neurotransmitters are stored in vesicles inside the presynaptic neuron and released into the synaptic cleft by a Ca²⁺-dependent mechanism and in response to a depolarizing stimulus. These neurotransmitters generate excitatory or inhibitory responses through binding to their specific receptors in the post-synaptic neuron or even the same presynaptic cell. A malfunction of these neurotransmitter communication systems is associated with different neurological or psychiatric disorders. For this reason, several pharmacological therapies aim to regulate release mechanisms, receptor binding, or neurotransmitter reuptake.

In this review, we will focus on the inhibitory neurotransmitter GABA (g-aminobutyric acid), specifically in the participation of their receptors in different neurodegenerative disorders and the different pharmacological strategies aimed at their regulation for the treatment of these pathologies.

GABA

GABA is the main inhibitory neurotransmitter of the CNS and one of the most abundant neurotransmitters in mammals. It was first described in the early 1900s, while its presence and participation as a neurotransmitter in the mammalian CNS was determined until the 1950s. During the following two decades, many studies established its mechanism of action, as well as its inhibitory activity in the cerebral cortex². We, currently, know that GABA is distributed in most areas of the brain and participates in 40% of the inhibitory synapses of adult vertebrates.

GABA metabolism

GABA is produced in the CNS, through the decarboxylation of glutamic acid, and catalyzed by glutamic acid decarboxylase (GAD). In general, the enzymatic activity of GAD is regulated by its expression levels and the degree of association with the cofactor pyridoxal phosphate (PLP)³. GAD has two isoforms, GAD₆₅ and GAD₆₇, both encoded by different genes, and different expression patterns, but with similar mechanisms regulating their function⁴,⁵. GAD₆₅ is located mainly in the synaptic terminals, where it induces vesicular GABA release in a Ca²⁺-dependent process. GAD₆₅ dissociates from PLP under physiological conditions and increases its activity depending on the demands of the synaptic release of GABA⁶. Therefore, the main regulatory mechanism for this isoform is the association with its cofactor and not it is level of expression³.

GAD₆₇ is expressed in the cytoplasm, it is associated with PLP under physiological conditions, and is regulated by its levels of expression. GAD₆₇ is involved in cell metabolic activity and is responsible for most of the GABA synthesis in the brain³. However, it has been suggested that cytoplasmic GABA can be released by a Ca²⁺-independent mechanism. If this is correct, then GAD₆₇ may participate in inhibitory neurotransmission by activating extra-synaptic GABA receptors.

After GABA release into the synaptic cleft, it is reuptake by the GAT-1 and GAT-3 transporters, these systems are expressed in both neurons and glial cells. GABA catabolism is carried out through the GABA transaminase (GABA-T). GABA-T is dependent on PLP, which, in turn, requires GABA and α-ketoglutarate as substrates, generating glutamic acid, and succinic semialdehyde (SSA) as by-products. SSA can be metabolized into γ-hydroxybutyric acid, which regulates GABA_B receptors, or can be dehydrogenated to succinate. When GABA is dehydrogenated to succinate via SSA dehydrogenase, it is incorporated into the Krebs cycle, where it participates in cellular energy metabolism. Many clinical and preclinical researches indicate the presence of GABAergic neurons in regions such as the amygdala, hippocampus, hypothalamus, prefrontal cortex, olfactory bulb, retina, and spinal cord.

GABA receptors

GABA exerts its inhibitory effect through two types of specific receptors called GABA_A and GABA_B, which show different pharmacological, structural, and molecular differences (Fig. 1). GABA_A receptors, or ionotropic, share its structural and functional properties with the ligand-gated ion channels or “Cys-loop” family, which include glycine (Gly), acetylcholine (ACh), and serotonin (5-HT₃) receptors⁷. The complexity of GABA_A receptors lies in the number of subunits they contain, and in the different combinations in which they are assembled. There are also variants generated by splicing or RNA editing⁸. To date, six subunits have been characterized in humans: six type a, three type b, three type g, and one each of type d, e, p, and q, giving this receptor a high degree of heterogeneity. On the other hand, three subunits r have been characterized, which unlike the others form functional homopentamers with different pharmacological properties. These subunits are considered a subfamily called GABA_C. In adult mammals, the most abundant isoform is composed of the subunits α₁β₂γ₂. However, the number of GABA_A receptor isoforms expressed in mammals is still unknown⁹ (Fig. 2).

Figure 1 Representative molecular structure of GABA and GABA receptors. GABA is a full agonist of two types of receptors, named according to their mechanism of action. GABA_A or ionotropic receptors, are protein channels activated by GABA and permeable to Cl-. These receptors are conformed by different subunits, six type α, three type β, three type γ, and one each of type δ, ε, π and θ. GABA_B, or metabotropic receptors, are receptors coupled to G proteins that modulate K⁺ and Ca²⁺ channels and are conform for only two subunits. The GABA structure was from https://es.wikipedia.org/wiki/Ácido_γ-aminobutírico (Last accessed on September 20, 2020). 

Figure 2 Structural conformation of GABA_A receptors. Note the number of subunits that contain, and in the different combinations in which they are assembled. Some of them are variants from splicing. giving this receptor a high degree of heterogeneity. On the other hand, three subunits ρ have been characterized, which unlike the others form functional homopentamers. In adult mammals, the most abundant receptor conformation, composed of the subunits α₁β₂γ₂. However, the number of GABA_A receptor isoforms expressed in mammals is still unknown. 

GABA_A receptors are selectively blocked by bicuculline and picrotoxin, and they are allosterically modulated by neurosteroids, barbiturates, and benzodiazepines. At present, there is a continuously increasing large number of regulatory molecules for this type of receptors, due to their pathological importance¹⁰. In the case of GABA_C receptors, they are not regulated by modulators and blockers of the GABA_A receptor. However, they are sensitive to picrotoxin and 1,2,5,6-Tetrahydropyridin-4-yl)methyl phosphonic acid or TPMPA, and specific GABA_C antagonist¹¹ (Fig. 3). GABA_A receptors activation leads to the inhibition of synaptic transmission, due to hyperpolarization in response to a Cl⁻ influx through these receptors¹². Interestingly, during development there is a delay in the expression of Cl⁻ mobilization systems, generating a high intracellular concentration of this anion¹³. Therefore, an increase of Cl⁻ conductance in response to the activation of GABA_A receptors generates a Cl⁻ outflux current and consequently, cell depolarization, so GABA acts as an excitatory AA during CNS development¹⁴.

Figure 3 Pharmacology properties of GABA_A receptors. At the right is the classical conformation of the GABA_A receptor, observe the modulation sites were several drugs neuromodulator act to modulate the GABA physiology. These drugs exert their action in different sites of the receptor. Benzodiazepines in the γ subunit; Picrotoxin actin the pore of the channel and blocked; the Barbiturics and neurosteroids in the α subunit and, Muscimol and GABAzine compete for the GABA binding site located between α and β subunit. On the left side, are functional homopentameric receptor formed by ρ. In this case, Picrotixine, as the α₁β₂γ₂ conformation, acts in the pore of the receptors, however, TACA and TPMA compete for the GABA binding site. 

GABA_B receptors, or metabotropic, belong to the family of G-protein-coupled receptors or GPCRs, which activate slow responses through the second messengers¹⁵, and have a limited structural diversity, unlike GABA_A receptors or metabotropic glutamate receptors (mGluRs). GABA_B receptors are heterodimers composed of the GABA_B1a or GABA_B1b subunits combined with the GABA_B2 subunit. Nevertheless, despite their poor structural diversity, native GABA_B receptors show a varied kinetic and pharmacological response (Fig. 4).

Figure 4 Structural conformation of GABA_B receptor. This receptor is heterodimers composed of the GABA_B1 or GABA_B1, belong to the G-protein-coupled receptors, which activate slow responses through the second messengers, that regulate K⁺ and Ca²⁺ channels. Nevertheless, despite their poor structural diversity, native GABA_B receptors show a varied kinetic and pharmacological response. 

The location of GABA_B receptors in the synaptic region is key to regulating neurotransmission. Depending on whether the receptor is presynaptic or postsynaptic, their activation generates an inhibition or disinhibition of synaptic activity. At the post-synaptic level, receptor activation induces a K⁺ conductance increase, which is responsible for the “slow” inhibitory events of GABA in the CNS¹⁶. The activation of this K⁺ conductance, coupled with negative regulation of Ca²⁺ influx at the presynaptic level, decreases the release of GABA by regulating the inhibitory effect mediated by this neurotransmitter¹⁷.

GABA and the CNS

The proper functioning of the CNS depends on the balance between the excitatory and inhibitory neurotransmitter systems. The excitatory system is regulated by glutamate, while the inhibitor system is regulated by GABA through interneurons, which modulate the excitatory level generated by glutamate release. These interneurons control the flow of information and the synchronization of the cerebral cortex, despite the existence of a 1:5 proportion with glutamatergic neurons. Suggesting, that the numerical balance between neurons and interneurons determines brain functionality, in the case of the cerebral cortex, the proportion 1:5 indicates that this region is mainly excitatory.

Studies in animal models indicate the presence of GABAergic neurons in regions such as the amygdala, hippocampus, hypothalamus, prefrontal cortex, olfactory bulb, retina, and spinal cord, this observation was corroborated also in human studies. This wide expression of the GABAergic cells indicates that this inhibitory neurotransmitter is involved in many functions in the CNS (Fig. 5), for instance, the thalamocortical pathway, which regulates primordial functions such as behavior, motor control, mood, sleep, and among others.

Figure 5 GABA pathways in the brain. The GABAergic system goes throughout the amygdala, hippocampus, hypothalamus, prefrontal cortex, olfactory bulb, including the spinal cord and even the retina. This wide expression of the GABAergic cells indicates the key role of this inhibitory neurotransmitter in functions in the CNS, such as behavior, motor control, mood, sleep, among others. Modified from Brain Chat. https:// thebrainchat.com/photos/gamma-aminobutyric-acid-gaba-is-a-naturally-occurring-amino-acid-that-works-as-a/1243388282530289/. 

The thalamocortical pathway consists of GABAergic neurons that project from the thalamic reticular nucleus (nRT) toward the ventral basalis (VB), generating an inhibitory “loop” for a self-modulation of the nRT. GABAergic neurons of the nRT receive the glutamatergic stimulation from the VB and the cortico-thalamic fibers that are projected from the layer six of the cerebral cortex. In this “loop context,” GABA_A receptors are in glutamatergic thalamocortical and cortico-thalamic neurons, as well as in the GABAergic neurons themselves¹⁸.

Thanks to benzodiazepines it has been understood the functional differences between the GABA_A receptors located in the nRT and the thalamic relay neurons. Since benzodiazepines increase the neurotransmission regulated by GABA_A receptors, these compounds exacerbate absence seizures; however, they show some therapeutic efficacy in clinical and animal models. This double function of benzodiazepines is because they increase the inhibition regulated by GABA_A receptors in the nRT, causing a decrease in the inhibitory stimulus of the nRT toward the thalamic relay neurons¹⁹. Experimental evidence indicates the participation of GABA_B receptors in the pathogenesis of absence seizures²⁰. GABA_B receptors regulate K⁺, activating low-threshold Ca²⁺ potentials and consequently, burst firing, and the oscillatory behavior of thalamic neurons. Agonists of these receptors exacerbate absence seizures in animal models in a greater proportion than GABA_A receptors.

Disorders associated with GABA receptors

Conclusion

Its complex structural diversity, associated with its differential expression pattern in the neural loops of the brain, makes it very difficult to understand its inhibitory control in different neural networks. Although our knowledge about the specific functions of GABAergic receptors in a neural loop is limited, there is evidence that suggests that the same subtype of GABAergic receptors can be expressed in different neuronal populations with different subcellular locations and in different areas of the brain, modulating the excitability and neuronal synchronization in different ways (Fig. 6). Therefore, understanding the role that GABA plays in the brain is possible only through the specific study of a receptor subtype, considering important variables such as synaptic activity, sex, or the circadian cycle, are partially explored along with GABA.

Figure 6 Its complex structural diversity, associated with its differential expression pattern in the neural loops of the brain, makes it very difficult to understand its inhibitory control in different neural networks. Although our knowledge about the specific functions of GABAergic receptors in a neural loop is limited, there is evidence that suggests that the same subtype of GABAergic receptors can be expressed in different neuronal populations with different subcellular locations and in different areas of the brain, modulating the excitability and neuronal synchronization in different ways (Fig. 6). Therefore, understanding the role that GABA plays in the brain is possible only through the specific study of a receptor subtype, a loop, or a neuronal type, considering important variables such as previous synaptic physiology, synaptic activity, sex, or the circadian cycle, are partially explored along with GABA.