GABA Transporter Research Articles

Spinal GABA transporter 1 contributes to evoked-pain related behavior but not resting pain after incision injury.

The inhibitory function of GABA at the spinal level and its central modulation in the brain are essential for pain perception. However, in post-surgical pain, the exact mechanism and modes of action of GABAergic transmission have been poorly studied. This work aimed to investigate GABA synthesis and uptake in the incisional pain model in a time-dependent manner. Here, we combined assays for mechanical and heat stimuli-induced withdrawal reflexes with video-based assessments and assays for non-evoked (NEP, guarding of affected hind paw) and movement-evoked (MEP, gait pattern) pain-related behaviors in a plantar incision model in male rats to phenotype the effects of the inhibition of the GABA transporter (GAT-1), using a specific antagonist (NO711). Further, we determined the expression profile of spinal dorsal horn GAT-1 and glutamate decarboxylase 65/67 (GAD65/67) by protein expression analyses at four time points post-incision. Four hours after incision, we detected an evoked pain phenotype (mechanical, heat and movement), which transiently ameliorated dose-dependently following spinal inhibition of GAT-1. However, the NEP-phenotype was not affected. Four hours after incision, GAT-1 expression was significantly increased, whereas GAD67 expression was significantly reduced. Our data suggest that GAT-1 plays a role in balancing spinal GABAergic signaling in the spinal dorsal horn shortly after incision, resulting in the evoked pain phenotype. Increased GAT-1 expression leads to increased GABA uptake from the synaptic cleft and reduces tonic GABAergic inhibition at the post-synapse. Inhibition of GAT-1 transiently reversed this imbalance and ameliorated the evoked pain phenotype.

GABA transporters gene expression associates with [18F]FDG‐PET signal in regions typically hypometabolic in Alzheimer’s disease

AbstractBackgroundIt is suggested that the [18F]fluorodeoxyglucose(FDG)‐PET signal reflects astrocyte and neuronal activity, with the excitatory glutamate uptake by astrocytes as the trigger for brain glucose metabolism. The inhibitory neurotransmitter γ‐aminobutyric acid (GABA) is taken up by neurons and astrocytes through the GABA transporters (GAT) 1 and 3. However, its role in the [18F]FDG‐PET signal in healthy aging and Alzheimer’s disease (AD) remains poorly investigated. Thus, we aimed to explore associations between neuronal and astrocytic GABA transporters gene expression with [18F]FDG‐PET in cognitively unimpaired (CU) and AD individuals.MethodWe obtained [18F]FDG‐PET imaging data of CU and AD from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) (Table 1). GAT1 and GAT3 gene expression in the brain was obtained from the Allen Brain Atlas (Table 1, Figure 1). We extracted SUVr (pons as reference region) and mRNA values using the RMINC package and performed Pearson’s correlation to test the association between [18F]FDG‐PET and GABA transporters mRNA (p‐value < 0.05).ResultIn brain regions typically hypometabolic in AD, we observed a strong positive association of [18F]FDG‐PET SUVr with GAT1 and GAT3 gene expression in CU (GAT1: r = 0.93; p < 0.0001; Figure 2A, GAT3: r = 0.94; p < 0.0001; Figure 2B) and AD individuals (GAT1: r = 0.9; p < 0.0001; Figure 2A, GAT3: r = 0.92; p < 0.0001; Figure 2B). By contrast, in brain regions typically resilient, we found a weak correlation with CU individuals (GAT1: r = 0.32; p = 0.016; Figure 2C, GAT3: r = 0.26; p = 0.04; Figure 2D) and no significant correlation in AD patients (GAT1: r = 0.24; p = 0.07; Figure 2C, GAT3: r = 0.24; p = 0.059; Figure 2D).ConclusionOur results demonstrate a significant strong positive association between [18F]FDG‐PET SUVr and GABA transporters gene expression in CU and AD in hypometabolic regions. These findings suggest that GABA transporters are involved with brain glucose metabolism in regions classically vulnerable to AD pathology.

GABA transporters gene expression associates with [18F]FDG‐PET signal in regions typically hypometabolic in Alzheimer’s disease

AbstractBackgroundIt is suggested that the [18F]fluorodeoxyglucose(FDG)‐PET signal reflects astrocyte and neuronal activity, with the excitatory glutamate uptake by astrocytes as the trigger for brain glucose metabolism. The inhibitory neurotransmitter γ‐aminobutyric acid (GABA) is taken up by neurons and astrocytes through the GABA transporters (GAT) 1 and 3. However, its role in the [18F]FDG‐PET signal in healthy aging and Alzheimer’s disease (AD) remains poorly investigated. Thus, we aimed to explore associations between neuronal and astrocytic GABA transporters gene expression with [18F]FDG‐PET in cognitively unimpaired (CU) and AD individuals.MethodWe obtained [18F]FDG‐PET imaging data of CU and AD from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) (Table 1). GAT1 and GAT3 gene expression in the brain was obtained from the Allen Brain Atlas (Table 1, Figure 1). We extracted SUVr (pons as reference region) and mRNA values using the RMINC package and performed Pearson’s correlation to test the association between [18F]FDG‐PET and GABA transporters mRNA (p‐value < 0.05).ResultIn brain regions typically hypometabolic in AD, we observed a strong positive association of [18F]FDG‐PET SUVr with GAT1 and GAT3 gene expression in CU (GAT1: r = 0.93; p < 0.0001; Figure 2A, GAT3: r = 0.94; p < 0.0001; Figure 2B) and AD individuals (GAT1: r = 0.9; p < 0.0001; Figure 2A, GAT3: r = 0.92; p < 0.0001; Figure 2B). By contrast, in brain regions typically resilient, we found a weak correlation with CU individuals (GAT1: r = 0.32; p = 0.016; Figure 2C, GAT3: r = 0.26; p = 0.04; Figure 2D) and no significant correlation in AD patients (GAT1: r = 0.24; p = 0.07; Figure 2C, GAT3: r = 0.24; p = 0.059; Figure 2D).ConclusionOur results demonstrate a significant strong positive association between [18F]FDG‐PET SUVr and GABA transporters gene expression in CU and AD in hypometabolic regions. These findings suggest that GABA transporters are involved with brain glucose metabolism in regions classically vulnerable to AD pathology. ”

Alzheimer’s disease biomarker‐related phospho‐tau 181 signals localize to demyelinated axons of parvalbumin‐positive GABAergic interneurons in App knock‐in mouse

AbstractBackgroundTau proteins phosphorylated at Thr181 (p‐tau 181) in cerebrospinal fluid and blood is a sensitive biomarker for Alzheimer’s disease (AD). An increase in p‐tau 181 level well correlates with Aβ pathology and precedes neurofibrillary tangle formation in the early stage of AD, though the relationship between p‐tau 181 and Aβ‐mediated pathology is less well understood. We have recently reported that p‐tau 181 signals are detected in the axonal structures and represent axonal abnormality around Aβ plaques in the cortex of App knock‐in mouse model of Aβ amyloidosis (AppNLGF mice). However, from which neuronal subtype(s) these p‐tau 181‐positive axons are originated remains elusive. In this study, we aimed to determine which neuronal subtype(s) express p‐tau 181 in the axons.MethodTo investigate the localization of p‐tau 181 signals in the brains of aged (24‐month‐old) AppNLGF and wild‐type mice, we prepared frozen brain sections and co‐immunostained with antibodies against p‐tau 181 and myeline basic protein (MBP), the glutamatergic neuronal vesicle marker vesicular glutamate transporter 1 (VGLUT1), the GABAergic neuronal vesicle marker vesicular GABA transporter (VGAT), the inhibitory neuron marker parvalbumin, the cholinergic neuronal vesicle marker vesicular acetylcholine transporter (VAChT), or the noradrenergic neuronal vesicle marker norepinephrine transporter (NET).ResultHistochemical analyses revealed that p‐tau 181 signals were not overlapped with axons of unmyelinated cholinergic nor noradrenergic neurons, but well colocalized with myelinated axons of parvalbumin‐positive GABAergic interneurons, but not of glutamatergic neurons. Interestingly, the density of unmyelinated axons was significantly decreased in AppNLGF mice, while that of glutamatergic, GABAergic or p‐tau 181‐positive axons was less affected. Instead, myelin sheaths surrounding p‐tau 181‐positive axons were significantly reduced in AppNLGF mice.ConclusionThis study suggests that p‐tau 181 signals may represent axonal abnormality of parvalbumin‐positive GABAergic interneurons with disrupted myelin sheaths in brains of mouse model of Aβ pathology.

Expression of the vesicular GABA transporter within neuromedin S+ neurons sustains behavioral circadian rhythms.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of a central circadian clock that orchestrates overt rhythms of physiology and behavior. Circadian timekeeping requires intercellular communication among SCN neurons, and multiple signaling pathways contribute to SCN network coupling. Gamma-aminobutyric acid (GABA) is produced by virtually all SCN neurons, and previous work demonstrates that this transmitter regulates coupling in the adult SCN but is not essential for the nucleus to sustain overt circadian rhythms. Here, we show that the deletion of the gene that codes for the GABA vesicular transporter Vgat from neuromedin-S (NMS)+ neurons-a subset of neurons critical for SCN function-causes arrhythmia of locomotor activity and sleep. Further, NMS-Vgat deletion impairs intrinsic clock gene rhythms in SCN explants cultured ex vivo. Although vasoactive intestinal polypeptide (VIP) is critical for SCN function, Vgat deletion from VIP-expressing neurons did not lead to circadian arrhythmia in locomotor activity rhythms. Likewise, adult SCN-specific deletion of Vgat led to mild impairment of behavioral rhythms. Our results suggest that while the removal of GABA release from the adult SCN does not affect the pacemaker's ability to sustain overt circadian rhythms, its removal from a critical subset of neurons within the SCN throughout development removes the nucleus ability to sustain circadian rhythms. Our findings support a model in which SCN GABA release is critical for the developmental establishment of intercellular network properties that define the SCN as a central pacemaker.

4-Phenylbutyrate promoted wild-type γ-aminobutyric acid type A receptor trafficking, reduced endoplasmic reticulum stress, and mitigated seizures in Gabrg2+/Q390X mice associated with Dravet syndrome.

γ-Aminobutyric acid type A (GABAA ) receptor subunit gene mutations are major causes of various epilepsy syndromes, including severe kinds such as Dravet syndrome. Although the GABAA receptor is a major target for antiseizure medications, treating GABAA receptor mutations with receptor channel modulators is ineffective. Here, we determined the effect of a novel treatment with 4-phenylbutyrate (PBA) in Gabrg2+/Q390X knockin mice associated with Dravet syndrome. We used biochemistry in conjunction with differential tagging of the wild-type and the mutant alleles, live brain slice surface biotinylation, microsome isolation, patch-clamp whole-cell recordings, and video-monitoring synchronized electroencephalographic (EEG) recordings in Gabrg2+/Q390X mice to determine the effect of PBA invitro with recombinant GABAA receptors and invivo with knockin mice. We found that PBA reduced the mutant γ2(Q390X) subunit protein aggregates, enhanced the wild-type GABAA receptor subunits' trafficking, and increased the membrane expression of the wild-type receptors. PBA increased the current amplitude of GABA-evoked current in human embryonic kidney 293T cells and the neurons bearing the γ2(Q390X) subunit protein. PBA also proved to reduce endoplasmic reticulum (ER) stress caused by the mutant γ2(Q390X) subunit protein, as well as mitigating seizures and EEG abnormalities in the Gabrg2+/Q390X mice. This research has unveiled a promising and innovative approach for treating epilepsy linked to GABAA receptor mutations through an unconventional antiseizure mechanism. Rather than directly modulating the affected mutant channel, PBA facilitates the folding and transportation of wild-type receptor subunits to the cell membrane and synapse. Combining these findings with our previous study, which demonstrated PBA's efficacy in restoring GABA transporter 1 (encoded by SLC6A1) function, we propose that PBA holds significant potential for a wide range of genetic epilepsies. Its ability to target shared molecular pathways involving mutant protein ER retention and impaired protein membrane trafficking suggests broad application in treating such conditions.

Histone methyltransferase Ezh2 coordinates mammalian axon regeneration via regulation of key regenerative pathways.

Current treatments for neurodegenerative diseases and neural injuries face major challenges, primarily due to the diminished regenerative capacity of neurons in the mammalian CNS as they mature. Here, we investigated the role of Ezh2, a histone methyltransferase, in regulating mammalian axon regeneration. We found that Ezh2 declined in the mouse nervous system during maturation but was upregulated in adult dorsal root ganglion neurons following peripheral nerve injury to facilitate spontaneous axon regeneration. In addition, overexpression of Ezh2 in retinal ganglion cells in the CNS promoted optic nerve regeneration via both histone methylation-dependent and -independent mechanisms. Further investigation revealed that Ezh2 fostered axon regeneration by orchestrating the transcriptional silencing of genes governing synaptic function and those inhibiting axon regeneration, while concurrently activating various factors that support axon regeneration. Notably, we demonstrated that GABA transporter 2, encoded by Slc6a13, acted downstream of Ezh2 to control axon regeneration. Overall, our study underscores the potential of modulating chromatin accessibility as a promising strategy for promoting CNS axon regeneration.

Perinatal exposure to bisphenol A impairs cognitive function via the gamma-aminobutyric acid signaling pathway in male rat offspring.

Bisphenol A (BPA) is a common synthetic endocrine disruptor that can be utilized in the fabrication of materials such as polycarbonates and epoxy resins. Numerous studies have linked BPA to learning and memory problems, although the precise mechanism remains unknown. Gamma-aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the vertebrate central nervous system, and it is intimately related to learning and memory. This study aims to evaluate whether altered cognitive behavior involves the GABA signaling pathway in male offspring of rats exposed to BPA during the prenatal and early postnatal periods. Pregnant rats were orally given BPA (0, 0.04, 0.4, and 4 mg/kg body weight (BW)/day) from the first day of pregnancy to the 21st day of breastfeeding. Three-week-old male rat offspring were selected for an open-field experiment and a new object recognition experiment to evaluate the effect of BPA exposure on cognitive behavior. Furthermore, the role of GABA signaling markers in the cognition affected by BPA was investigated at the molecular level using western blotting and real-time polymerase chain reaction (RT-PCR). The research demonstrated that BPA exposure impacted the behavior and memory of male rat offspring and elevated the expression of glutamic acid decarboxylase 67 (GAD67), GABA type A receptors subunit (GABAARα1), and GABA vesicle transporter (VGAT) in the hippocampus while decreasing the expression levels of GABA transaminase (GABA-T) and GABA transporter 1 (GAT-1). These findings indicate that the alteration in the expression of GABA signaling molecules may be one of the molecular mechanisms by which perinatal exposure to BPA leads to decreased learning and memory in male rat offspring.

Action Sequence Learning Is Impaired in Genetically Modified Mice with the Suppressed GABAergic Transmission from the Thalamic Reticular Nucleus to the Thalamus.

The thalamic reticular nucleus (TRN) is a thin sheet of GABAergic neurons surrounding the thalamus, and it regulates the activity of thalamic relay neurons. The TRN has been reported to be involved in sensory gating, attentional regulation, and some other functions. However, little is known about the contribution of the TRN to sequence learning. In the present study, we examined whether the TRN is involved in reward-based learning of action sequence with no eliciting stimuli (operant conditioning), by analyzing the performance of male and female Avp-Vgat-/- mice (Vgatflox/flox mice crossed to an Avp-Cre driver line) on tasks conducted in an operant box having three levers. Our histological and electrophysiological data demonstrated that in adult Avp-Vgat-/- mice, vesicular GABA transporter (VGAT) was absent in most TRN neurons and the GABAergic transmission from the TRN to the thalamus was largely suppressed. The performance on a task in which mice needed to press an active lever for food reward showed that simple operant learning of lever pressing and learning of win-stay and lose-shift strategies are not affected in Avp-Vgat-/- mice. In contrast, the performance on a task in which mice needed to press three levers in a correct order for food reward showed that learning of the order of lever pressing (action sequence learning) was impaired in Avp-Vgat-/- mice. These results suggest that the TRN plays an important role in action sequence learning.

ANALYSIS OF THE FUNCTIONAL STATE OF ASTROGLIA IN THE HIPPOCAMPUS OF RATS GENETICALLY PRONE TO AUDIOGENIC SEIZURES

It was shown that abnormalities in astrocyte functions can cause alterations in neuronal excitability and promote the development of epilepsy, however, this question claims further investigation. The aim of the present work was to analyze the functional state of astrocytes in the hippocampus of Krushinsky-Molodkina (KM) rats genetically predisposed to the reflex audiogenic seizures. Adult naïve animals with fully-established audiogenic sensitivity were recruited in our experiments. Rats of parental Wistar strain were used as a control. Obtained results revealed unchanged protein expression of such astocytic markers as GFAP, ALDH1L1, and NFIA suggesting the absence of the reactive astrogliosis in the hippocampus of KM rats. SPARC and aquaporin 4 expression also did not differ from the control indicating no violations in astroglia regulation of synaptogenesis and water balance. Analysis of glial proteins responsible for reuptake and metabolism of neurotransmitters revealed normal expression of GABA transporter GAT-3 and glutamine synthetase, while glutamate transporters EAAT1 and 2 were significantly elevated pointing on enhanced activity of glutamate removal from synapses. In addition, decreased expression of the key glycolytic enzyme aldolase C, probably, indicated insufficient activity of glucose metabolism in astrocytes. Thus, obtained data pointed on genetically determined alterations of astroglia functions in the hippocampus of rats with inherited reflex epilepsy.

Dynamical changes in the expression of GABAergic and purinergic components occur during the polarization of THP-1 monocytes to proinflammatory macrophages

The monocytes are key components of innate immunity, as they can differentiate into phagocytic cells or macrophages with proinflammatory or anti-inflammatory phenotypes. The gamma-aminobutyric acid (GABA) and adenosine triphosphate (ATP), two known neurotransmitters, are two environmental signals that contribute to the differentiation of monocytes into macrophages and their subsequent polarization into proinflammatory M1 and anti-inflammatory M2 macrophages. Although monocytes and macrophages express proteins related to GABA and ATP-mediated response (GABAergic and purinergic systems, respectively), it is unknown whether changes in their expression occur during monocyte activation or their differentiation and polarization into macrophages. Therefore, we evaluated the expression levels of GABAergic and purinergic signaling components in the THP-1 monocyte cell line and their changes during monocyte activation, differentiation, and polarization to M1 proinflammatory macrophages. Our results showed that activated monocytes are characterized by increased expression of two GABAergic components, the GABA transporter 2 (GAT-2) and the glutamic acid decarboxylase (GAD)-67, an enzyme involved in GABA synthesis. Also, monocytes showed a pronounced expression of the purinergic receptors P2X4 and P2X7. Interestingly, during differentiation, monocytes increased the expression of the β2 subunit of GABA A-type receptor (GABA-AR), while the purinergic receptors P2X1 and P2X1del were reduced. In contrast, proinflammatory M1 macrophages showed a reduced expression in the α4 subunit of GABA-AR and GAD67, while P2X4 and P2X7 were overexpressed. These results indicate that dynamical changes in the GABAergic and purinergic components occur during the transition from monocytes to macrophages. Since GABA and ATP are two neurotransmitters, our results suggest that monocytes and macrophages respond to neurotransmitter-induced stimulation and may represent a path of interaction between the nervous and immune systems during peripheral inflammation and neuroinflammation development.

Preferential expression of SCN1A in GABAergic neurons improves survival and epileptic phenotype in a mouse model of Dravet syndrome

The SCN1A gene encodes the alpha subunit of a voltage-gated sodium channel (Nav1.1), which is essential for the function of inhibitory neurons in the brain. Mutations in this gene cause severe encephalopathies such as Dravet syndrome (DS). Upregulation of SCN1A expression by different approaches has demonstrated promising therapeutic effects in preclinical models of DS. Limiting the effect to inhibitory neurons may contribute to the restoration of brain homeostasis, increasing the safety and efficacy of the treatment. In this work, we have evaluated different approaches to obtain preferential expression of the full SCN1A cDNA (6 Kb) in GABAergic neurons, using high-capacity adenoviral vectors (HC-AdV). In order to favour infection of these cells, we considered ErbB4 as a surface target. Incorporation of the EGF-like domain from neuregulin 1 alpha (NRG1α) in the fiber of adenovirus capsid allowed preferential infection in cells lines expressing ErbB4. However, it had no impact on the infectivity of the vector in primary cultures or in vivo. For transcriptional control of transgene expression, we developed a regulatory sequence (DP3V) based on the Distal-less homolog enhancer (Dlx), the vesicular GABA transporter (VGAT) promoter, and a portion of the SCN1A gene. The hybrid DP3V promoter allowed preferential expression of transgenes in GABAergic neurons both in vitro and in vivo. A new HC-AdV expressing SCN1A under the control of this promoter showed improved survival and amelioration of the epileptic phenotype in a DS mouse model. These results increase the repertoire of gene therapy vectors for the treatment of DS and indicate a new avenue for the refinement of gene supplementation in this disease.Key messagesAdenoviral vectors can deliver the SCN1A cDNA and are amenable for targeting.An adenoviral vector displaying an ErbB4 ligand in the capsid does not target GABAergic neurons.A hybrid promoter allows preferential expression of transgenes in GABAergic neurons.Preferential expression of SCN1A in GABAergic cells is therapeutic in a Dravet syndrome model.

The Co-Expression Pattern of Calcium-Binding Proteins with γ-Aminobutyric Acid and Glutamate Transporters in the Amygdala of the Guinea Pig: Evidence for Glutamatergic Subpopulations.

The amygdala has large populations of neurons utilizing specific calcium-binding proteins such as parvalbumin (PV), calbindin (CB), or calretinin (CR). They are considered specialized subsets of γ-aminobutyric acid (GABA) interneurons; however, many of these cells are devoid of GABA or glutamate decarboxylase. The neurotransmitters used by GABA-immunonegative cells are still unknown, but it is suggested that a part may use glutamate. Thus, this study investigates in the amygdala of the guinea pig relationships between PV, CB, or CR-containing cells and GABA transporter (VGAT) or glutamate transporter type 2 (VGLUT2), markers of GABAergic and glutamatergic neurons, respectively. The results show that although most neurons using PV, CB, and CR co-expressed VGAT, each of these populations also had a fraction of VGLUT2 co-expressing cells. For almost all neurons using PV (~90%) co-expressed VGAT, while ~1.5% of them had VGLUT2. The proportion of neurons using CB and VGAT was smaller than that for PV (~80%), while the percentage of cells with VGLUT2 was larger (~4.5%). Finally, only half of the neurons using CR (~53%) co-expressed VGAT, while ~3.5% of them had VGLUT2. In conclusion, the populations of neurons co-expressing PV, CB, and CR are in the amygdala, primarily GABAergic. However, at least a fraction of neurons in each of them co-express VGLUT2, suggesting that these cells may use glutamate. Moreover, the number of PV-, CB-, and CR-containing neurons that may use glutamate is probably larger as they can utilize VGLUT1 or VGLUT3, which are also present in the amygdala.

UBR-1 ubiquitin ligase regulates the balance between GABAergic and glutamatergic signaling.

Excitation/inhibition (E/I) balance is carefully maintained by the nervous system. The neurotransmitter GABA has been reported to be co-released with its sole precursor, the neurotransmitter glutamate. The genetic and circuitry mechanisms to establish the balance between GABAergic and glutamatergic signaling have not been fully elucidated. Caenorhabditis elegans DVB is an excitatory GABAergic motoneuron that drives the expulsion step in the defecation motor program. We show here that in addition to UNC-47, the vesicular GABA transporter, DVB also expresses EAT-4, a vesicular glutamate transporter. UBR-1, a conserved ubiquitin ligase, regulates DVB activity by suppressing a bidirectional inhibitory glutamate signaling. Loss of UBR-1 impairs DVB Ca2+ activity and expulsion frequency. These impairments are fully compensated by the knockdown of EAT-4 in DVB. Further, glutamate-gated chloride channels GLC-3 and GLC-2/4 receive DVB's glutamate signals to inhibit DVB and enteric muscle activity, respectively. These results implicate an intrinsic cellular mechanism that promotes the inherent asymmetric neural activity. We propose that elevated glutamate in ubr-1 mutants, being the cause of the E/I shift, potentially contributes to Johanson Blizzard syndrome.

Discovery of novel multifunctional ligands targeting GABA transporters, butyrylcholinesterase, β-secretase, and amyloid β aggregation as potential treatment of Alzheimer's disease

Alzheimer's disease (AD) is a global health problem in the medical sector that will increase over time. The limited treatment of AD leads to the search for a new clinical candidate. Considering the multifactorial nature of AD, a strategy targeting number of regulatory proteins involved in the development of the disease is an effective approach. Here, we present a discovery of new multi-target-directed ligands (MTDLs), purposely designed as GABA transporter (GAT) inhibitors, that successfully provide the inhibitory activity against butyrylcholinesterase (BuChE), β-secretase (BACE1), amyloid β aggregation and calcium channel blockade activity. The selected GAT inhibitors, 19c and 22a – N-benzylamide derivatives of 4-aminobutyric acid, displayed the most prominent multifunctional profile. Compound 19c (mGAT1 IC50 = 10 μM, mGAT4 IC50 = 12 μM and BuChE IC50 = 559 nM) possessed the highest hBACE1 and Aβ40 aggregation inhibitory activity (IC50 = 1.57 μM and 99 % at 10 μM, respectively). Additionally, it showed a decrease in both the elongation and nucleation constants of the amyloid aggregation process. In contrast compound 22a represented the highest activity and a mixed-type of eqBuChE inhibition (IC50 = 173 nM) with hBACE1 (IC50 = 9.42 μM), Aβ aggregation (79 % at 10 μM) and mGATs (mGAT1 IC50 = 30 μM, mGAT4 IC50 = 25 μM) inhibitory activity. Performed molecular docking studies described the mode of interactions with GATs and enzymatic targets. In ADMET in vitro studies both compounds showed acceptable metabolic stability and low neurotoxicity. Successfully, compounds 19c and 22a at the dose of 30 mg/kg possessed statistically significant antiamnesic properties in a mouse model of amnesia caused by scopolamine and assessed in the novel object recognition (NOR) task or the passive avoidance (PA) task.

THU125 Altered Pro-opiomelanocortin (POMC) Neuron Function Characterizes Energy Expenditure Dysfunction And Increased Body Weight Linked To Fragile X Gene Mutation

Abstract Disclosure: R.E. Ruggiero-Ruff: None. P. Villa: None. S. Abu Hijleh: None. B. Avalos: None. N. DiPatrizio: None. D. Coss: None. Obesity is a world-wide epidemic that has become a major public health concern. Obesity increases the risk of cardiovascular disease, type 2 diabetes, stroke, and dementia. Children and adolescents with Fragile X syndrome (FXS), the most common cause of inherited intellectual disability, have higher rates of obesity compared to peers. FXS is caused by a mutation of the Fragile X messenger ribonucleoprotein gene (FMR1), which leads to the loss of FMRP, a ubiquitously expressed mRNA binding protein. Food intake and energy expenditure are regulated by orexigenic neuropeptide Y (NPY)/agouti-related protein (AgRP) and anorexigenic pro-opiomelanocortin (POMC) neurons from the hypothalamus. While our understanding of mechanisms of intellectual disability in FXS patients is beginning to emerge, the role of FMR1 gene in the hypothalamus is unknown. In this study, we used a knock-out (KO) mouse model to analyze if obesity in FXS patients stems from dysregulation of the hypothalamic circuitry regulating food intake and energy expenditure. Consistent with what is seen in the human population, KO males weigh more than their WT littermates. There were no differences in growth hormone or basal or fasting glucose levels. There was no difference in food intake, however KO males exhibited lower locomotor activity in the dark cycle, during their active phase. Since POMC neurons regulate energy expenditure in addition to food intake, we analyzed POMC neuron number, using immunohistochemistry. We determined fewer POMC neurons in KO male mice compared to WT. AgRP/NPY neurons modulate POMC neurons through GABAergic neuronal transmission. We determined that KO males have increased obligatory GABAa receptor subunit on POMC neurons. We also analyzed GABAa receptor and vesicular GABA transporter (VGAT) apposition on POMC neurons to identify synaptic receptors and indeed, KO males had significantly higher levels of GABAa-VGAT appositions, indicating increased synaptic input. In summary, our results suggest a role for FMR1 in the regulation of POMC neuronal activity in the regulation of locomotor activity and point to a role in modulating motor activity and energy expenditure in obese Fragile X patients. Presentation: Thursday, June 15, 2023

Anxiolytic potential of resveratrol and rufinamide combination by modulating GABA-ergic transmission: insights from experiments, molecular docking and dynamics simulations.

Resveratrol is a polyphenolic phytocompound known to possess anxiolytic-like effects but its impact on central gammaaminobutyric acid (GABA) modulation has never been explored. The purpose of this study was to analyze the anxiolytic-like effects of resveratrol alone and in combination with rufinamide, an antiepileptic drug which has never been studied for its anxiolytic potential. The BALB/c mice were tested in a battery of behavior testing after administration of resveratrol (50 mg/kg) and rufinamide (50 mg/kg) alone and in combination. Moreover, molecular docking studies were also carried out to understand the interaction of resveratrol and rufinamide with GABA aminotransferase, GABA receptor and GABA-A transporter type 1. Resveratrol alone exerted notable anxiolytic-like effects and improved outcomes in few experiments but rufinamide alone did not yield any beneficial outcomes. However, the animal co-administered with resveratrol and rufinamide behaved exceptionally well (p<0.05) and preferred open, illuminated and exposed areas of open field, light/dark and elevated plus maze. Further, these animals showed reduced anxiety towards anxiogenic stimuli i.e. holes and marbles in hole board and marble bury tests, respectively. Resveratrol and rufinamide showed moderate to strong binding affinities with GABA proteins, indicating the potential to treat anxiety-like neurological disorders. Moreover, resveratrol and rufinamide were analyzed using molecular docking to determine their interaction with GABA receptors, transporters, and transaminase. The results suggest that their anxiolytic-like effects may be due to inhibiting GABA reuptake transporter 1 protein, leading to increased synaptic levels of GABA neurotransmitter, as seen in stable molecular dynamics results with the 7SK2 GABA transporter protein.

Shared GABA transmission pathology in dopamine agonist- and antagonist-induced dyskinesia

Dyskinesia is involuntary movement caused by long-term medication with dopamine-related agents: the dopamine agonist 3,4-dihydroxy-L-phenylalanine (L-DOPA) to treat Parkinson's disease (L-DOPA-induced dyskinesia [LID]) or dopamine antagonists to treat schizophrenia (tardive dyskinesia [TD]). However, it remains unknown why distinct types of medications for distinct neuropsychiatric disorders induce similar involuntary movements. Here, we search for a shared structural footprint using magnetic resonance imaging-based macroscopic screening and super-resolution microscopy-based microscopic identification. We identify the enlarged axon terminals of striatal medium spiny neurons in LID and TD model mice. Striatal overexpression of the vesicular gamma-aminobutyric acid transporter (VGAT) is necessary and sufficient for modeling these structural changes; VGAT levels gate the functional and behavioral alterations in dyskinesia models. Our findings indicate that lowered type 2 dopamine receptor signaling with repetitive dopamine fluctuations is a common cause of VGAT overexpression and late-onset dyskinesia formation and that reducing dopamine fluctuation rescues dyskinesia pathology via VGAT downregulation.

How do missense mutations of a highly conserved glycine residue in the human GABA transporter 1 trigger epilepsy?

How do missense mutations of a highly conserved glycine residue in the human GABA transporter 1 trigger epilepsy?

Synergistic neuroprotective effects of two natural medicinal plants against CORT-induced nerve cell injury by correcting neurotransmitter deficits and inflammation imbalance

BackgroundLilium henryi Baker (Liliaceae) and Rehmannia glutinosa (Gaertn.) DC. (Plantaginaceae) were the traditional natural medicinal plants for the treatment of depression, but the antidepression mechanism of two plants co-decoction (Also known as Lily bulb and Rehmannia decoction (LBRD) drug-containing serum (LBRDDS) has not been elucidated in the in vitro model of depression. Material and methodsHere, UHPLC-Q-TOF/MS was used to identify the active components of LBRDDS and the potential effector substance was identified by bioinformatics analysis. CORT-induced nerve cells cytotoxicity was used to investigate the neuroprotection effect of LBRDDS and the underlying pharmacological mechanisms were explored by multiple experimental methods such as molecular docking, immunofluorescence, gain- or loss-of function experiments. ResultsBioactive compounds in LBRDDS absorbed from intestinal tract were transformed or metabolized by the gut microbiota including palmitic acid, adrenic acid, linoleic acid, arachidonic acid and docosapentaenoic acid. Network pharmacology analysis and molecular docking of showed fatty acid metabolism, neurotransmitter synthesis and neuroinflammation may be potential therapeutic targets of LBRDDS. LBRDDS can improve the activity of model cells, reduce cytotoxicity of lactate dehydrogenase, recover neurotransmitter imbalance, relieve inflammatory damage, down-regulate the expression of miRNA-144-3p, increase the mRNAs and protein expression level of Gad-67 and VGAT, and promote the synthesis and transport of GABA. ConclusionTherefore, LBRDDS exerts neuroprotective effects by correcting neurotransmitter deficits and inflammation imbalance in the CORT-induced nerve cell injury model.