Dysbiosis of the microbiome correlates with many neurological disorders, yet very little is known about the chemistry that controls the production of neuromodulatory molecules by gut microbes. Here, we found that an enzyme glutamate decarboxylase (BfGAD) of a gut microbe Bacteroides fragilis forms multiple neuromodulatory molecules such as γ-aminobutyric acid (GABA), hypotaurine, taurine, homotaurine, and β-alanine. We evolved BfGAD and doubled its taurine productivity. Additionally, we increased its specificity towards the substrate L-glutamate. Here, we provide a chemical strategy via which the BfGAD activity could be fine-tuned. In future, this strategy could be used to modulate the production of neuromodulatory molecules by gut microbes.

Glucose transporter-2 (GLUT2) monitors cellular glucose uptake. Astrocyte GLUT2 controls glucose counterregulatory hormone secretion. In vivo gene silencing and laser-catapult-microdissection tools were used here to investigate whether ventromedial hypothalamic nucleus (VMN) GLUT2 may regulate dorsomedial (VMNdm) and/or ventrolateral (VMNvl) γ-aminobutyric acid (GABA) neurotransmission to control this endocrine outflow in female rats. VMN GLUT2 gene knockdown suppressed or stimulated hypoglycemia-associated glutamate decarboxylase (GAD)1 and GAD2 mRNA expression in VMNdm versus VMNvl GABAergic neurons, respectively. GLUT2 siRNA pretreatment also modified co-expressed transmitter marker gene profiles in each cell population. VMNdm GABA neurons exhibited GLUT2 knockdown-sensitive up-regulated 5\'-AMP-activated protein kinase-alpha1 (AMPKα1) and -alpha2 (AMPKα2) transcripts during hypoglycemia. Hypoglycemic augmentation of VMNvl GABA neuron AMPKα2 was refractory to GLUT2 siRNA. GLUT2 siRNA blunted (VMNdm) or exacerbated (VMNvl) hypoglycemic stimulation of GABAergic neuron steroidogenic factor-1 (SF-1) mRNA. Results infer that VMNdm and VMNvl GABA neurons may exhibit divergent, GLUT2-dependent GABA neurotransmission patterns in the hypoglycemic female rat. Data also document differential GLUT2 regulation of VMNdm versus VMNvl GABA nerve cell SF-1 gene expression. Evidence for intensification of hypoglycemic hypercorticosteronemia and -glucagonemia by GLUT2 siRNA infers that VMN GLUT2 function imposes an inhibitory tone on these hormone profiles in this sex.

Transcranial focused ultrasound stimulation (tFUS) has emerged as a promising neuromodulation technique that delivers acoustic energy with high spatial resolution for inducing long-term potentiation (LTP)- or depression (LTD)-like plasticity. The variability in the primary effects of tFUS-induced plasticity could be due to different stimulation patterns, such as intermittent versus continuous, and is an aspect that requires further detailed exploration. In this study, we developed a platform to evaluate the neuromodulatory effects of intermittent and continuous tFUS on motor cortical plasticity before and after tFUS application. Three groups of rats were exposed to either intermittent, continuous, or sham tFUS. We analyzed the neuromodulatory effects on motor cortical excitability by examining changes in motor-evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS). We also investigated the effects of different stimulation patterns on excitatory and inhibitory neural biomarkers, examining c-Fos and glutamic acid decarboxylase (GAD-65) expression using immunohistochemistry staining. Additionally, we evaluated the safety of tFUS by analyzing glial fibrillary acidic protein (GFAP) expression. The current results indicated that intermittent tFUS produced a facilitation effect on motor excitability, while continuous tFUS significantly inhibited motor excitability. Furthermore, neither tFUS approach caused injury to the stimulation sites in rats. Immunohistochemistry staining revealed increased c-Fos and decreased GAD-65 expression following intermittent tFUS. Conversely, continuous tFUS downregulated c-Fos and upregulated GAD-65 expression. In conclusion, our findings demonstrate that both intermittent and continuous tFUS effectively modulate cortical excitability. The neuromodulatory effects may result from the activation or deactivation of cortical neurons following tFUS intervention. These effects are considered safe and well-tolerated, highlighting the potential for using different patterns of tFUS in future clinical neuromodulatory applications.

Although classically recognized as a neurotransmitter, gamma aminobutyric acid (GABA) has also been identified in colonic tumors. Moreover, the gut microbiome represents another potential source of GABA. Both GABAA and GABAB receptors have been implicated in contributing to the effects of GABA in colorectal cancer, with both pro- and anti-tumorigenic functions identified. However, their subunit composition is often overlooked. Studies to date have not addressed whether the GABA-producing potential of the microbiome changes over the course of colon tumor development or whether receptor subunit expression patterns are altered in colon cancer. Therefore, we investigated the clusters of orthologous group frequencies of glutamate decarboxylase (GAD) in feces from two murine models of colon cancer and found that the frequency of microbial GAD was significantly decreased early in the tumorigenic process. We also determined that microbial-derived GABA inhibited proliferation of colon cancer cells in vitro and that this effect of GABA on SW480 cells involved both GABAA and GABAB receptors. GABA also inhibited prostaglandin E2 (PGE2)-induced proliferation and interleukin-6 (IL-6) expression in these cells. Gene expression correlations were assessed using the \"Cancer Exploration\" suite of the TIMER2.0 web tool and identified that GABA receptor subunits were differentially expressed in human colon cancer. Moreover, GABAA receptor subunits were predominantly positively associated with PGE2 synthase, cyclooxygenase-2 and IL-6. Collectively, these data demonstrate decreased potential of the microbiome to produce GABA during tumorigenesis, a novel anti-tumorigenic pathway for GABA, and that GABA receptor subunit expression adds a further layer of complexity to GABAergic signaling in colon cancer.

Bacterial pathogens inject effector proteins inside plant cells to manipulate cellular functions and achieve a successful infection. The soil-borne pathogen Ralstonia solanacearum (Smith), the causal agent of bacterial wilt disease, secretes > 70 different effectors inside plant cells, although only a handful of them have been thoroughly characterized. One of these effectors, named RipI, is required for full R. solanacearum pathogenicity. RipI associates with plant glutamate decarboxylases (GADs) to promote the accumulation of gamma-aminobutyric acid (GABA), which serves as bacterial nutrient. In this work, we found that RipI can also suppress plant immune responses to bacterial elicitors, which seems to be unrelated to the ability of RipI to induce GABA accumulation and plant cell death. A detailed characterization of the RipI features that contribute to its virulence activities identified two residues at the C-terminal domain that mediate RipI interaction with plant GADs and the subsequent promotion of GABA accumulation. These residues are also required for the appropriate homeostasis of RipI in plant cells and the induction of cell death, although they are partially dispensable for the suppression of plant immune responses. Altogether, we decipher and uncouple the virulence activities of an important bacterial effector at the biochemical level.

We present a rare case of low titre GAD65 antibody-associated autoimmune encephalitis and status epilepticus in a young woman. She initially presented with left arm dystonic movements, contractures and status epilepticus. Due to the concern of autoimmune encephalitis and seizures, the patient received intravenous immunoglobulin empirically. After the detection of low serum GAD65 antibodies, the patient underwent immunomodulation therapy with significant improvement. This case demonstrated that in autoimmune encephalitis, it is important to monitor serum GAD65 antibodies levels and consider immunotherapy, despite mildly elevated serum levels. The patient\'s history of left arm dystonic movements without impaired awareness may have been due to limb dystonia, a presenting symptom of stiff person syndrome (SPS), despite SPS more commonly affecting axial muscles. This case further demonstrates that GAD65 antibody-related syndromes can manifest with different neurological phenotypes including co-occurrence of epilepsy with possible focal SPS despite low GAD65 antibodies titres.

β-Alanine, a valuable β-type amino acid, is experiencing increased demand due to its multifaceted applications in food flavoring, nutritional supplements, pharmaceuticals, and the chemical industry. Nevertheless, the sustainable biosynthesis of β-alanine currently faces challenges due to the scarcity of robust strains, attributed to the complexities of modulating multiple genes and the inherent physiological constraints. Here, systems metabolic engineering was implemented in Escherichia coli to overcome these limitations. First, an efficient l-aspartate-α-decarboxylase (ADC) was recruited for β-alanine biosynthesis. To conserve phosphoenolpyruvate flux, we subsequently modified the endogenous glucose assimilation system by inactivating the phosphotransferase system (PTS) and introducing an alternative non-PTS system, which increased β-alanine production to 1.70 g/L. The supply of key precursors, oxaloacetate and l-aspartate, was synergistically improved through comprehensive modulation, including strengthening main flux and blocking bypass metabolism, which significantly increased the β-alanine titer to 3.43 g/L. Next, the expression of ADC was optimized by promoter and untranslated region (UTR) engineering. Further transport engineering, which involved disrupting β-alanine importer CycA and heterologously expressing β-alanine exporter NCgI0580, improved β-alanine production to 8.48 g/L. Additionally, corn steep liquor was used to develop a cost-effective medium. The final strain produced 74.03 g/L β-alanine with a yield of 0.57 mol/mol glucose during fed-batch fermentation.

Immune checkpoint inhibitors (ICIs) are widely used in cancer treatment; however, they can lead to immune-related adverse events, including immune checkpoint inhibitor-induced type 1 diabetes mellitus (ICI-T1DM). While fulminant T1DM is common in East Asia, ICI-T1DM has predominantly been reported in Western countries. In this report, we present the case of a 66-year-old Japanese man with type 2 diabetes mellitus undergoing dialysis for diabetic nephropathy. The patient was diagnosed with left upper lobe lung cancer, and treatment with nivolumab and ipilimumab was initiated. After 48 days, the patient experienced impaired consciousness and difficulty moving. His blood glucose levels were 815 mg/dL, and metabolic acidosis was detected, leading to a diagnosis of diabetic ketoacidosis. The patient was subsequently treated with continuous intravenous insulin. However, his C-peptide levels rapidly depleted, and new-onset ICI-T1DM was diagnosed. Although most Japanese patients with ICI-T1DM test negative for glutamic acid decarboxylase (GAD) antibodies, this case exhibited a strong positivity. Thus, we reviewed the literature on 15 similar Japanese cases, revealing a mean HbA1c level at onset of 8.7% and a mean time from ICI administration to onset of 9.7 weeks, which was shorter than that in GAD-negative cases. Moreover, human leukocyte antigen typing revealed five cases of DRB1*04:05-DQB1*04:01, including the present case, and one case of DRB1*09:01-DQB1*03:03, both of which were susceptible to T1DM haplotypes. These findings suggest that GAD antibody positivity may be associated with acute onset and disease progression in some cases of Japanese patients with ICI-T1DM. Given that the prediction of new-onset ICI-T1DM is challenging, monitoring GAD antibody levels might be useful. However, further studies with large sample sizes and validation across different racial and ethnic populations are warranted.

Even a transient period of hearing loss during the developmental critical period can induce long-lasting deficits in temporal and spectral perception. These perceptual deficits correlate with speech perception in humans. In gerbils, these hearing loss-induced perceptual deficits are correlated with a reduction of both ionotropic GABAA and metabotropic GABAB receptor-mediated synaptic inhibition in auditory cortex, but most research on critical period plasticity has focused on GABAA receptors. Therefore, we developed viral vectors to express proteins that would upregulate gerbil postsynaptic inhibitory receptor subunits (GABAA, Gabra1; GABAB, Gabbr1b) in pyramidal neurons, and an enzyme that mediates GABA synthesis (GAD65) presynaptically in parvalbumin-expressing interneurons. A transient period of developmental hearing loss during the auditory critical period significantly impaired perceptual performance on two auditory tasks: amplitude modulation depth detection and spectral modulation depth detection. We then tested the capacity of each vector to restore perceptual performance on these auditory tasks. While both GABA receptor vectors increased the amplitude of cortical inhibitory postsynaptic potentials, only viral expression of postsynaptic GABAB receptors improved perceptual thresholds to control levels. Similarly, presynaptic GAD65 expression improved perceptual performance on spectral modulation detection. These findings suggest that recovering performance on auditory perceptual tasks depends on GABAB receptor-dependent transmission at the auditory cortex parvalbumin to pyramidal synapse and point to potential therapeutic targets for developmental sensory disorders.

Autoimmune polyendocrine syndrome type 1 (APS-1) is a rare monogenic disease caused by mutations in the autoimmune regulator gene. Although the disease-associated autoantibodies mostly target endocrine organs, autoantibodies from patients with APS-1 bind also to rat brain structures. The patients often have GAD65-antibodies, that can cause autoimmune encephalitis. However, neurological manifestations of APS-1 have not been systematically explored. We conducted a retrospective chart review on 44 Finnish patients with APS-1 (median age 38 years, 61% females) and collected all their neurological diagnoses. To assess the prevalence of serum antineuronal antibodies in APS-1, serum samples of 24 patients (median age 36 years, 63% females) were analyzed using a fixed cell-based assay. Of the 44 APS-1 patients, 10 (23%) had also received a diagnosis of a neurological disease. Of these neurological comorbidities, migraine (n = 7; 16%), central nervous system infections (n = 3; 7%), and epilepsy (n = 2; 5%) were the most prevalent. Other diagnoses recorded for single patients were axonal sensorimotor polyneuropathy, essential tremor, idiopathic intracranial hypertension, ischemic stroke, and trigeminal neuralgia. Serum antineuronal antibodies were detected in 42% of patients tested (10/24, 50% females, median age 42 years), GAD65 antibodies being the most common finding. Antibodies against glycine and aquaporin 4 were found in low titers. In four patients, relatively high titers of GAD65 antibodies without coexisting type 1 diabetes were found, but none presented with GAD65-encephalitis. Our study suggests an association between APS-1 and neurological disorders, the mechanisms of which are to be further investigated.