Rab27a targeting to melanosomes requires nucleotide exchange but not effector binding

Rab GTPases are important determinants of organelle identity and regulators of vesicular transport pathways. Consequently, each Rab occupies a highly specific subcellular localization. However, the precise mechanisms governing Rab targeting remain unclear. Guanine nucleotide exchange factors (GEFs), putative membrane-resident targeting factors and effector binding have all been implicated as critical regulators of Rab targeting. Here, we address these issues using Rab27a targeting to melanosomes as a model system. Rab27a regulates motility of lysosome-related organelles and secretory granules. Its effectors have been characterized extensively, and we have identified Rab3GEP as the non-redundant Rab27a GEF in melanocytes (Figueiredo AC et al. Rab3GEP is the non-redundant guanine nucleotide exchange factor for Rab27a in melanocytes. J Biol Chem 2008;283:23209-23216). Using Rab27a mutants that show impaired binding to representatives of all four Rab27a effector subgroups, we present evidence that effector binding is not essential for targeting of Rab27a to melanosomes. In contrast, we observed that knockdown of Rab3GEP resulted in mis-targeting of Rab27a, suggesting that Rab3GEP activity is required for correct targeting of Rab27a. However, the identification of Rab27a mutants that undergo efficient GDP/GTP exchange in the presence of Rab3GEP in vitro but are mis-targeted in a cellular context indicates that nucleotide loading is not the sole determinant of subcellular targeting of Rab27a. Our data support a model in which exchange activity, but not effector binding, represents one essential factor that contributes to membrane targeting of Rab proteins.publishersversionpublishe

Chemogenetic attenuation of cortical seizures in nonhuman primates

「てんかん」の発生を時間的・空間的にピンポイントで抑える画期的な治療法を開発 --世界で初めてサルでの有効性を実証、臨床応用に向け大きく前進--. 京都大学プレスリリース. 2023-03-01.Epilepsy is a disorder in which abnormal neuronal hyperexcitation causes several types of seizures. Because pharmacological and surgical treatments occasionally interfere with normal brain function, a more focused and on-demand approach is desirable. Here we examined the efficacy of a chemogenetic tool—designer receptors exclusively activated by designer drugs (DREADDs)—for treating focal seizure in a nonhuman primate model. Acute infusion of the GABAA receptor antagonist bicuculline into the forelimb region of unilateral primary motor cortex caused paroxysmal discharges with twitching and stiffening of the contralateral arm, followed by recurrent cortical discharges with hemi- and whole-body clonic seizures in two male macaque monkeys. Expression of an inhibitory DREADD (hM4Di) throughout the seizure focus, and subsequent on-demand administration of a DREADD-selective agonist, rapidly suppressed the wide-spread seizures. These results demonstrate the efficacy of DREADDs for attenuating cortical seizure in a nonhuman primate model

Chemogenetic suppression of pharmacologically induced frontal lobe epilepsy in a macaque monkey

Rationale: Medical treatments and surgical lesions of epilepsy sometimes lead to unexpected side effects by affecting the surrounding normal tissues, and therefore, a more focused and on-demand approach is desirable. DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) is a recently developed “chemogenetics” tool that combines genetically introduced functional proteins (artificial receptors) and pharmacologically introduced chemicals (actuator drugs), offering focused and on-demand control of neuronal activities. We have recently developed a potent and selective actuator drug, Deschloroclozapine (DCZ), whose radiolabeled form can be used as a PET tracer to monitor DREADD expression in vivo. This study examined the utility of DREADDs on epilepsy treatment using a pharmacological model of epilepsy in a non-human primate (NHP).Methods: We injected virus vectors carrying hM4Di, an inhibitory DREADD gene (AAV2.1-hSyn-FLAG-hM4Di-IRES-AcGFP) to the putative hand/arm-region of the motor cortex in a cynomolgus macaque. Five weeks after the vector injection, hM4Di expression was confirmed using PET imaging with [11C]DCZ. We performed additional surgery to open a cranial window and placed an epidural electrocorticogram (ECoG) electrode array and a chronic chamber to approach the target region. Acute injections of bicuculline methiodide (6~12 µg) into the hM4Di-expressing region were conducted to start artificial epilepsy. DCZ (100~200 µg/kg) or control vehicle (2% DMSO in saline) were injected intramuscularly while we monitored the ECoG and video for seizure and body responses. Nissl and anti-GFP antibody staining were performed after the animal was euthanized, and cardially fixated.Results: Bicuculline injection caused paroxysmal cortical discharges with twitching and stiffening of the right arm. Recurrent cortical discharges (status epilepticus) spreading over a wider area of the cortex soon followed coinciding with hemi- and whole-body convulsions. Intramuscular injection of DCZ (100~200 µg/kg), but not the control vehicle (2% DMSO in saline), attenuated the body convulsions and spreading of recurrent seizures. Post-mortem histology confirmed the preserved cell condition of the DCZ-expressing cortex, but with some reduction of the layer 5 large pyramidal cells in the bicuculline-injected subregion.Conclusions: Although further study is needed, these preliminary results are the first to demonstrate in an NHP model that epilepsy can be suppressed using the DREADD system, suggesting its promising utility for future clinical applications.AES 202

Neurodegenerative processes accelerated by protein malnutrition and decelerated by essential amino acids in a tauopathy mouse model

Protein malnutrition is epidemiologically suggested as a potential risk factor for senile dementia, although molecular mechanisms linking dietary proteins and amino acids to neurodegeneration remain unknown. Here, we show that a low-protein diet resulted in down-regulated expression of synaptic components and a modest acceleration of brain atrophy in mice modeling neurodegenerative tauopathies. Notably, these abnormal phenotypes were robustly rescued by the administration of seven selected essential amino acids. The up-regulation of inflammation-associated gene expression and progressive brain atrophy in the tauopathy model were profoundly suppressed by treatment with these essential amino acids without modifications of tau depositions. Moreover, the levels of kynurenine, an initiator of a pathway inducing neuroinflammatory gliosis and neurotoxicity in the brain, were lowered by treatment through inhibition of kynurenine uptake in the brain. Our findings highlight the importance of specific amino acids as systemic mediators of brain homeostasis against neurodegenerative processes