AADC deficiency causes severe motor and intellectual disability as a result of reduced catecholamine levels. Kojima et al. report beneficial effects of gene therapy in six patients with heterogeneous ...genetic backgrounds. Gene delivery into putamen improved motor function in all patients, plus verbal and cognitive skills in one moderate-phenotype patient.
Abstract
In patients with aromatic l-amino acid decarboxylase (AADC) deficiency, a decrease in catecholamines and serotonin levels in the brain leads to developmental delay and movement disorders. The beneficial effects of gene therapy in patients from 1 to 8 years of age with homogeneous severity of disease have been reported from Taiwan. We conducted an open-label phase 1/2 study of population including adolescent patients with different degrees of severity. Six patients were enrolled: four males (ages 4, 10, 15 and 19 years) and one female (age 12 years) with a severe phenotype who were not capable of voluntary movement or speech, and one female (age 5 years) with a moderate phenotype who could walk with support. The patients received a total of 2 × 1011 vector genomes of adeno-associated virus vector harbouring DDC via bilateral intraputaminal infusions. At up to 2 years after gene therapy, the motor function was remarkably improved in all patients. Three patients with the severe phenotype were able to stand with support, and one patient could walk with a walker, while the patient with the moderate phenotype could run and ride a bicycle. This moderate-phenotype patient also showed improvement in her mental function, being able to converse fluently and perform simple arithmetic. Dystonia disappeared and oculogyric crisis was markedly decreased in all patients. The patients exhibited transient choreic dyskinesia for a couple of months, but no adverse events caused by vector were observed. PET with 6-18Ffluoro-l-m-tyrosine, a specific tracer for AADC, showed a persistently increased uptake in the broad areas of the putamen. In our study, older patients (>8 years of age) also showed improvement, although treatment was more effective in younger patients. The genetic background of our patients was heterogeneous, and some patients suspected of having remnant enzyme activity showed better improvement than the Taiwanese patients. In addition to the alleviation of motor symptoms, the cognitive and verbal functions were improved in a patient with the moderate phenotype. The restoration of dopamine synthesis in the putamen via gene transfer provides transformative medical benefit across all patient ages, genotypes, and disease severities included in this study, with the most pronounced improvements noted in moderate patients.
10.1093/brain/awy331_video1
awy331media1
5991361892001
The lateral habenula (LHb) regulates the activity of monoaminergic neurons in the brainstem. This area has recently attracted a surge of interest in psychiatry because studies have reported the ...pathological activation of the habenula in patients with major depression and in animal models. The LHb plays a significant role in the pathophysiology of depression; however, how habenular neurons are activated to cause various depression symptoms, such as reduced motivation and sleep disturbance, remain unclear. We hypothesized that dysfunctional astrocytes may cause LHb hyperactivity due to the defective uptake activity of extracellular glutamate, which induces depressive-like behaviors. We examined the activity of neurons in habenular pathways and performed behavioral and sleep analyses in mice with pharmacological and genetic inhibition of the activity of the glial glutamate transporter GLT-1 in the LHb. The habenula-specific inhibition of GLT-1 increased the neuronal firing rate and the level of c-Fos expression in the LHb. Mice with reduced GLT-1 activity in the habenula exhibited a depressive-like phenotype in the tail suspension and novelty-suppressed feeding tests. These animals also displayed increased susceptibility to chronic stress, displaying more frequent avoidant behavior without affecting locomotor activity in the open-field test. Intriguingly, the mice showed disinhibition of rapid eye movement sleep, which is a characteristic sleep pattern in patients with depression. These results provide evidence that disrupting glutamate clearance in habenular astrocytes increases neuronal excitability and depressive-like phenotypes in behaviors and sleep.
Haemophilia B, a congenital haemorrhagic disease caused by mutations in coagulation factor IX gene (F9), is considered an appropriate target for genome editing technology. Here, we describe treatment ...strategies for haemophilia B mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system. Administration of adeno-associated virus (AAV) 8 vector harbouring Staphylococcus aureus Cas9 (SaCas9) and single guide RNA (sgRNA) to wild-type adult mice induced a double-strand break (DSB) at the target site of F9 in hepatocytes, sufficiently developing haemophilia B. Mutation-specific gene editing by simultaneous induction of homology-directed repair (HDR) sufficiently increased FIX levels to correct the disease phenotype. Insertion of F9 cDNA into the intron more efficiently restored haemostasis via both processes of non-homologous end-joining (NHEJ) and HDR following DSB. Notably, these therapies also cured neonate mice with haemophilia, which cannot be achieved with conventional gene therapy with AAV vector. Ongoing haemophilia therapy targeting the antithrombin gene with antisense oligonucleotide could be replaced by SaCas9/sgRNA-expressing AAV8 vector. Our results suggest that CRISPR/Cas9-mediated genome editing using an AAV8 vector provides a flexible approach to induce DSB at target genes in hepatocytes and could be a good strategy for haemophilia gene therapy.
The nonapeptide oxytocin is considered beneficial to mental health due to its anxiolytic, prosocial and antistress effects, but evidence for anxiogenic actions of oxytocin in humans has recently ...emerged. Using region-specific manipulations of the mouse oxytocin receptor (Oxtr) gene (Oxtr), we identified the lateral septum as the brain region mediating fear-enhancing effects of Oxtr. These effects emerge after social defeat and require Oxtr specifically coupled to the extracellular signal-regulated protein kinase pathway.
Although corticospinal neurons are known to be distributed in both the primary motor and somatosensory cortices (S1), details of the projection pattern of their fibers to the lumbar cord gray matter ...remain largely uncharacterized, especially in rodents. We previously investigated the cortical area projecting to the gray matter of the fourth lumbar cord segment (L4) (L4 Cx) in mice. In the present study, we injected an anterograde tracer into multiple sites to cover the entire L4 Cx. We found that (1) the rostromedial part of the L4 Cx projects to the intermediate and ventral zones of the lumbar cord gray matter, (2) the lateral part projects to the medial dorsal horn, and (3) the caudal part projects to the lateral dorsal horn. We also found that the border between the rostromedial and caudolateral parts corresponds to the border between the agranular and granular cortex. Analysis of the somatotopic patterns formed by the cortical projection cells and the primary sensory neurons innervating the skin of the hindlimb and its related area suggests that the lateral part corresponds to the S1 hindlimb area and the caudal part to the S1 trunk area. Examination of thalamic innervation by the L4 Cx revealed that the caudolateral L4 Cx focally projects to the ventrobasal complex (VB) and the posterior complex (PO), while the medial L4 Cx widely projects to the PO but little to the VB. These findings suggest that the L4 Cx is parceled into subregions defined by the cytoarchitecture and subcortical projection.
We investigated fiber distribution patterns of corticospinal neurons within the cortical area projecting to the lumbar cord gray matter of mice. The lumbar‐cord‐projecting area fundamentally innervates the gray matter in the same way as the cervical‐cord‐projecting area does.
Autism spectrum disorder (ASD) is a continuum of neurodevelopmental disorders and needs new therapeutic approaches. Recently, oxytocin (OXT) showed potential as the first anti-ASD drug. Many reports ...have described the efficacy of intranasal OXT therapy to improve the core symptoms of patients with ASD; however, the underlying neurobiological mechanism remains unknown. The OXT/oxytocin receptor (OXTR) system, through the lateral septum (LS), contributes to social behavior, which is disrupted in ASD. Therefore, we selectively express hM3Dq in OXTR-expressing (OXTR+) neurons in the LS to investigate this effect in ASD mouse models developed by environmental and genetic cues. In mice that received valproic acid (environmental cue), we demonstrated successful recovery of impaired social memory with three-chamber test after OXTR+ neuron activation in the LS. Application of a similar strategy to Nl3
knock-in mice (genetic cue) also caused successful recovery of impaired social memory in single field test. OXTR+ neurons in the LS, which are activated by social stimuli, are projected to the CA1 region of the hippocampus. This study identified a candidate mechanism for improving core symptoms of ASD by artificial activation of DREADDs, as a simulation of OXT administration to activate OXTR+ neurons in the LS.
Our previous genome-wide association study to explore genetic loci associated with lean nonalcoholic fatty liver disease (NAFLD) in Japan suggested four candidate loci, which were mapped to chr6, ...chr7, chr12 and chr13. The present study aimed to identify the locus involved functionally in NAFLD around the association signal observed in chr13. Chromosome conformation capture assay and a database survey suggested the intermolecular interaction among DNA fragments in association signals with the adjacent four coding gene promoters. The four genes were further screened by knockdown (KD) in mice using shRNA delivered by an adeno-associated virus vector (AAV8), and KD of G protein-coupled receptor 180 (Gpr180) showed amelioration of hepatic lipid storage. Gpr180 knockout (KO) mice also showed ameliorated hepatic and plasma lipid levels without influencing glucose metabolism after high-fat diet intake. Transcriptome analyses showed downregulation of mTORC1 signaling and cholesterol homeostasis, which was confirmed by weakened phosphorylation of mTOR and decreased activated SREBP1 in Gpr180KO mice and a human hepatoma cell line (Huh7). AAV8-mediated hepatic rescue of GPR180 expression in KO mice showed recovery of plasma and hepatic lipid levels. In conclusion, ablation of GPR180 ameliorated plasma and hepatic lipid levels, which was mediated by downregulation of mTORC1 signaling.
Gene transfer of dopamine-synthesizing enzymes into the striatal neurons has led to behavioral recovery in animal models of Parkinson's disease (PD). We evaluated the safety, tolerability, and ...potential efficacy of adeno-associated virus (AAV) vector–mediated gene delivery of aromatic L-amino acid decarboxylase (AADC) into the putamen of PD patients. Six PD patients were evaluated at baseline and at 6 months, using multiple measures, including the Unified Parkinson's Disease Rating Scale (UPDRS), motor state diaries, and positron emission tomography (PET) with 6-18Ffluoro-L-m-tyrosine (FMT), a tracer for AADC. The short-duration response to levodopa was measured in three patients. The procedure was well tolerated. Six months after surgery, motor functions in the OFF-medication state improved an average of 46% based on the UPDRS scores, without apparent changes in the short-duration response to levodopa. PET revealed a 56% increase in FMT activity, which persisted up to 96 weeks. Our findings provide class IV evidence regarding the safety and efficacy of AADC gene therapy and warrant further evaluation in a randomized, controlled, phase 2 setting.
Two-photon imaging in behaving animals has revealed neuronal activities related to behavioral and cognitive function at single-cell resolution. However, marmosets have posed a challenge due to ...limited success in training on motor tasks. Here we report the development of protocols to train head-fixed common marmosets to perform upper-limb movement tasks and simultaneously perform two-photon imaging. After 2-5 months of training sessions, head-fixed marmosets can control a manipulandum to move a cursor to a target on a screen. We conduct two-photon calcium imaging of layer 2/3 neurons in the motor cortex during this motor task performance, and detect task-relevant activity from multiple neurons at cellular and subcellular resolutions. In a two-target reaching task, some neurons show direction-selective activity over the training days. In a short-term force-field adaptation task, some neurons change their activity when the force field is on. Two-photon calcium imaging in behaving marmosets may become a fundamental technique for determining the spatial organization of the cortical dynamics underlying action and cognition.
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