GABAergic neuron-specific whole-brain transduction by AAV-PHP.B incorporated with a new GAD65 promoter

Abstract

GABAergic interneurons play a critical role in tuning neural networks in the central nervous system, and their defects are associated with neuropsychiatric disorders. Currently, the mDlx enhancer is solely used for adeno-associated virus (AAV) vector-mediated transgene delivery into cortical interneurons. Here, we developed a new inhibitory neuron-specific promoter (designated as the mGAD65 promoter), with a length of 2.5 kb, from a mouse genome upstream of exon 1 of the Gad2 gene encoding glutamic acid decarboxylase (GAD) 65. Intravenous infusion of blood–brain barrier-penetrating AAV-PHP.B expressing an enhanced green fluorescent protein under the control of the mGAD65 promoter transduced the whole brain in an inhibitory neuron-specific manner. The specificity and efficiency of the mGAD65 promoter for GABAergic interneurons, which was assessed at the motor cortex, were almost identical to or slightly higher than those of the mDlx enhancer. Immunohistochemical analysis revealed that the mGAD65 promoter preferentially transduced parvalbumin (PV)-expressing interneurons. Notably, the mGAD65 promoter transduced chandelier cells more efficiently than the mDlx enhancer and robustly labeled their synaptic boutons, called the cartridge, targeting the axon initial segments of excitatory pyramidal neurons. To test the ability of the mGAD65 promoter to express a functional molecule, we virally expressed G-CaMP, a fluorescent Ca2+ indicator, in the motor cortex, and this enabled us to monitor spontaneous and drug-induced Ca2+ activity in GABAergic inhibitory neurons. These results suggest that the mGAD65 promoter is useful for AAV-mediated targeting and manipulation of GABAergic neurons with the dominance of cortical PV-expressing neurons, including chandelier cells.

GABAergic Gene Regulatory Elements Used in Adeno-Associated Viral Vectors

“…Direct injection of rAAVs carrying I56i, h12R, or h56D into the murine cerebral cortex and hippocampus resulted in the transduction of 80–90% of cortical and hippocampal GABAergic neurons after each injection, respectively ( 12 , 14 ). Intravenous injection of rAAV-I56i labeled 57% of murine cortical GABAergic neurons ( 10 ). All of the aforementioned DLX enhancers (I12b, I56i, h12R, and h56D) directed transgene expression to murine cortical and hippocampal GABAergic neurons with approximately 90% or greater specificity [( 10 – 12 , 14 ); see Table 1 ].…”

“…Intravenous injection of rAAV-I56i labeled 57% of murine cortical GABAergic neurons ( 10 ). All of the aforementioned DLX enhancers (I12b, I56i, h12R, and h56D) directed transgene expression to murine cortical and hippocampal GABAergic neurons with approximately 90% or greater specificity [( 10 – 12 , 14 ); see Table 1 ]. Similar levels of GABAergic neuronal specificity and coverage were observed in gerbils and in non-human primates ( 12 , 14 ).…”

“…Despite the excellent ability of these enhancers to target GABAergic neurons in anterior brain regions, neither intravenous injection of rAAV-I56i ( 10 , 16 ), nor intra-inferior collicular injection of rAAV-h65D ( 14 ) induced transgene expression in posterior brain regions (such as the midbrain, cerebellum, and brainstem). The non-functionality of DLX enhancers in the posterior brain poses a limitation for studies of candidate therapies requiring posterior brain or CNS-wide GABAergic neuronal targeting; other regulatory elements will likely be necessary in these cases.…”

“…An rAAV carrying a 3 kb region of the murine Gad2 promoter (3,000 base pairs upstream from the start codon) was used to examine enhanced green fluorescent protein (eGFP) transgene expression in mouse brain; this rAAV displayed 20% specificity to excitatory neurons in the murine cerebral cortex ( 10 ). However, another construct carrying a deletion of Gad2 exon 1 abolished excitatory neuron expression and induced labeling of 60% of cortical GABAergic neurons with 96% GABA neuron specificity after intravenous injection of AAV-PHP.B ( 10 ).…”

“…An rAAV carrying a 3 kb region of the murine Gad2 promoter (3,000 base pairs upstream from the start codon) was used to examine enhanced green fluorescent protein (eGFP) transgene expression in mouse brain; this rAAV displayed 20% specificity to excitatory neurons in the murine cerebral cortex ( 10 ). However, another construct carrying a deletion of Gad2 exon 1 abolished excitatory neuron expression and induced labeling of 60% of cortical GABAergic neurons with 96% GABA neuron specificity after intravenous injection of AAV-PHP.B ( 10 ). rAAVs carrying this truncated Gad2 promoter (denoted as rAAV-mGad65 by the authors of the study) showed broad expression in the mouse CNS, including in anterior areas such as the cerebral cortex, hippocampus, and striatum, and in posterior areas including the midbrain, cerebellum, and brainstem.…”