An enhancer-AAV approach selectively targeting dentate granule cells of the mouse hippocampus.

The mammalian brain contains a diverse array of cell types, including dozens of neuronal subtypes with distinct anatomical and functional characteristics. The brain leverages these neuron-type specializations to perform diverse circuit operations and thus execute different behaviors properly. Through the use of Cre lines, access to specific neuron types has improved over past decades. Despite their extraordinary utility, development and cross-breeding of Cre lines is time consuming and expensive, presenting a significant barrier to entry for investigators. Furthermore, cell-based therapeutics developed in Cre mice are not clinically translatable. Recently, several adeno-associated virus (AAV) vectors utilizing neuron-type-specific regulatory transcriptional sequences (enhancer-AAVs) were developed that overcome these limitations. Using a publicly available RNA sequencing (RNA-seq) dataset, we evaluated the potential of several candidate enhancers for neuron-type-specific targeting in the hippocampus. Here, we demonstrate that a previously identified enhancer-AAV selectively targets dentate granule cells over other excitatory neuron types in the hippocampus of wild-type adult mice.

A novel enhancer-AAV approach selectively targeting dentate granule cells.

The mammalian brain contains the most diverse array of cell types of any organ, including dozens of neuronal subtypes with distinct anatomical and functional characteristics. The brain leverages these neuron-type-specializations to perform diverse circuit operations and thus execute different behaviors properly. Through the use of Cre lines, access to specific neuron types has steadily improved over past decades. Despite their extraordinary utility, development and cross-breeding of Cre lines is time-consuming and expensive, presenting a significant barrier to entry for many investigators. Furthermore, cell-based therapeutics developed in Cre mice are not clinically translatable. Recently, several AAV vectors utilizing neuron-type-specific regulatory transcriptional sequences (enhancer-AAVs) were developed which overcome these limitations. Using a publicly available RNAseq dataset, we evaluated the potential of several candidate enhancers for neuron-type-specific targeting in the hippocampus. Here we identified a promising enhancer-AAV for targeting dentate granule cells and validated its selectivity in wild-type adult mice.

Kinetics of ATP and TNP-ATP binding to the active site of CheA from Thermotoga maritima.

The mechanism of nucleotide binding to the active site of Thermotoga maritima CheA was investigated using stopped-flow fluorescence experiments that monitored binding of ATP and TNP-ATP to the catalytic domain (P4) of CheA that had been engineered to include a tryptophan residue as a fluorescent reporter group at the active site (P4(F487W)). Rapid decreases in protein intrinsic fluorescence and increases in TNP-ATP fluorescence were observed during binding reactions, and time courses were analyzed to define the kinetic mechanisms for ATP and TNP-ATP binding. This analysis indicated that binding of ATP(Mg(2+)) to P4(F487W) involves a single reversible step with a k(on) of 0.92 +/- 0.09 microM(-1) s(-1), a k(off) of 1.9 +/- 0.4 s(-1), and a K(d) of 1.5-2.1 microM (all values determined at 4 degrees C). Binding of TNP-ATP(Mg(2+)) to P4(F487W) involves a more complicated mechanism, requiring at least three sequential steps. Computer simulations and nonlinear regression analysis were used to estimate the rate constants of the forward and reverse reactions for each of the three steps in the reaction scheme [Formula: see text] Similar analysis indicated that an alternative reaction scheme, involving a rate-limiting conformational change in P4 prior to TNP-ATP binding, did an equally good job of accounting for all of the kinetics results:[Formula: see text] In both models, steps 2 and 3 have slow reversal rates that contribute to the high affinity of the active site for TNP-ATP (K(d) = 0.015 microM). These results highlight the dramatic effect of the TNP moieties on CheA-nucleotide interactions, and they provide the first detailed information about the kinetic mechanism underlying interaction of a protein histidine kinase with this tight-binding inhibitor.

The two active sites of Thermotoga maritima CheA dimers bind ATP with dramatically different affinities.

CheA is a central component of the chemotaxis signal transduction pathway that allows prokaryotic cells to control their movements in response to environmental cues. This dimeric protein histidine kinase autophosphorylates via an intersubunit phosphorylation reaction in which each protomer of the dimer binds ATP, at an active site located in its P4 domain and then catalyzes transfer of the gamma-phosphoryl group of ATP to the His(45) side chain within the P1 domain of the trans protomer. Here we utilize the fluorescent nucleotide analogue TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate] to investigate the two ATP-binding sites of the Thermotoga maritima CheA dimer (TmCheA) and the single site of the isolated TmP4 domain (a monomer). We define the affinity of CheA for TNP nucleotides and, by competition, for unmodified ATP. The two ATP-binding sites of the TmCheA dimer exhibit dramatically different affinities for TNP-ATP (K(d1)(TNP) approximately 0.0016 muM and K(d2)(TNP) approximately 22 muM at 4 degrees C in the presence of Mg(2+)) as well as for ATP (K(d1)(ATP) approximately 6 muM and K(d2)(ATP) approximately 5000 muM at 4 degrees C in the presence of Mg(2+)) and in their ability to influence the fluorescence of bound TNP-ATP. The ATP-binding site of the isolated TmP4 domain interacts with ATP and TNP-ATP in a manner similar to that of the high-affinity site of the TmCheA dimer. These results suggest that the two active sites of TmCheA homodimers exhibit large differences in their interactions with ATP. We consider possible implications of these differences for the CheA autophosphorylation mechanism and for CheA function in bacterial cells.