ABSTRACT
Recent emphasis has been placed on the role that cerebellar dysfunctions could have in the genesis of cognitive deficits in Duchenne muscular dystrophy (DMD). However, relevant genotype-phenotype analyses are missing to define whether cerebellar defects underlie the severe cases of intellectual deficiency that have been associated with genetic loss of the smallest product of the dmd gene, the Dp71 dystrophin. To determine for the first time whether Dp71 loss could affect cerebellar physiology and functions, we have used patch-clamp electrophysiological recordings in acute cerebellar slices and a cerebellum-dependent behavioral test battery addressing cerebellum-dependent motor and non-motor functions in Dp71-null transgenic mice. We found that Dp71 deficiency selectively enhances excitatory transmission at glutamatergic synapses formed by climbing fibers (CFs) on Purkinje neurons, but not at those formed by parallel fibers. Altered basal neurotransmission at CFs was associated with impairments in synaptic plasticity and clustering of the scaffolding postsynaptic density protein PSD-95. At the behavioral level, Dp71-null mice showed some improvements in motor coordination and were unimpaired for muscle force, static and dynamic equilibrium, motivation in high-motor demand and synchronization learning. Dp71-null mice displayed altered strategies in goal-oriented navigation tasks, however, suggesting a deficit in the cerebellum-dependent processing of the procedural components of spatial learning, which could contribute to the visuospatial deficits identified in this model. In all, the observed deficits suggest that Dp71 loss alters cerebellar synapse function and cerebellum-dependent navigation strategies without being detrimental for motor functions.
Subject(s)
Cerebellum/metabolism , Dystrophin/deficiency , Motor Activity , Synapses/metabolism , Animals , Biomechanical Phenomena , Cerebellum/physiopathology , Dystrophin/metabolism , Exploratory Behavior , Genotype , Glutamic Acid/metabolism , Male , Maze Learning , Mice, Inbred C57BL , Mice, Knockout , Motivation , Neuronal Plasticity , Purkinje Cells/metabolism , Synaptic TransmissionABSTRACT
The universal N6-threonylcarbamoyladenosine (t6A) modification at position A37 of ANN-decoding tRNAs is essential for translational fidelity. In bacteria the TsaC enzyme first synthesizes an l-threonylcarbamoyladenylate (TC-AMP) intermediate. In cooperation with TsaB and TsaE, TsaD then transfers the l-threonylcarbamoyl-moiety from TC-AMP onto tRNA. We determined the crystal structure of the TsaB-TsaE-TsaD (TsaBDE) complex of Thermotoga maritima in presence of a non-hydrolysable AMPCPP. TsaE is positioned at the entrance of the active site pocket of TsaD, contacting both the TsaB and TsaD subunits and prohibiting simultaneous tRNA binding. AMPCPP occupies the ATP binding site of TsaE and is sandwiched between TsaE and TsaD. Unexpectedly, the binding of TsaE partially denatures the active site of TsaD causing loss of its essential metal binding sites. TsaE interferes in a pre- or post-catalytic step and its binding to TsaBD is regulated by ATP hydrolysis. This novel binding mode and activation mechanism of TsaE offers good opportunities for antimicrobial drug development.
Subject(s)
Adenosine/analogs & derivatives , Bacterial Proteins/chemistry , RNA, Transfer/metabolism , Thermotoga maritima/enzymology , Adenosine/biosynthesis , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Archaeal Proteins/chemistry , Bacterial Proteins/metabolism , Binding Sites , Catalytic Domain , Enzymes/chemistry , Enzymes/metabolism , Models, Molecular , Protein Conformation , Protein Structure, Quaternary , RNA, Transfer/chemistryABSTRACT
In cerebellar cortex, mGlu4 receptors located on parallel fibers play an essential role in normal motor function, but the molecular mechanisms involved are not yet completely understood. Using a strategy combining biochemical and electrophysiological approaches in the rodent cerebellum, we demonstrate that presynaptic mGlu4 receptors control synaptic transmission through an atypical activation of Gαq proteins. First, the Gαq subunit, PLC and PKC signaling proteins present in cerebellar extracts are retained on affinity chromatography columns grafted with different sequences of the cytoplasmic domain of mGlu4 receptor. The i2 loop and the C terminal domain were used as baits, two domains that are known to play a pivotal role in coupling selectivity and efficacy. Second, in situ proximity ligation assays show that native mGlu4 receptors and Gαq subunits are in close physical proximity in cerebellar cortical slices. Finally, electrophysiological experiments demonstrate that the molecular mechanisms underlying mGlu4 receptor-mediated inhibition of transmitter release at cerebellar Parallel Fiber (PF) - Molecular Layer Interneuron (MLI) synapses involves the Gαq-PLC signaling pathway. Taken together, our results provide compelling evidence that, in the rodent cerebellar cortex, mGlu4 receptors act by coupling to the Gαq protein and PLC effector system to reduce glutamate synaptic transmission.
