Neuronal functions of RIM3γ and RIM4γ

Abstract

The large isoforms of the Rab3 interacting molecule (RIM) family, RIM1α/β and RIM2α/β are integral components of the cytomatrix of the presynaptic active zone. Through multiple interactions with other active zone proteins they are involved in regulating several steps of presynaptic neurotransmitter release. The RIM protein family contains two additional isoforms, RIM3γ and RIM4γ, whose functions remain to be elucidated. <br /> In this study we could show that RIM3γ and RIM4γ are key regulators in neuronal growth involved in the establishment of axons and dendrites, formation of synapses and dendritic spines. Furthermore, the loss of RIM3γ leads to a dispersion of the Golgi apparatus whereas the loss of RIM4γ induces the condensation of the Golgi in the cell's soma. These findings provide first hints that the small γ-Rims are involved in neuronal growth by regulating processes of vesicular traffic. Live cell imaging of Rab-protein marked vesicles in RIM3γ and RIM4γ knock-down neurons revealed changes in the velocity and the path length of Rab11 and Rab8 vesicles, confirming that the loss RIM3γ and RIM4γ has an effect on intracellular transport routes. <br /> In an affinity purification mass spectrometry based search for novel γ-RIM interaction partners we identified a large set of proteins that have been associated with neuronal growth through the remodeling of the cytoskeleton or vesicular traffic and in addition a smaller group of synaptic proteins. A comparison of the individual affinity purification mass spectrometry approaches performed in different tissue fractions revealed that a cluster of new γ-RIM interaction partners was repeatedly detected. This cluster contained proteins involved in the remodeling of the cytoskeleton, site directed transport of vesicles and proteins that belong to signaling cascades important for axonal and dendritic growth. We performed first validation experiments using in vitro binding assays to decipherer the direct interactions of this complex. These experiments confirmed in vitro an interaction between γ-RIMs and the cytoskeleton regulator IQGAP3, the adhesion molecule plakophilin4 and Syd-1 an important initiator of synapse assembly. <br /> To get further insights in the cellular functions of RIM3γ and RIM4γ we generated constitutive knock-out mouse lines for both proteins. RIM3γ and RIM4γ knock-out mice are viable and not distinguishable from wild type litters until 20 days after birth. Around an age of three weeks RIM4γ knock-out mice develop a strong episodic motor phenotype, most prominent in the hind limbs, whereas RIM3γ knock-out mice show no obvious phenotype. Behavioral tests of motor coordination revealed that RIM4γ knock-out mice suffer also during phenotypic inconspicuous periods from milder coordinative disturbances suggesting impairments in cerebellar functions. Interestingly, after the induction of neuronal activity in an animal model of epilepsy RIM4γ transcripts were strongly upregulated in the hippocampus 12 hours after status epilepticus whereas RIM3γ transcripts were downregulated in the hippocampal subregion dentate gyrus after 36 hours. These findings suggest that the γ-RIMs might be either involved in pathological changes occurring during epileptogenesis or in neuroprotective mechanisms in response to increased network activity. <br /> Taken together, the results of this study provide new insights into the function of RIM3γ and RIM4γ in the development and the function of a healthy brain and form the basis for future studies on the precise understanding of the role of RIM3γ and RIM4γ during the pathogenesis of neurological disorders.