Neuronal activity-dependent membrane traffic at the neuromuscular junction

Abstract

During development and also in adulthood, synaptic connections are modulated by neuronal activity. To follow such modifications in vivo, new genetic tools are designed. The nontoxic C-terminal fragment of tetanus toxin (TTC) fused to a reporter gene such as LacZ retains the retrograde and transsynaptic transport abilities of the holotoxin itself. In this work, the hybrid protein is injected intramuscularly to analyze in vivo the mechanisms of intracellular and transneuronal traffics at the neuromuscular junction (NMJ). Traffic on both sides of the synapse are strongly dependent on presynaptic neural cell activity. In muscle, a directional membrane traffic concentrates ␤-galactosidase-TTC hybrid protein into the NMJ postsynaptic side. In neurons, the probe is sorted across the cell to dendrites and subsequently to an interconnected neuron. Such fusion protein, sensitive to presynaptic neuronal activity, would be extremely useful to analyze morphological changes and plasticity at the NMJ. T he precision with which neuronal circuits are assembled during development is critical in defining the behavioral repertoire of a mature organism. Analyzing brain functioning in normal and pathological conditions will require to integrate functional information onto anatomical data at a cellular and molecular level. To that goal, genetic tools are being constructed in parallel with improving performances in molecular imaging techniques. The atoxic C-terminal fragment of tetanus toxin (TTC) fused to a reporter gene such as LacZ can traffic retrogradely and transcellularly inside a restricted neural network. After injection into the tongue, the enzymatic activity could be detected in the hypoglossal nucleus and also in connected neurons of the brainstem areas (1). However, the molecular mechanisms involved in protein transfer between two synaptically connected cells are still unknown and should be investigated at several levels of integration. To explore the in vivo intracellular and transneuronal traffic molecular details at the neuromuscular junction (NMJ), we have chosen to inject the purified hybrid protein ␤-galactosidase (␤-gal)-TTC intramuscularly and follow the transport details by confocal and electron microscopy. A high amount of tracer protein thus is made available locally to progress into endocytic itineraries. Visualization of spreading thus is facilitated in a simple neuromuscular system.In this report, we show that ␤-gal-TTC allows selection and visualization of a specific membrane traffic, demonstrating in vivo the feasibility of tracing endocytic pathways at an NMJ. On both sides of the synapse, normal motoneuronal activity has strong effects on the probe intracellular distribution. In muscle, motoneuronal activity polarizes the membrane traffic to the active NMJ compartment. In neuron, the hybrid protein is sorted rapidly across the cell to dendrites and subsequently to an interconnected neuron, suggesting the existence of a retrograde intraneuronal feedback traffic. Materials and MethodsIn Vivo Intramuscular I...

Pax6andEngrailed 1Regulate Two Distinct Aspects of Renshaw Cell Development

“…The transsynaptic trafficking of TTC fragment fusion proteins and WGA is known to be sensitive to presynaptic neuronal activity (Yoshihara et al, 1999;Miana-Mena et al, 2002). These tracers therefore can be used to reveal the presence of functional synapses between V1 INs and motor neurons.…”

“…These tracers therefore can be used to reveal the presence of functional synapses between V1 INs and motor neurons. Although WGA also can be transported from postsynaptic dendrites to presynaptic axons, this route of transfer is very inefficient (Miana-Mena et al, 2002). Two complementary approaches were used to assess synaptic contacts between V1 INs and motor neurons: (1) transsynaptic labeling of motor neurons with a floxed WGA reporter mouse (Yoshihara et al, 1999; Y. Yoshihara, unpublished results) and (2) retrograde transsynaptic labeling of V1 INs from defined populations of motor neurons by the use of TTC fusion proteins (Coen et al, 1997).…”

A Non-Viral Vector for Targeting Gene Therapy to Motoneurons in the CNS

“…TTC fusion protein lacks the translocation domain of native tetanus toxin, which is presumably required for cytosolic entry of tetanus neurotoxin to the cytosol [31]. In this respect, ß-gal-TTC protein injection studies have shown that the protein is never observed to be free in the lumen of vesicles or in the cytosol, its transport always being associated with membranes [32]. In the DNA injection study presented in this paper, the signal detected in the CNS was so weak that electron microscopy analyses of its subcellular location could not be performed.…”

Motor neuron targeting of IGF-1 attenuates age-related external Ca2+-dependent skeletal muscle contraction in senescent mice

“…The injection interval was chosen for two reasons: 1) to limit the exposure of the muscle to the protein in order to minimize potential direct effects on the muscle and 2) the hIGF-1-TTC fusion protein is detectable in the spinal cord for 21 days post-injection (Payne, et al, 2006). TTC was chosen as the carrier for hIGF-1 based on its rapid uptake from muscle and subsequent retrograde axonal delivery to spinal cord motor neurons (Coen, et al, 1997,Fishman and Carrigan, 1987,Fishman and Savitt, 1989,Miana-Mena, et al, 2002. Indeed, in a previous report, our laboratory has shown that the hIGF-1-TTC protein undergoes quick retrograde axonal transport to the spinal cord following intramuscular injection (Payne, et al, 2006).…”