The functional role of laminins-α4 and -β2 in development of the neuromuscular junction

Abstract

The precise development and organisation of the neuromuscular junction (NMJ) is critical for the production of efficient neurotransmission signals. Disruption of these specialisations may result in severely altered transmission properties at the NMJ.Signalling and adhesion molecules have long been established to be key mediators in the distribution of synaptic specialisations. The basal lamina acts to maintain stability of the associated tissue and contains a number of these signalling and adhesion molecules. The laminins, a family of large multimeric proteins, are one of the key components of the basal lamina that act as specific cell regulators and provide mechanical stability. Laminins have been shown to form an interconnecting network that assists in stabilisation of the basal lamina and provides attachment sites for other basal lamina components. The synapse specific laminin chains, -α4, -α5, and -β2, play an essential role in differentiation and organisation of the NMJ. These chains form the laminin isoforms, laminin-221 (α2β2γ1), laminin-421 (α4β2γ1), and laminin-521 (α5β2γ1).Laminin-β2, found in each of the three synapse specific isoforms, has been shown to organise presynaptic specialisations at the developing NMJ. In vitro laminin-β2 is able to interact directly with N-type and P/Q-type voltage-gated calcium This study examined the role of laminin-β2 in the organisation of N-and P/Q-type VGCCs at active zones of developing postnatal day 8 (P8) and matured, postnatal day 18 (P18) NMJs.The contribution of each channel to the release of transmitter was assessed using intracellular electrode recordings of end-plate potentials (EPPs). The VGCC toxins, ω-conotoxin-GVIA and ω-agatoxin-IVA, were used to specifically target N-and P/Q-type NMJs displayed significantly higher levels of synaptic depression under high frequency stimuli and altered paired pulse facilitation compared to wild-type littermates. Analysis of the binomial parameters of neurotransmitter release demonstrated a decrease in quantal release as a result of a decrease in the number of active release sites, but not in the average probability of transmitter release from these sites. Our functional findings suggest alterations in the short-term plasticity of the NMJ and possibly defective recycling of ii synaptic vesicles and/or the calcium handling at lama4 -/-NMJs. We propose that alterations to synapsin I and its associated molecules may be partly responsible for the changes in neurotransmission observed at lama4 -/-NMJs. Our findings demonstrate altered distribution and expression of presynaptic components associated with active zones, specifically an increase in the number of synaptic vesicles and a decrease in the density of the fluorescently labelled Bassoon at the active zone region. Our results suggest that the fewer active release sites may compensate for the deficits of the lama4 -/-NMJs by alterations to pre-and postsynaptic specialisations.In conclusion, this thesis has identified that laminins-α4 and -β2 play fundamental role...