The role of synaptic laminins-α4 and -β2 in maturation and maintenance of the neuromuscular synapse

Lee, Kah Meng (2016). The role of synaptic laminins-α4 and -β2 in maturation and maintenance of the neuromuscular synapse PhD Thesis, School of Biomedical Sciences, The University of Queensland. doi:10.14264/uql.2016.784

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Author Lee, Kah Meng
Thesis Title The role of synaptic laminins-α4 and -β2 in maturation and maintenance of the neuromuscular synapse
School, Centre or Institute School of Biomedical Sciences
Institution The University of Queensland
DOI 10.14264/uql.2016.784
Publication date 2016-09-26
Thesis type PhD Thesis
Supervisor Peter Noakes
Nickolas Lavidis
Total pages 206
Language eng
Subjects 1116 Medical Physiology
1109 Neurosciences
Formatted abstract
The proper development, maturation and maintenance of the neuromuscular junction (NMJ) are critical for ensuring efficient neurotransmission. Proteins found within the basal lamina of the synaptic cleft, namely the laminins family, are heavily involved in maintaining normal structure and function of the NMJ. In particular, individual laminin chains such as laminin-β2, -α4 and -α5 play essential roles in organisation and maintenance of the NMJ. These laminin chains interact together to form three synapse specific laminin heterotrimers; laminin-221 (α2β2γ1), laminin-421 (α4β2γ1) and laminin-521 (α5β2γ1).

Laminin-β2 is the common laminin chain found in each of these synapse specific laminin heterotrimers. In-vitro it has been shown to play a key role in the organisation of the presynaptic elements at the NMJ via its interaction and clustering of the N- and P/Q-type voltage-gated calciums (VGCCs). During development of the NMJ, both N- and P/Q-type VGCCs are involved in mediating neurotransmitter release. As the NMJ matures, P/Q-type becomes the dominant channel involved in release while N-type takes on the role of fine-tuning calcium influx. Laminin-α4, on the other hand, ensures proper alignment of presynaptic active zones to postjunctional folds at the NMJ, with its loss resulting in misalignment of these specialisations. Furthermore, this laminin chain has been shown to be involved in the maintenance of the NMJ with the observation of premature ageing features at 6 months of age (6MO) in laminin-α4 deficient mice (lama4-/-), which are commonly observed at NMJs from 18 months old (18MO) and onwards. Altered laminins expression such as laminin-α4 has been noted at the NMJ of hind-limb muscles from human amytrophic lateral sclerosis (ALS) donors. ALS is a neurodegenerative disease which is characterised with the dying of motor neurons and degeneration of the NMJ. This finding suggests a possible link between altered laminin expression at the NMJ and the progression of NMJ degeneration in ALS.

This study examined the role of laminin-β2 in maturation of the NMJ at both pre- and postsynaptic regions utilising immunohistochemical and electrophysiological methods. Immunohistochemical results showed retained clusters of N-type VGCCs without upregulation of P/Q-type VGCCs clusters at mature NMJs of laminin-β2 deficient mice (lamb2-/-), suggesting a failure in switching from N- to P/Q-type VGCCs during NMJ maturation. Immature postsynaptic endplates were also noted at lamb2-/- NMJs by postnatal day 35 (P35) suggesting halted postsynaptic maturation of the NMJ. Functionally, no improvement in transmission was noted from postnatal day 21 (P21) to P35, with maintained lower frequency of spontaneous release, decreased quantal content and higher failures in evoked responses. Next, this study investigated the role of laminin-α4 in maintenance of the NMJ from adulthood to ageing. Functionally, lama4-/- NMJs displayed perturbed transmission properties that were non-progressive from 3 months old (3MO) young adult to 18-22MO aged NMJs. Similar trends were also observed in their hind-limb grip force, with lama4-/- mice consistently displaying weaker grip force at all ages investigated. Importantly, findings demonstrated that aged wild-type NMJs showed a decline in transmission properties to levels resembling that of lama4-/-. These alterations coincided with a decline in hind-limb grip force at 18-22MO wild-type. This finding was further strengthened by the change in laminin-α4 expression prior to any prominent decline in neurotransmission and hind-limb grip force observed in wild-type mice. The final study aimed to investigate the role of laminins at the NMJs of TDP43Q331K, a mouse model of ALS. Immunohistochemical findings found mislocalised and absent expression of laminin-α4 at the NMJs of TDP43Q331K during presymptomatic stage (3MO) which coincided with a drop in transmission properties such as lower quantal content, higher intermittence and decreased frequency in spontaneous release. Furthermore, altered innervation patterns such as polyinnervation, thinning and swollen axons as well as partial denervation were also noted in these NMJs.

In conclusion, this thesis has identified the significant roles laminins-α4 and -β2 play at the NMJ during development, maturation and maintenance. Results suggest that laminin-β2 is not only an important regulator of the presynaptic maturation through the switching of VGCCs and clustering of P/Q-type VGCCs, but also equally important for maturation of the postsynaptic apparatus. I propose that laminin-β2 is important for the maturation of each component at the NMJ. The altered responses in transmission properties presented by aged wild-type NMJs which resembled adult lama4-/-, preceded by altered expression of laminin-α4 chain, strongly suggest that laminin-α4 is not only important for maintaining proper alignment of pre- to postsynaptic NMJ, but also essentially important for maintaining a healthy adult NMJ. Finally, I observed early alterations in expression of laminin-α4 at the NMJs in diseased mouse model of ALS prior to any appearance of neuromotor impairment, suggesting a potential involvement of laminins in NMJ degeneration associated with neuromuscular disorders such as ALS.
Keyword Neuromuscular junction
Laminins
Voltage-gated calcium channels
Neurotransmission
Maturation
Ageing
Maintenance
Amyotrophic lateral sclerosis

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Created: Tue, 20 Sep 2016, 19:45:59 EST by Kah Meng Lee on behalf of Learning and Research Services (UQ Library)