Loss and gain of Drosophila TDP-43 impair synaptic efficacy and motor control leading to age-related neurodegeneration by loss-of-function phenotypes
Danielle C. DiaperYoshitsugu AdachiBen SutcliffeDickon M. HumphreyChristopher ElliottAlan SteptoZoe N. LudlowLies Vanden BroeckPatrick CallaertsBart DermautAmmar Al‐ChalabiChristopher E. ShawIain M. RobinsonFrank Hirth
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Abstract:
Cytoplasmic accumulation and nuclear clearance of TDP-43 characterize familial and sporadic forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, suggesting that either loss or gain of TDP-43 function, or both, cause disease formation. Here we have systematically compared loss- and gain-of-function of Drosophila TDP-43, TAR DNA Binding Protein Homolog (TBPH), in synaptic function and morphology, motor control, and age-related neuronal survival. Both loss and gain of TBPH severely affect development and result in premature lethality. TBPH dysfunction caused impaired synaptic transmission at the larval neuromuscular junction (NMJ) and in the adult. Tissue-specific knockdown together with electrophysiological recordings at the larval NMJ also revealed that alterations of TBPH function predominantly affect pre-synaptic efficacy, suggesting that impaired pre-synaptic transmission is one of the earliest events in TDP-43-related pathogenesis. Prolonged loss and gain of TBPH in adults resulted in synaptic defects and age-related, progressive degeneration of neurons involved in motor control. Toxic gain of TBPH did not downregulate or mislocalize its own expression, indicating that a dominant-negative effect leads to progressive neurodegeneration also seen with mutational inactivation of TBPH. Together these data suggest that dysfunction of Drosophila TDP-43 triggers a cascade of events leading to loss-of-function phenotypes whereby impaired synaptic transmission results in defective motor behavior and progressive deconstruction of neuronal connections, ultimately causing age-related neurodegeneration.Keywords:
Loss function
Frontotemporal lobar degeneration
Immunological synapse
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Abstract The neuromuscular junction (NMJ) is the highly specialised peripheral synapse formed between lower motor neuron terminals and muscle fibres. Post-synaptic acetylcholine receptors (AChRs), which are found in high density in the muscle membrane, bind to acetylcholine released into the synaptic cleft of the NMJ, ultimately facilitating the conversion of motor action potentials to muscle contractions. NMJs have been studied for many years as a general model for synapse formation, development and function, and are known to be early sites of pathological changes in many neuromuscular diseases. However, information is limited on the diversity of NMJs in different muscles, whether muscle fibre type impacts NMJ morphology and growth, and the relevance of these parameters to neuropathology. Here, this crucial gap was addressed using a robust and standardised semi-automated workflow called NMJ-morph to quantify features of pre- and post-synaptic NMJ architecture in an unbiased manner. Five wholemount muscles from wild-type mice were dissected and compared at immature (post-natal day, P7) and early adult (P31-32) timepoints. Post-synaptic AChR morphology was found to be more variable between muscles than that of the motor neuron terminal and there were greater differences in the developing NMJ than at the mature synapse. Post-synaptic architecture, but not neuronal morphology or post-natal synapse growth, correlates with fibre type and is largely independent of muscle fibre diameter. Counter to previous observations, this study indicates that smaller NMJs tend to innervate muscles with higher proportions of fast twitch fibres and that NMJ growth rate is not conserved across all muscles. Furthermore, healthy pre- and post-synaptic NMJ morphological parameters were collected for five anatomically and functionally distinct mouse muscles, generating reference data that will be useful for the future assessment of neuromuscular disease models. Graphical Abstract
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Abstract Synaptic size, synaptic remodelling, polyneuronal innervation, and synaptic efficacy of neuromuscular junctions were studied as a function of growth in cutaneous pectoris muscles of postmetamorphic Rana pipiens . Recently metamorphosed frogs grew rapidly, and this growth was accompanied by hypertrophy of muscle fibers, myogenesis, and increases in the size and complexity of neuromuscular junctions. There were pronounced gradients in pre‐ and postsynaptic size across the width of the muscle, with neuromuscular junctions and muscle fibers near the medial edge being smaller than in more lateral regions. The incidence of polyneuronal innervation, measured physiologically and histologically, was also higher near the medial edge. Growth‐associated declines in all measures of polyneuronal innervation indicated that synapse elimination occurs throughout life. Electrophysiology also demonstrated regional differences in synaptic efficacy and showed that doubly innervated junctions have lower synaptic efficacy than singly innervated junctions. Repeated, in vivo observations revealed extensive growth and remodelling of motor nerve terminals and confirmed that synapse elimination is a slow process. It was concluded that some processes normally associated with embryonic development persist long into adulthood in frog muscles.
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Neuromuscular junction (NMJ) is the specialized chemical synapse that mediates the transmission of the electrical impulse running along motor neuron axons to skeletal muscle fibers. NMJ is the best characterized chemical synapse and its study along many years of research has provided most of the general knowledge of synapse development, structure and functionality. Electrophysiology is the most accurate experimental procedure to study NMJ physiology and it largely contributed to the elucidation of synaptic transmission basic principles. Many electrophysiological techniques have been developed to study NMJ physiology and physiopathology. In this paper, we describe an ex vivo tissue preparation for electrophysiology that can be applied to investigate nerve-muscle transmission functionality in mice. It is routinely used in our laboratory to study presynaptic neurotoxins, antitoxins, and to monitor NMJ degeneration and regeneration. This is a broadly applicable technique which can also be adopted to investigate alterations of NMJ activity in mouse models of neuromuscular diseases, including peripheral neuropathies, motor neuron disorders and myasthenic syndromes.
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Summary The neuromuscular junctions (NMJs) of postnatal rat soleus muscles were examined by immunohistochemical staining for S100, a marker of Schwann cells (SCs), and for protein gene product 9.5, a neuronal marker, to elucidate the involvement of SCs in synapse elimination. The morphological maturation of S100-immunoreactive terminal SCs at NMJs proceeded with the gradual increase in their number. The number of terminal SCs per NMJ was one or two at postnatal day (P) 7, reaching the adult number at P28, when it became three or four. Confocal laser scanning microscopic analysis of multi-innervated NMJs, whose number decreased between P7 and P14, revealed a change in the ratio between terminal SCs and axons with age. At P7, the ratio between axons and terminal SCs per NMJ was 52 : 1, which was exactly the reverse of that in adults, while at P14 this had changed to 2 : 2. A structural change appeared to occur at the same time at the preterminal region, this being prior to the establishment of a 1 : 1 relationship between axon and SC sheath which was detected at P14, with the 52 : 1 relationship seeming to occur at P7. Thus, synapse elimination seems to proceed, at least for one week, with the gradual loss of axons which are at different stages of maturation with respect to their spatial relationship with SCs. From our results it seems unlikely that SCs play an active role in selecting a single axon to survive.
Axon terminal
Schwann cell
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Frontotemporal lobar degeneration
Pathogenesis
Exome
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Reinnervation
Synaptogenesis
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Abstract During late embryonic and early postnatal development, synaptic connections are extensively modified so that some functional connections are weakened and eliminated from a neural circuit while others are strengthened and maintained. The mechanisms that underlie synapse elimination are beginning to be understood from studies of the neuromuscular junction. A recent paper (1) provides some intriguing insights into the role proteases may play in the developmental disassembly of neuromuscular synapses.
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Motor Endplate
Synaptic cleft
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Synapse formation
Developmental Biology
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