Binding of beta-bungarotoxin to synaptic membrane fractions of chick brain.

AbstractInvestigations of the human neuromuscular junction (NMJ) have predominately utilised experimental animals, model organisms, or monolayer cell cultures that fail to represent the physiological complexity of the synapse. Consequently, there remains a paucity of data regarding the development of the human NMJ and a lack of systems that enable investigation of the motor unit. This work addresses this need, providing the methodologies to bioengineer 3D models of the human motor unit. Spheroid culture of iPSC derived motor neuron progenitors augmented the transcription of OLIG2, ISLET1 and SMI32 motor neuron mRNAs ~ 400, ~ 150 and ~ 200-fold respectively compared to monolayer equivalents. Axon projections of adhered spheroids exceeded 1000 μm in monolayer, with transcription of SMI32 and VACHT mRNAs further enhanced by addition to 3D extracellular matrices in a type I collagen concentration dependent manner. Bioengineered skeletal muscles produced functional tetanic and twitch profiles, demonstrated increased acetylcholine receptor (AChR) clustering and transcription of MUSK and LRP4 mRNAs, indicating enhanced organisation of the post-synaptic membrane. The number of motor neuron spheroids, or motor pool, required to functionally innervate 3D muscle tissues was then determined, generating functional human NMJs that evidence pre- and post-synaptic membrane and motor nerve axon co-localisation. Spontaneous firing was significantly elevated in 3D motor units, confirmed to be driven by the motor nerve via antagonistic inhibition of the AChR. Functional analysis outlined decreased time to peak twitch and half relaxation times, indicating enhanced physiology of excitation contraction coupling in innervated motor units. Our findings provide the methods to maximise the maturity of both iPSC motor neurons and primary human skeletal muscle, utilising cell type specific extracellular matrices and developmental timelines to bioengineer the human motor unit for the study of neuromuscular junction physiology.

Download Full-text

Regulation of Endogenous Phosphorylation of Specific Proteins in Synaptic Membrane Fractions from Rat Brain by Adenosine 3′:5′-Monophosphate

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)43227-4 ◽

1973 ◽

Vol 248 (23) ◽

pp. 8295-8305 ◽

Cited By ~ 11

Author(s):

Tetsufumi Ueda ◽

Hiroo Maeno ◽

Paul Greengard

Keyword(s):

Rat Brain ◽

Synaptic Membrane ◽

Specific Proteins

Download Full-text

Immunochemical characterization of brain synaptic membrane glutamate-binding proteins.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)46207-7 ◽

1990 ◽

Vol 265 (27) ◽

pp. 16195-16204 ◽

Cited By ~ 2

Author(s):

M J Eaton ◽

J W Chen ◽

K N Kumar ◽

Y Cong ◽

E K Michaelis

Keyword(s):

Binding Proteins ◽

Synaptic Membrane ◽

Immunochemical Characterization ◽

Glutamate Binding

Download Full-text

L-glutamate stimulation of Na+ efflux from brain synaptic membrane vesicles.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)68745-4 ◽

1981 ◽

Vol 256 (19) ◽

pp. 10084-10087

Author(s):

H.H. Chang ◽

E.K. Michaelis

Keyword(s):

Membrane Vesicles ◽

Synaptic Membrane ◽

Na Efflux ◽

Stimulation Of

Download Full-text

Kinesin Mutations Cause Motor Neuron Disease Phenotypes by Disrupting Fast Axonal Transport in Drosophila

Genetics ◽

10.1093/genetics/144.3.1075 ◽

1996 ◽

Vol 144 (3) ◽

pp. 1075-1085 ◽

Cited By ~ 14

Author(s):

Daryl D Hurd ◽

William M Saxton

Keyword(s):

Motor Neuron ◽

Axonal Transport ◽

Action Potentials ◽

Light And Electron Microscopy ◽

Synaptic Membrane ◽

Slow Axonal Transport ◽

Fast Axonal Transport ◽

Motor Neuron Diseases ◽

Transmitter Secretion ◽

Axon Terminals

Abstract Previous work has shown that mutation of the gene that encodes the microtubule motor subunit kinesin heavy chain (Khc) in Drosophila inhibits neuronal sodium channel activity, action potentials and neurotransmitter secretion. These physiological defects cause progressive distal paralysis in larvae. To identify the cellular defects that cause these phenotypes, larval nerves were studied by light and electron microscopy. The axons of Khc mutants develop dramatic focal swellings along their lengths. The swellings are packed with fast axonal transport cargoes including vesicles, synaptic membrane proteins, mitochondria and prelysosomal organelles, but not with slow axonal transport cargoes such as cytoskeletal elements. Khc mutations also impair the development of larval motor axon terminals, causing dystrophic morphology and marked reductions in synaptic bouton numbers. These observations suggest that as the concentration of maternally provided wild-type KHC decreases, axonal organelles transported by kinesin periodically stall. This causes organelle jams that disrupt retrograde as well as anterograde fast axonal transport, leading to defective action potentials, dystrophic terminals, reduced transmitter secretion and progressive distal paralysis. These phenotypes parallel the pathologies of some vertebrate motor neuron diseases, including some forms of amyotrophic lateral sclerosis (ALS), and suggest that impaired fast axonal transport is a key element in those diseases.

Download Full-text