Modeling Hypoxia-Induced Neuropathies Using a Fast and Scalable Human Motor Neuron Differentiation System

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.

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Sterol auto-oxidation adversely affects human motor neuron viability and is a neuropathological feature of amyotrophic lateral sclerosis

Scientific Reports ◽

10.1038/s41598-020-80378-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

James C. Dodge ◽

Jinlong Yu ◽

S. Pablo Sardi ◽

Lamya S. Shihabuddin

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Motor Neuron ◽

Cholesterol Homeostasis ◽

Cholesterol Oxidation ◽

Therapeutic Approaches ◽

Cellular Survival ◽

Sporadic Als ◽

Human Motor ◽

Als Patients ◽

Lateral Sclerosis

AbstractAberrant cholesterol homeostasis is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), a fatal neuromuscular disease that is due to motor neuron (MN) death. Cellular toxicity from excess cholesterol is averted when it is enzymatically oxidized to oxysterols and bile acids (BAs) to promote its removal. In contrast, the auto oxidation of excess cholesterol is often detrimental to cellular survival. Although oxidized metabolites of cholesterol are altered in the blood and CSF of ALS patients, it is unknown if increased cholesterol oxidation occurs in the SC during ALS, and if exposure to oxidized cholesterol metabolites affects human MN viability. Here, we show that in the SOD1G93A mouse model of ALS that several oxysterols, BAs and auto oxidized sterols are increased in the lumbar SC, plasma, and feces during disease. Similar changes in cholesterol oxidation were found in the cervical SC of sporadic ALS patients. Notably, auto-oxidized sterols, but not oxysterols and BAs, were toxic to iPSC derived human MNs. Thus, increased cholesterol oxidation is a manifestation of ALS and non-regulated sterol oxidation likely contributes to MN death. Developing therapeutic approaches to restore cholesterol homeostasis in the SC may lead to a treatment for ALS.

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Sonic hedgehog synergizes with the extracellular matrix protein vitronectin to induce spinal motor neuron differentiation

Development ◽

10.1242/dev.127.2.333 ◽

2000 ◽

Vol 127 (2) ◽

pp. 333-342 ◽

Cited By ~ 6

Author(s):

S. Pons ◽

E. Marti

Keyword(s):

Extracellular Matrix ◽

Motor Neuron ◽

Sonic Hedgehog ◽

Motor Neurons ◽

Neural Tube ◽

Matrix Protein ◽

Floor Plate ◽

Binding Domain ◽

Extracellular Matrix Protein ◽

Neuron Differentiation

Patterning of the vertebrate neural tube depends on intercellular signals emanating from sources such as the notochord and the floor plate. The secreted protein Sonic hedgehog and the extracellular matrix protein Vitronectin are both expressed in these signalling centres and have both been implicated in the generation of ventral neurons. The proteolytic processing of Sonic hedgehog is fundamental for its signalling properties. This processing generates two secreted peptides with all the inducing activity of Shh residing in the highly conserved 19 kDa amino-terminal peptide (N-Shh). Here we show that Vitronectin is also proteolitically processed in the embryonic chick notochord, floor plate and ventral neural tube and that this processing is spatiotemporally correlated with the generation of motor neurons. The processing of Vitronectin produces two fragments of 54 kDa and 45 kDa, as previously described for Vitronectin isolated from chick yolk. The 45 kDa fragment lacks the heparin-binding domain and the integrin-binding domain, RGD, present in the non-processed Vitronectin glycoprotein. Here we show that N-Shh binds to the three forms of Vitronectin (70, 54 and 45 kDa) isolated from embryonic tissue, although is preferentially associated with the 45 kDa form. Furthermore, in cultures of dissociated neuroepithelial cells, the combined addition of N-Shh and Vitronectin significantly increases the extent of motor neuron differentiation, as compared to the low or absent inducing capabilities of either N-Shh or Vitronectin alone. Thus, we conclude that the differentiation of motor neurons is enhanced by the synergistic action of N-Shh and Vitronectin, and that Vitronectin may be necessary for the proper presentation of the morphogen N-Shh to one of its target cells, the differentiating motor neurons.

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Peptide modified polymer poly (glycerol- dodecanedioate co-fumarate) for efficient control of motor neuron differentiation

Biomedical Materials ◽

10.1088/1748-6041/10/6/065013 ◽

2015 ◽

Vol 10 (6) ◽

pp. 065013 ◽

Cited By ~ 7

Author(s):

Xizi Dai ◽

Yen-Chih Huang ◽

Jared Leichner ◽

Madhvan Nair ◽

Wei-Chiang Lin ◽

...

Keyword(s):

Motor Neuron ◽

Neuron Differentiation ◽

Modified Polymer ◽

Efficient Control

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Loss of TBK1 activity leads to TDP-43 proteinopathy through lysosomal dysfunction in human motor neurons

10.1101/2021.10.11.464011 ◽

2021 ◽

Author(s):

Jin Hao ◽

Michael F Wells ◽

Gengle Niu ◽

Irune Guerra San Juan ◽

Francesco Limone ◽

...

Keyword(s):

Motor Neuron ◽

Neurodegenerative Disease ◽

Motor Neurons ◽

Inhibition Mechanism ◽

Loss Of Function ◽

Neurodegenerative Process ◽

Lysosomal Dysfunction ◽

Human Motor ◽

Lateral Sclerosis ◽

Lysosomal Acidification

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron loss accompanied by cytoplasmic localization of TDP-43 proteins and their insoluble accumulations. Haploinsufficiency of TBK1 has been found to associate with or cause ALS. However, the cell-autonomous mechanisms by which reduced TBK1 activity contributes to human motor neuron pathology remain elusive. Here, we generated a human cellular model harboring loss-of-function mutations of TBK1 by gene editing and found that TBK1 deficiency was sufficient to cause TDP-43 pathology in human motor neurons. In addition to its functions in autophagy, we found that TBK1 interacted with endosomes and was required for normal endosomal maturation and subsequent lysosomal acidification. Surprisingly, TDP-43 pathology resulted more from the dysfunctional endo-lysosomal pathway than the previously recognized autophagy inhibition mechanism. Restoring TBK1 levels ameliorated lysosomal dysfunction and TDP-43 pathology and maintained normal motor neuron homeostasis. Notably, using patient-derived motor neurons, we found that haploinsufficiency of TBK1 sensitized neurons to lysosomal stress, and chemical regulators of endosomal maturation rescued the neurodegenerative process. Together, our results revealed the mechanism of TBK1 in maintaining TDP-43 and motor neuron homeostasis and suggested that modulating endosomal maturation was able to rescue neurodegenerative disease phenotypes caused by TBK1 deficiency.

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