Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Altman, Topaz; Maimon, Roy; Ionescu, Ariel; Pery, Tal Gradus; Perlson, Eran

doi:10.3791/60993

Cited by 8 publications

(6 citation statements)

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“…1:1000, goat anti mouse 594, Invitrogen), washed and mounted with ProLong Gold (Life Technologies). 2, where 7mm-wells MFC were punched for spinal cord explant culture 59 .…”

Section: Methodsmentioning

confidence: 99%

Axonal TDP-43 condensates drive neuromuscular junction disruption through inhibition of local synthesis of nuclear encoded mitochondrial proteins

et al. 2021

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Mislocalization of the predominantly nuclear RNA/DNA binding protein, TDP-43, occurs in motor neurons of ~95% of amyotrophic lateral sclerosis (ALS) patients, but the contribution of axonal TDP-43 to this neurodegenerative disease is unclear. Here, we show TDP-43 accumulation in intra-muscular nerves from ALS patients and in axons of human iPSC-derived motor neurons of ALS patient, as well as in motor neurons and neuromuscular junctions (NMJs) of a TDP-43 mislocalization mouse model. In axons, TDP-43 is hyper-phosphorylated and promotes G3BP1-positive ribonucleoprotein (RNP) condensate assembly, consequently inhibiting local protein synthesis in distal axons and NMJs. Specifically, the axonal and synaptic levels of nuclear-encoded mitochondrial proteins are reduced. Clearance of axonal TDP-43 or dissociation of G3BP1 condensates restored local translation and resolved TDP-43-derived toxicity in both axons and NMJs. These findings support an axonal gain of function of TDP-43 in ALS, which can be targeted for therapeutic development.

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“…1:1000, goat anti mouse 594, Invitrogen), washed and mounted with ProLong Gold (Life Technologies). 2, where 7mm-wells MFC were punched for spinal cord explant culture 59 .…”

Section: Methodsmentioning

confidence: 99%

Axonal TDP-43 condensates drive neuromuscular junction disruption through inhibition of local synthesis of nuclear encoded mitochondrial proteins

et al. 2021

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“…The axonal transport toolkit is continuously expanding 28 , 29 , and ex vivo protocols have been developed to assess transport dynamics using cultured mouse ventral horn explants 49 or excised mouse nerve-muscle preparations 50 .…”

Section: Discussionmentioning

confidence: 99%

Expanding the Toolkit for <em>In Vivo</em> Imaging of Axonal Transport

Tosolini

Villarroel‐Campos

Schiavo

et al. 2021

JoVE

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Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative. This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.1. Pre-surgery preparation 1. Express H C T (H C T 441 , residues 875-1315) fused to an improved cysteine-rich tag in bacteria as a glutathione-S-transferase fusion protein as per 45 .

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“… a Rabieh et al, 2016; b Uzel et al, 2016; Osaki et al., 2018; c Uzel et al, 2016; d Machado et al, 2019; e Santhanam et al, 2018; f Lu et al, 2012; Altman et al., 2020; g Nijssen et al, 2018; h Lee et al, 2015.…”

Section: Als‐on‐a‐chip: Dissection Of Disease Mechanisms and Therapy Developmentmentioning

confidence: 99%

“…Finally, mitochondrial density is elevated at the NMJ (Ly & Verstreken, 2006) and NMJ formation leads to the accumulation of mitochondria at the NMJ, which might be relevant for motor neuron function (Altman et al., 2019). A detailed protocol for tracking and analyzing axonal transport of fluorescently labeled organelles in motor neurons has been published, including a simplified microfluidic chip manual (Altman et al., 2020). This method will be valuable for studying the effect of impaired mitochondrial transport on NMJ function.…”

Section: Als‐on‐a‐chip: Dissection Of Disease Mechanisms and Therapy Developmentmentioning

confidence: 99%

Neuromuscular junction‐on‐a‐chip: ALS disease modeling and read‐out development in microfluidic devices

Jongh

Spijkers

Pasteuning-Vuhman

et al. 2021

Journal of Neurochemistry

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Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Cited by 8 publications

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Axonal TDP-43 condensates drive neuromuscular junction disruption through inhibition of local synthesis of nuclear encoded mitochondrial proteins

Axonal TDP-43 condensates drive neuromuscular junction disruption through inhibition of local synthesis of nuclear encoded mitochondrial proteins

Expanding the Toolkit for <em>In Vivo</em> Imaging of Axonal Transport

Neuromuscular junction‐on‐a‐chip: ALS disease modeling and read‐out development in microfluidic devices

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