Dataset Information

Microphysiological 3D model of amyotrophic lateral sclerosis (ALS) from human iPS-derived muscle cells and optogenetic motor neurons.

ABSTRACT: Amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease involving loss of motor neurons (MNs) and muscle atrophy, still has no effective treatment, despite much research effort. To provide a platform for testing drug candidates and investigating the pathogenesis of ALS, we developed an ALS-on-a-chip technology (i.e., an ALS motor unit) using three-dimensional skeletal muscle bundles along with induced pluripotent stem cell (iPSC)-derived and light-sensitive channelrhodopsin-2-induced MN spheroids from a patient with sporadic ALS. Each tissue was cultured in a different compartment of a microfluidic device. Axon outgrowth formed neuromuscular junctions on the muscle fiber bundles. Light was used to activate muscle contraction, which was measured on the basis of pillar deflections. Compared to a non-ALS motor unit, the ALS motor unit generated fewer muscle contractions, there was MN degradation, and apoptosis increased in the muscle. Furthermore, the muscle contractions were recovered by single treatments and cotreatment with rapamycin (a mechanistic target of rapamycin inhibitor) and bosutinib (an Src/c-Abl inhibitor). This recovery was associated with up-regulation of autophagy and degradation of TAR DNA binding protein-43 in the MNs. Moreover, administering the drugs via an endothelial cell barrier decreased the expression of P-glycoprotein (an efflux pump that transports bosutinib) in the endothelial cells, indicating that rapamycin and bosutinib cotreatment has considerable potential for ALS treatment. This ALS-on-a-chip and optogenetics technology could help to elucidate the pathogenesis of ALS and to screen for drug candidates.

SUBMITTER: Osaki T

PROVIDER: S-EPMC6179377 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Microphysiological 3D model of amyotrophic lateral sclerosis (ALS) from human iPS-derived muscle cells and optogenetic motor neurons.

Osaki Tatsuya T Uzel Sebastien G M SGM Kamm Roger D RD

Science advances 20181010 10

Amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease involving loss of motor neurons (MNs) and muscle atrophy, still has no effective treatment, despite much research effort. To provide a platform for testing drug candidates and investigating the pathogenesis of ALS, we developed an ALS-on-a-chip technology (i.e., an ALS motor unit) using three-dimensional skeletal muscle bundles along with induced pluripotent stem cell (iPSC)-derived and light-sensitive channelrhodopsin- ...[more]

PMID: 30324134

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Project description:BACKGROUND:Amyotrophic lateral sclerosis (ALS) represents a devastating, progressive, heterogeneous, and the most common motor neuron (MN) disease. To date, no cure has been available for the condition. Studies with transgenic mice have yielded significant results that help us understand the underlying mechanisms of ALS. Nonetheless, none of more than 30 large clinical trials over the past 20 years proved successful, which led some researchers to challenge the validity of the preclinical models. METHODS:Human-induced pluripotent cells (iPSCs) were established by introducing Sendai virus into fibroblast cells. We established TDP-43 HES by inserting CAG-TDP43 (G298S) cassette or the CAG-EGFP cassette into PPP1R12C-locus of human embryonic stem cells (ESC, H9) by TALEN-mediated homologous recombination. iPSCs or HESC were differentiated to motor neurons and non-motor neuron as control. Relevant biomarkers were detected in different differentiated stages. TDP-43 aggregates, neurofilament, and mitochondria analyses were performed. RESULTS:In this study, using iPSCs-derived human MN from an ALS patient with a TDP43 G298S mutation and two sporadic ALS patients, we showed that both sporadic and familial ALS were characterized by TDP-43 aggregates in the surviving MN. Significantly higher neurofilament (NF) inclusion was also found in ALS MN compared with wild-type (WT) GM15 controls (P?<?0.05). The neurite mitochondria density was significantly lower in ALS MN than that in the control MNs. Transgenesis of TDP-43 G298S into AAVS locus in human embryonic stem cells reproduced phenotype of patient-derived G289S MN. By challenging MNs with a proteasome inhibitor, we found that MNs were more vulnerable to MG132, with some accompanying phenotype changes, such as TDP43 translocation, NF inclusion, mitochondria distribution impairment, and activation of caspase3. CONCLUSIONS:Our results suggested that changes in TDP43 protein, NF inclusion, and distribution impairment of mitochondria are common early pathology both in familial and sporadic ALS. These findings will help us gain insight into the pathogenesis of the condition and screen relevant drugs for the disease.

Project description:Amyotrophic lateral sclerosis (ALS), a common late-onset neurodegenerative disease, is associated with fronto-temporal dementia (FTD) in 3-10% of patients. A mutation in CHMP2B was recently identified in a Danish pedigree with autosomal dominant FTD. Subsequently, two unrelated patients with familial ALS, one of whom also showed features of FTD, were shown to carry missense mutations in CHMP2B. The initial aim of this study was to determine whether mutations in CHMP2B contribute more broadly to ALS pathogenesis.Sequencing of CHMP2B in 433 ALS cases from the North of England identified 4 cases carrying 3 missense mutations, including one novel mutation, p.Thr104Asn, none of which were present in 500 neurologically normal controls. Analysis of clinical and neuropathological data of these 4 cases showed a phenotype consistent with the lower motor neuron predominant (progressive muscular atrophy (PMA)) variant of ALS. Only one had a recognised family history of ALS and none had clinically apparent dementia. Microarray analysis of motor neurons from CHMP2B cases, compared to controls, showed a distinct gene expression signature with significant differential expression predicting disassembly of cell structure; increased calcium concentration in the ER lumen; decrease in the availability of ATP; down-regulation of the classical and p38 MAPK signalling pathways, reduction in autophagy initiation and a global repression of translation. Transfection of mutant CHMP2B into HEK-293 and COS-7 cells resulted in the formation of large cytoplasmic vacuoles, aberrant lysosomal localisation demonstrated by CD63 staining and impairment of autophagy indicated by increased levels of LC3-II protein. These changes were absent in control cells transfected with wild-type CHMP2B.We conclude that in a population drawn from North of England pathogenic CHMP2B mutations are found in approximately 1% of cases of ALS and 10% of those with lower motor neuron predominant ALS. We provide a body of evidence indicating the likely pathogenicity of the reported gene alterations. However, absolute confirmation of pathogenicity requires further evidence, including documentation of familial transmission in ALS pedigrees which might be most fruitfully explored in cases with a LMN predominant phenotype.

Dataset Information

Microphysiological 3D model of amyotrophic lateral sclerosis (ALS) from human iPS-derived muscle cells and optogenetic motor neurons.

Publications

Microphysiological 3D model of amyotrophic lateral sclerosis (ALS) from human iPS-derived muscle cells and optogenetic motor neurons.

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