A new impedance based approach to test the activity of recombinant protein – Semaphorins as a test case

Birger, Anastasya; Besser, Elazar; Reubinoff, Benjamin; Behar, Oded

doi:10.1016/j.ejcb.2015.06.006

Cited by 3 publications

(3 citation statements)

References 16 publications

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“…U87MG human glioblastoma cells, which express NRP1 ( Fig. 3-2 A ) and were used previously specifically in this assay ( Birger et al, 2015 ), were transfected to overexpress miR126-5p or miR142 as a control. One day after transfection, cells were resuspended and plated in xCELLigence multiwell electric plates.…”

Section: Resultsmentioning

confidence: 99%

miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non–Cell-Autonomous Mechanism in ALS

et al. 2018

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Axon degeneration and disruption of neuromuscular junctions (NMJs) are key events in amyotrophic lateral sclerosis (ALS) pathology. Although the disease's etiology is not fully understood, it is thought to involve a non–cell-autonomous mechanism and alterations in RNA metabolism. Here, we identified reduced levels of miR126-5p in presymptomatic ALS male mice models, and an increase in its targets: axon destabilizing Type 3 Semaphorins and their coreceptor Neuropilins. Using compartmentalized in vitro cocultures, we demonstrated that myocytes expressing diverse ALS-causing mutations promote axon degeneration and NMJ dysfunction, which were inhibited by applying Neuropilin1 blocking antibody. Finally, overexpressing miR126-5p is sufficient to transiently rescue axon degeneration and NMJ disruption both in vitro and in vivo. Thus, we demonstrate a novel mechanism underlying ALS pathology, in which alterations in miR126-5p facilitate a non–cell-autonomous mechanism of motor neuron degeneration in ALS.SIGNIFICANCE STATEMENT Despite some progress, currently no effective treatment is available for amyotrophic lateral sclerosis (ALS). We suggest a novel regulatory role for miR126-5p in ALS and demonstrate, for the first time, a mechanism by which alterations in miR126-5p contribute to axon degeneration and NMJ disruption observed in ALS. We show that miR126-5p is altered in ALS models and that it can modulate Sema3 and NRP protein expression. Furthermore, NRP1 elevations in motor neurons and muscle secretion of Sema3A contribute to axon degeneration and NMJ disruption in ALS. Finally, overexpressing miR126-5p is sufficient to transiently rescue NMJ disruption and axon degeneration both in vitro and in vivo.

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Section: Resultsmentioning

confidence: 99%

miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non–Cell-Autonomous Mechanism in ALS

et al. 2018

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“…To verify the activity of Sema3A, we utilized an impedance-based approach, which we recently developed 18 . In this system, cells are seeded onto specialized plates, which integrate with microelectronic sensor arrays.…”

Section: Resultsmentioning

confidence: 99%

ALS-related human cortical and motor neurons survival is differentially affected by Sema3A

et al. 2018

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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by cell death of upper and lower motor neurons (MNs). The cause of MN cell loss is not completely understood but involves both cell autonomous and non-cell autonomous mechanisms. Numerous molecules have been implicated to be involved in the death of MNs. One such candidate is semaphorin 3A (Sema3A). In ALS patients, Sema3A was shown to be significantly upregulated in the motor cortex and downregulated in the spinal cord. In the mouse, Sema3A was shown to be an axon repellent molecule for MNs. Sema3A could also induce death of different neuronal types that are also repelled by it, including sensory, sympathetic, retinal, and cortical neurons. In contrast, astrocyte-specific knockout of Sema3A results in motor neuron cell death, consistent with the idea that Sema3A is a survival factor for mouse motor neurons. Here, we tested the response of human cortical neurons and spinal cord MNs to Sema3A. We found that Sema3A enhances the survival of spinal cord MNs. In contrast, Sema3A reduces the survival of cortical neurons. Thus, both upregulation of Sema3A in the cortex, or downregulation in the spinal cord of ALS patients is likely to directly contribute to MNs cell loss in ALS patients.

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“…To test this possibility, we used a secreted version of the Sema4B ectodomain fused to the Fc of human IgG. We then added a concentration of 50 ng/ml of Sema4B-Fc based on our prior study ( Birger et al, 2015 ). As a control, we used 50 ng/ml of Fc-only protein cultured on pure neuronal cultures and monitored neuronal survival after 7 days.…”

Section: Resultsmentioning

confidence: 99%

Improved neuron protection following cortical injury in the absence of Semaphorin4B

Sweetat

Casden

Behar

2022

Front. Cell. Neurosci.

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Injury to the central nervous system induces neuronal cell death and astrogliosis, an astrocyte-mediated response that has both a beneficial and detrimental impact on surrounding neuronal cells. The circumstance however, in which astrogliosis improves neuronal survival after an injury is not fully characterized. We have recently shown that Semaphorin4B (Sema4B) in the cortex is mostly expressed by astrocytes, and in its absence, astrocyte activation after an injury is altered. Here we find that in Sema4B knockout mice, neuronal cell death is reduced; as a result, more neurons survive near the injury site. Sema4B protein applied directly to neurons does not affect neuronal survival. In contrast, survival of wild-type neurons is increased when plated on glial culture isolated from the Sema4B knockout mice, as compared to Sema4B heterozygous cultures. Furthermore, this increased survival is also observed with conditioned medium collected from glial cultures of Sema4B knockout mice compared to heterozygous mice. This indicates that the increased survival is glial cell-dependent and mediated by a secreted factor(s). Together, our results imply that following injury, the lack of Sema4B expression in glial cells improves neuronal survival either as a result of reduced toxic factors, or perhaps increased survival factors under these conditions.

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A new impedance based approach to test the activity of recombinant protein – Semaphorins as a test case

Cited by 3 publications

References 16 publications

miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non–Cell-Autonomous Mechanism in ALS

miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non–Cell-Autonomous Mechanism in ALS

ALS-related human cortical and motor neurons survival is differentially affected by Sema3A

Improved neuron protection following cortical injury in the absence of Semaphorin4B

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