Literature DB >> 32550441

The autophagy gene product BEC-1 supports normal aging and neurodevelopment in Caenorhabditis elegans III.

Nicholas Ashley1, Andrea Holgado1.   

Abstract

Entities:  

Year:  2019        PMID: 32550441      PMCID: PMC7252317          DOI: 10.17912/micropub.biology.000101

Source DB:  PubMed          Journal:  MicroPubl Biol        ISSN: 2578-9430


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Homozygous (A-C) Representative fluorescent micrographs of D-type motor neuron expressing GFP in (A) control, (B) homozygous bec-1(ok691) mutant, and (C) heterozygous bec-1(ok691) mutant nematodes on day 5 of adulthood. (D) Quantification of D-type motor neuron cell bodies on days 0,1,3,5, and 7 of adulthood was recorded. Data plotted are mean ± 1 SEM, n=60. Statistical analysis was performed using a non-parametric Kruskal-Wallis (p< .001).

Description

The loss of autophagy function in the motor cortex has been associated with progression of neurodegenerative symptoms in Parkinson’s disease (Kaila and Lang 2015; Fahn et al. 2004). To analyze possible effects of the bec-1(ok691) mutation on neuronal density, we followed the transgene juIs76 as it produced GFP marked D-type motor neurons (Figure 1 A-C). Previous research has found lineage timing of GABAergic (VD) motor neuron differentiation in C. elegans to occur before animals reach adulthood (Jin et al., 1994). These studies show a delay in development of ventral D-type motor neurons in bec-1(ok691) homozygous mutants (Figure 1 D). Maturation and development of VD motor neurons to the levels of controls were seen on day 5 of adulthood in bec-1(ok691) homozygous mutants. However, our discovery in delayed lineage timing of VD motor neurons suggests a potential role of BEC-1 in neurodevelopment. This is consistent with findings in mouse models, where ortholog Beclin 1 plays an essential role in cell differentiation during development (Cecconi and Levine, 2008). Instead of observing rapid neurodegeneration of VD motor neurons, resulting from the bec-1(ok691) mutation, we observed a rapid decrease in lifespan (Ashley and Holgado, 2019) as VD motor neurons were differentiated. This conclusion should be considered as preliminary as we have not verified by an alternative line of investigation (e.g., a second allele or transgene rescue) that the observed phenotypes are specific to bec-1(ok691).There is additional evidence that suggests autophagy’s role in mechanisms of cell editing in early developmental stages of C. elegans (Di Bartolomeo et al., 2010).
Figure 1.

Homozygous (A-C) Representative fluorescent micrographs of D-type motor neuron expressing GFP in (A) control, (B) homozygous bec-1(ok691) mutant, and (C) heterozygous bec-1(ok691) mutant nematodes on day 5 of adulthood. (D) Quantification of D-type motor neuron cell bodies on days 0,1,3,5, and 7 of adulthood was recorded. Data plotted are mean ± 1 SEM, n=60. Statistical analysis was performed using a non-parametric Kruskal-Wallis (p< .001).

Methods

Synchronizing: Mixed stage nematodes grown on NGM plates at 20 °C were floated off using 1 mL of M9 reagent and collected in 1.5 mL tubes. Tubes containing animals were centrifuged at 9.3 × g for 1 minute. After centrifugation, the supernatant was discarded and the worm pellet was kept and treated with 1 mL of Alkaline Bleach (2.0% bleach (VWR), 0.5N NaOH) for 7 minutes at room temperature with occasional mixing. Once the 7-minute treatment concluded, bleached animals were centrifuged at 9. 3 × g for 2 minutes to collect eggs. Pelleted eggs were washed 3 times with 1 mL of M9 and centrifuged for 1 min. at 9.3 × g. After centrifugation, the supernatant was discarded and the pelleted eggs were suspended. Two drops of resuspended eggs were placed onto seeded NGM plates. Neuron Cell Body Count: Individuals were mounted on 2% agarose padded microscope slides in 2 drops of mineral oil. Using an Olympus fluorescent microscope (BX41), GFP positive neuronal cell bodies were counted on days 0,1, 3, 5, and 7 of adulthood. Neuron cell body count was reported as the average number of cell bodies per animal over time.

Reagents

Strains CZ1200 and VC517 were obtained from the C. elegans Genetics Center. CZ1200 contains the transgene juIs76[unc25p::GFP] which drives the expression of GFP in d-type motor neurons. Strain AMH50 was produced in our laboratory by crossing CZ1200 with VC517 bec-1(ok691)/nT1[qIs51]. AMH50 possess the balanced lethal mutation bec-1(ok691)/nT1[qIs51] and the transgene juIs76, {bec-1(ok691)IV/nT1[qIs51](IV;V);juIs76[unc-25p::GFP] II}.
  6 in total

Review 1.  The role of autophagy during development in higher eukaryotes.

Authors:  Sabrina Di Bartolomeo; Francesca Nazio; Francesco Cecconi
Journal:  Traffic       Date:  2010-10       Impact factor: 6.215

2.  The autophagy gene product BEC-1 supports normal aging and neurodevelopment in Caenorhabditis elegans I.

Authors:  Nicholas Ashley; Andrea Holgado
Journal:  MicroPubl Biol       Date:  2019-06-14

Review 3.  Parkinson's disease.

Authors:  Lorraine V Kalia; Anthony E Lang
Journal:  Lancet       Date:  2015-04-19       Impact factor: 79.321

4.  Levodopa and the progression of Parkinson's disease.

Authors:  Stanley Fahn; David Oakes; Ira Shoulson; Karl Kieburtz; Alice Rudolph; Anthony Lang; C Warren Olanow; Caroline Tanner; Kenneth Marek
Journal:  N Engl J Med       Date:  2004-12-09       Impact factor: 91.245

5.  Control of type-D GABAergic neuron differentiation by C. elegans UNC-30 homeodomain protein.

Authors:  Y Jin; R Hoskins; H R Horvitz
Journal:  Nature       Date:  1994 Dec 22-29       Impact factor: 49.962

Review 6.  The role of autophagy in mammalian development: cell makeover rather than cell death.

Authors:  Francesco Cecconi; Beth Levine
Journal:  Dev Cell       Date:  2008-09       Impact factor: 12.270

  6 in total
  1 in total

1.  The autophagy gene product BEC-1 supports normal aging and neurodevelopment in Caenorhabditis elegans: Integration.

Authors:  Nicholas Ashley; Andrea Holgado
Journal:  MicroPubl Biol       Date:  2019-06-15
  1 in total

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