PLA2G6 and α-synuclein interaction in human RPE cells

Publication Date

July 2019

Location

LSU Health Medical Education Building

Document Type

Abstract

Start Date

26-7-2019 9:00 AM

End Date

26-7-2019 12:00 PM

Description

Phospholipase A2 group VI activity is critical for the survival of dopaminergic cells. Mutations in the Calmodulin binding site are proposed to induce a dysregulation of PLA2G6 activity and a progressive degeneration of dopaminergic neurons located in Substatia Nigra pars compacta (SNpc) thus causing Parkinson’s- like symptoms at early age (Paisan-Ruiz et al., 2009; Karkheiran et al., 2015; Zhou et al., 2016). Despite the importance of PLA2G6 in the abnormalities observed in idiopathic and familial PD, the specific function that determines its pathogenicity remains unclear and only a few of its products have been investigated, for example, DHA and Cardiolipin. To produce a model to study dysfunction of PLA2G6, we designed a morpholino oligo that protects the initial ATG codon in exon 2 (MO-ATG1) and forces the translation to start in a cryptic site at exon 4 (MO-ATG2). The result is a truncated PLA2G6 (tPLA2G6) that does not reside in the membrane as the normal long isoform of the protein. We tested the idea in retinal pigment epithelial (RPE) cells as a first step. Under the hypothesis that PLA2G6 activity dysregulation interferes with the survival and function of retinal pigment epithelial (RPE) cells, our goal was to test species of PLA2G6 synthesized in the presence of MO-ATG1 by the means of Real-time PCR, Western blot assay, and the changes induced by the toxic isoform tPLA2G6 using immunocytochemistry. We found that MO-ATG1 induced and increased in the PLA2G6 transcript, however, the levels of the protein were noticeably reduced. MO-ATG2 alone did not affect the levels of transcript or protein as predicted, but in combination with MO-ATG1, the reduction of both species was potentiated as well as the colocalization with α-synuclein suggesting a silencing of all the possible isoforms may induce accumulation of α-synuclein. The addition of BEL, a suicidal substrate that inhibits PLA2G6 activity, exacerbates the appearance of colocalization vesicles and signal of both PLA2G6 and α-synuclein in the perinuclear zone, suggesting an increased expression of both proteins. Together, these results show for the first time that 1) morpholino strategy used to silence PLA2G6 is a useful tool to model L-PLA2G6 dysregulation and 2) deficits in the activity of the enzyme induce colocalization of this protein with α-synuclein suggesting a possible mechanism of interaction. In future studies, these results will serve as bases to test PLA2G6 dysregulation and interaction with α-synuclein in dopaminergic neurons and astroglial cells.

Comments

Mentor: Dr. Calandria, Neuroscience Center of Excellence

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Jul 26th, 9:00 AM Jul 26th, 12:00 PM

PLA2G6 and α-synuclein interaction in human RPE cells

LSU Health Medical Education Building

Phospholipase A2 group VI activity is critical for the survival of dopaminergic cells. Mutations in the Calmodulin binding site are proposed to induce a dysregulation of PLA2G6 activity and a progressive degeneration of dopaminergic neurons located in Substatia Nigra pars compacta (SNpc) thus causing Parkinson’s- like symptoms at early age (Paisan-Ruiz et al., 2009; Karkheiran et al., 2015; Zhou et al., 2016). Despite the importance of PLA2G6 in the abnormalities observed in idiopathic and familial PD, the specific function that determines its pathogenicity remains unclear and only a few of its products have been investigated, for example, DHA and Cardiolipin. To produce a model to study dysfunction of PLA2G6, we designed a morpholino oligo that protects the initial ATG codon in exon 2 (MO-ATG1) and forces the translation to start in a cryptic site at exon 4 (MO-ATG2). The result is a truncated PLA2G6 (tPLA2G6) that does not reside in the membrane as the normal long isoform of the protein. We tested the idea in retinal pigment epithelial (RPE) cells as a first step. Under the hypothesis that PLA2G6 activity dysregulation interferes with the survival and function of retinal pigment epithelial (RPE) cells, our goal was to test species of PLA2G6 synthesized in the presence of MO-ATG1 by the means of Real-time PCR, Western blot assay, and the changes induced by the toxic isoform tPLA2G6 using immunocytochemistry. We found that MO-ATG1 induced and increased in the PLA2G6 transcript, however, the levels of the protein were noticeably reduced. MO-ATG2 alone did not affect the levels of transcript or protein as predicted, but in combination with MO-ATG1, the reduction of both species was potentiated as well as the colocalization with α-synuclein suggesting a silencing of all the possible isoforms may induce accumulation of α-synuclein. The addition of BEL, a suicidal substrate that inhibits PLA2G6 activity, exacerbates the appearance of colocalization vesicles and signal of both PLA2G6 and α-synuclein in the perinuclear zone, suggesting an increased expression of both proteins. Together, these results show for the first time that 1) morpholino strategy used to silence PLA2G6 is a useful tool to model L-PLA2G6 dysregulation and 2) deficits in the activity of the enzyme induce colocalization of this protein with α-synuclein suggesting a possible mechanism of interaction. In future studies, these results will serve as bases to test PLA2G6 dysregulation and interaction with α-synuclein in dopaminergic neurons and astroglial cells.