Hemizygous variants in protein phosphatase 1 regulatory subunit 3F (PPP1R3F) are associated with a neurodevelopmental disorder characterized by developmental delay, intellectual disability and autistic features

Abstract

Protein phosphatase 1 regulatory subunit 3F (PPP1R3F) is a member of the glycogen targeting subunits (GTSs), which belong to the large group of regulatory subunits of protein phosphatase 1 (PP1), a major eukaryotic serine/threonine protein phosphatase that regulates diverse cellular processes. Here, we describe the identification of hemizygous variants in PPP1R3F associated with a novel X-linked recessive neurodevelopmental disorder in 13 unrelated individuals. This disorder is characterized by developmental delay, mild intellectual disability, neurobehavioral issues such as autism spectrum disorder, seizures and other neurological findings including tone, gait and cerebellar abnormalities. PPP1R3F variants segregated with disease in affected hemizygous males that inherited the variants from their heterozygous carrier mothers. We show that PPP1R3F is predominantly expressed in brain astrocytes and localizes to the endoplasmic reticulum in cells. Glycogen content in PPP1R3F knockout astrocytoma cells appears to be more sensitive to fluxes in extracellular glucose levels than in wild-type cells, suggesting that PPP1R3F functions in maintaining steady brain glycogen levels under changing glucose conditions. We performed functional studies on nine of the identified variants and observed defects in PP1 binding, protein stability, subcellular localization and regulation of glycogen metabolism in most of them. Collectively, the genetic and molecular data indicate that deleterious variants in PPP1R3F are associated with a new X-linked disorder of glycogen metabolism, highlighting the critical role of GTSs in neurological development. This research expands our understanding of neurodevelopmental disorders and the role of PP1 in brain development and proper function.

Keywords: PPP1R3F; X-linked; developmental delay; glycogen metabolism; intellectual disability; protein phosphatase 1; seizureautism.

Figure 1

Segregation of PPP1R3F variants in patient families. (A) Schematic diagram showing the domain structure of R3F and locations of identified variants from patient series. (B) Pedigrees and PPP1R3F variant segregation in the 13 families. m, mutant allele, −, normal allele, Y, Y chromosome. Asterisks denote females that have reported childhood seizures or seizure with learning disabilities.

Figure 2

R3F expression pattern and its role in regulating glycogen levels. (A) Immunoblotting detection of R3F in various mouse tissue lysates with GAPDH as a loading control. (B) Comparison of Ppp1r3f mRNA levels in mouse astrocytes and oligodendrocytes relative to neurons. The expression level in the neuron is set as 1. (C) Immunoblotting detection of R3F in brain lysates prepared from mice of indicated ages. Three mice were analyzed for each time point. (D) Quantification of relative R3F and GAPDH band intensities in D. ^*P < 0.05. (E) Relative glycogen levels (glycogen/protein) in WT and PPP1R3F KO cells at indicated hours after switching from high glucose medium to low glucose medium. ^*P < 0.05. (F) Relative glycogen levels (glycogen/protein) in WT and R3F KO cells at indicated minutes after switching from low glucose medium to high glucose medium. ^*P < 0.05. (G). Immunoblotting analysis of PP1, GS, R3F and β-actin levels in WT and KO H4 cells collected at indicated hours after switching from high glucose medium to low glucose medium.

Figure 3

COS-7 cells were co-expressed with mCherry-SEC61-C18 and Myc-R3F. Immunofluorescence staining of R3F was performed with an anti-Myc antibody. Cell nuclei were revealed with DAPI staining. Scale bar: 10 μm. The rightmost column shows magnified images from the boxed region of each variant. Experiments were repeated three times.

Figure 4

Functional characterization of R3F and identified variants. (A) Co-immunoprecipitation of R3F variants with PP1 and GS (upper panel). R3F variants were immunoprecipitated with the anti-Myc antibody from 293 T cells and immunoblotted with antibodies against PP1, GS, GS pSer641 and R3F. Protein levels in cell lysates are detected by immunoblotting and are shown in the lower panel. (B–E) Band intensities of immunoprecipitated PP1 (A), GS pSer641 (B), GS total (C) and R3F variants (E) were quantitated and plotted as ratios to the IgG heavy chain band. ^*P < 0.05 compared with WT.

Figure 5

Effects of R3F variants on glycogen metabolism in H4 KO cells. Relative glycogen levels (glycogen/protein) in H4 KO cells stably expressing the indicated R3F variants at 8 h after cells were switched from a high glucose medium to a low glucose medium. Each dot represents the value from an independent replicate. ^*P < 0.05 compared with WT.