Researchers from La Trobe University in Australia and the Paul Langerhans Institute Dresden, partner in the German Center for Diabetes Research (DZD e.V.), in Germany have identified that the protein Atp6ap2 is essential for the correct functioning of pancreatic beta cells.
When this protein was switched off in the beta cells of mice their blood glucose levels dramatically increased.
The results of this international research project, to which the Max Delbrück Center for Molecular Medicine in Berlin, Germany also contributed, have now been published in the renowned journal Proceedings of the National Academy of Sciences (PNAS).
One in eleven adults worldwide suffer from diabetes and the number of diabetes patients is rising rapidly. People with type-1 diabetes lack insulin, the hormone that regulates blood glucose.
Insulin is produced by specialised cells in the pancreas known as beta cells.
When these cells lose their function or are mistakenly destroyed, the body loses its ability to make insulin leading to diabetes and to patient’s life-long requirement for insulin injections.
Researchers from La Trobe University in Australia and the Paul Langerhans Institute Dresden in Germany have identified that a protein called Atp6ap2 is essential for the correct functioning of beta cells.
“When we switched off this protein in the beta cells of mice their blood glucose levels dramatically increased,” explains Dr. Katrina Binger, lecturer in the department of Biochemistry and Genetics at La Trobe University and first author of the study.
“On closer inspection, we found this was because Atp6ap2-deficient beta cells had become swollen with large bubbles or ‘vesicles,” which led to a loss of insulin in these cells and the onset of type 1 diabetes.”
To uncover the reason for the abnormal bubble formation the scientists used innovative imaging techniques to characterize their origin and measure their acidity.
This analysis showed that removal of Atp6ap2 lead to a constipation in junk-removal processes, suggesting that Atp6ap2 is required for the transport of vesicles and/or insulin-containing granules within beta cells – its absence causing in the accumulation and abnormal growth of the bubbles.
“Based on these findings, we are now curious to investigate what molecules Atp6ap2 interacts within beta cells, as this may give further insight into the causes of beta cell failure and diabetes,” says Prof. Dr. Andreas Birkenfeld, senior author of the study from the University Study Center of Metabolic Disease at the University Hospital Dresden and research group leader at the Paul Langerhans Institute Dresden of Helmholtz Zentrum München at the University Hospital and Faculty of Medicine Carl Gustav Carus of TU Dresden.
Atp6ap2 is also known as the (pro)renin receptor and was only a few years ago the focus of cardiovascular research as it was proposed to be a drug target for high blood pressure.
However, in the meantime, it has become clear that Atp6ap2 is essential for the proper functioning of many different cell types.
Commenting on the findings, Dr. Binger concludes: “Our study contributes to a growing body of evidence that Atp6ap2 is important for the correct functioning of our cells. Here we show that without this protein beta cells fail, resulting in the onset of type-1 diabetes.
It is simply too dangerous to block it.”
Rapidly progressing cerebral atrophy in a patient carrying an ATP6AP2 splice site variant.
We identified a de novo intronic ATP6AP2 variant in a boy with XLID and fulminant early postnatal neurodegeneration (patient 1). The patient was diagnosed at the age of 2 weeks with seizures and mild facial dysmorphism, with a short forehead and bitemporal narrowing. At 1 month his occipito-frontal head circumference was at the mean, but it decreased to ≤1.5 standard deviations (SDs) at 6 months and ≤2 SDs at 3 years. He developed intractable seizures and generalized limb spasticity. Sequential MRI at 5 weeks, 9 months, and 28 months of age showed rapidly decreasing cortical gray and white matter volumes, a thin, poorly developing corpus callosum, and myelination deficits (Figure 1A). Candidate gene sequencing revealed a deletion of 2 conserved nucleotides in intron 3 in ATP6AP2 (c.301-11_301-10delTT) (Figure 1, B and C), a variant not present in genome databases of normal genetic variation in human populations. Furthermore, Sanger sequencing of flanking exons and introns (3–5) and the putative promoter region of ATP6AP2 did not reveal additional abnormalities. Human Splicing Finder (20) analysis predicted that the deletion disrupts a branch point motif (CTCTTAA) at position c.301-14 expected to increase exon 4 skipping. Accordingly, reverse transcription PCR (RT-PCR) of patient fibroblasts showed 20% full-length ATP6AP2 (fl-ATP6AP2) and 80% ATP6AP2Δe4 transcripts (Figure 1, D and E).
Figure 1Fulminant neurodegeneration in patient carrying ATP6AP2 (c.301-11_301-10delTT) variant.
(A) Sequential brain MRI of patient 1 carrying the ATP6AP2 (c.301-11_301-10delTT) variant at 5 weeks, 9 months, and 28 months of age. Sagittal (left) and axial (right) T1-weighted MR images at 5 weeks showed a thin, poorly developing corpus callosum (arrow) and diffuse volume loss of the brain parenchyma, enlarged ventricles, and prominent sulci. At 9 months the MRI revealed delayed myelination, a persistently thin hypoplastic corpus callosum (arrow), and parenchymal volume loss. At 28 months the MRI showed a markedly thin corpus callosum (arrow), minimal myelination throughout the brain, and significant parenchymal gray and white matter volume losses. (B) ATP6AP2 genomic organization and position of c.301-11_301-10delTT variant. Red arrows: primers used for RT-PCR. (C) Chromatogram of genomic DNA sequencing shows TT deletion in ATP6AP2 in the affected individual. (D) Chromatogram of cDNA sequencing shows exon 4 deletion. (E) RT-PCR analysis of patient fibroblast with fl-ATP6AP2 and a shorter product representing approximately 80% of ATP6AP2 transcripts. Lane 1, patient; lane 2, control; lane 3, H2O.
In addition, we performed MRI on a patient from the previously reported family (patient 2, proband IV-18; ref. 18) diagnosed with seizures and moderate mental retardation carrying ATP6AP2 variant (c.321C>T, p.D107D; Figure 2I) resulting in about 50% ATP6AP2Δe4 transcripts (12). MRI at the age of 15 revealed an abnormally thin corpus callosum and diffuse volume loss of cortical gray and white matter, although to a substantially lesser degree than in patient 1 (Figure 2, A–H). These results suggested neurodevelopmental deficits and/or neurodegenerative processes that prominently affect cortical regions in these patients.
Figure 2Cerebral atrophy in a patient carrying the ATP6AP2 c.321C>T (p.D107D) variant.
(A–H) Brain MRI of the 15-year-old male patient carrying the c.321C>T variant in exon 4 (OMIM 300423) and of an age-matched normal subject. Patient axial T2-weighted MR brain images (A and B) and normal subject brain images (E and F) for comparison. Patient coronal T2-weighted MR image (C) and normal subject (G). Patient MR images (A–C) exhibit diffuse parenchymal volume loss of gray and white matter. This is observed in the white matter tracts that travel adjacent to the prominently enlarged lateral ventricles (white arrows), as well as the prominent sulci (white arrowheads) in the cerebral hemispheres and cerebellum. Supratentorial and infratentorial parenchymal volume loss involves the cerebral hemispheres and cerebellum symmetrically. Sagittal T1-weighted MR brain image (D) compared with normal subject (H) demonstrates significantly reduced white matter volume of the corpus callosum (curved white arrows). There is also significant white matter volume loss in the patient cerebellum (D) with increased cerebrospinal fluid (asterisk) in the prominent posterior fossa mega cisterna magna. (I) ATP6AP2 genomic locus and positions of variant c.321C>T (p.D107D). (J and K) Schematics of ATP6AP2 and ATP6AP2Δe4.
Atp6ap2 is required for cortical development.
We therefore studied neural Atp6ap2 functions in cortical development. We first analyzed its cortical expression in embryonic day 12 (E12) mice. Atp6ap2 was detectable throughout the developing cortex with notable apical enrichment in radial glial cells (RGCs) along the ventricular surface (Figure 3B).
Characteristically RGCs are polarized, displaying a specialized apical membrane domain separated from the basolateral domain by adherens junctions. Apical-basal polarity is crucial for the distribution of cell fate determinants during RGC mitosis to balance the generation of progenitors and neurons (21).
The subcellular distribution of Atp6ap2 therefore may suggest its involvement in RGC polarity and ultimately cell fate choice, consistent with recent data in retinal progenitors, suggesting that Atp6ap2 interacts with polarity complex protein Par3 (partitioning defective 3 homolog) and that Atp6ap2 knockout disrupts the development of retinal lamination (22).
More information: Katrina J. Binger et al. Atp6ap2 deletion causes extensive vacuolation that consumes the insulin content of pancreatic β cells, Proceedings of the National Academy of Sciences (2019). DOI: 10.1073/pnas.1903678116
Journal information: Proceedings of the National Academy of Sciences
Provided by La Trobe University