PIK3CA gain-of-function mutation in Schwann cells leads to severe neuropathy and aerobic glycolysis through a non-cell autonomous effect

Abstract

PIK3CA-related disorders are rare genetic disorders due to somatic gain-of-function mutations in PIK3CA during embryonic development, a pathway involved in cell growth, proliferation, and metabolism. Accumulating evidence from patients with PIK3CA-related disorders indicates that peripheral nerves are frequently affected, leading to severe neurological symptoms. However, the exact underlying mechanism of these disorders remains unclear. To address this, we developed a mouse model with a PIK3CA gain-of-function mutation specifically in Schwann cells, which successfully mirrored the clinical features observed in patients. In this model, we observed that PIK3CA-mutated cells communicate with neighboring healthy cells, such as adipocytes and hair follicles, through a unique crosstalk mechanism that triggers their growth, proliferation, and anagen phase expansion. Additionally, we demonstrated that PIK3CA mutation in peripheral nerves leads to a metabolic shift through glycolytic activation. We investigated the effects of alpelisib, an approved pharmacological inhibitor of PIK3CA, in the model. Early administration of alpelisib significantly improved the signs and symptoms in the mice. However, when treatment was delayed, its efficacy was diminished due to the drug's inability to penetrate the myelin sheath effectively. In summary, our study offers a valuable mouse model for studying PIK3CA-related neuropathy, uncovers a unique communication between healthy and affected tissues, and highlights the potential benefits of early pharmacological intervention using alpelisib.

Keywords: PIK3CA; nerve anomalies; somatic mutation.

Fig. 1.

A mouse model of PIK3CA-related peripheral nerve overgrowth. (A) Representative photography of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice at the age of P60. (B) Male and female body weights of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice. (C) Twelve-hour fasted glycemia in PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice. (D) Kaplan–Meier survival curves of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice. (E) Open field functional test and evaluation of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice at the age of P21. (F) Open field functional test and evaluation of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice at the age of P90. (G) Coronal whole-body T2-weighted MRI of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice. Neurolemma volume quantification. (H) Representative pictures and semithin sections of sciatic nerves of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice at P90 with nerve section area quantification. (I) Semithin sections of sciatic nerves of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice at P90 with myelinated fibers and density quantification (myelinated fibers on area). (J) Western blot and quantification of p110, AKT phosphorylation at Ser⁴⁷³ and S6RP in sciatic nerves of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice. (K) Quantification of GFP-positive Schwann cell area of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice using Amnis ImageStream. (L) Axial T2-weighted MRI of the head and quantification of the skin thickness of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice aged of P90. (M) Representative H&E staining of skin section of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice aged of P90. Arrows indicate hair follicles. (N) Quantification of the different skin layers of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice aged of P90. (O) Representative H&E staining in skin biopsies from healthy controls and in patients with PIK3CA-related nerve anomalies. Arrows indicate hair follicles.

Fig. 2.

PI3KCA gain-of-function mutations in Schwann cells activate the AKT pathway in surrounding adipocytes and hair follicles. (A) Representative immunostaining of GFP in the skin of PIK3CA^WT-Cre− and PIK3CA^WT-Cre+ mice. (B) Flow cytometry of GFP and S100 costaining of cells isolated from the skin of PIK3CA^WT-Cre− and PIK3CA^WT-Cre+ mice. (C) Representative H&E staining of skin biopsies from healthy controls and patients with PIK3CA-related neuropathies. (D) In situ hybridization using BaseScope assay of PIK3CA H1047R probe in the skin of healthy controls and patients with PIK3CA-related nerve disorders. Arrows show pink dots of hybridization. (E) Quantification of the number of spots localized either in the hair follicle, adipose tissue, or nerves. (F) Representative immunostaining of P-AKT^Thr308 in the skin of healthy controls and patients with PIK3CA-related neuropathies and (G) Quantification.

Fig. 3.

PIK3CA^MPZ-Cre+ mice present severe endocrine disruption with features of aerobic glycolysis. (A) Insulin circulating levels in PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice. (B) Representative immunofluorescence costaining of GLUT1 and S100β in sciatic nerves of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice. (C) Representative immunofluorescence costaining of GLUT3 and S100β in sciatic nerves of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice. Testis was used as a positive control. (D) Representative immunofluorescence costaining of GLUT4 and Perilipin-1 in surrounding areas of the sciatic nerves of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice and quantification. (E) Graphic example of metabolite modification observed in the plasma of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice. AU: Arbitrary units. (F) 18F-fluorodeoxyglucose uptake in sciatic and brachial of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice. (G) Gamma scintigraphy of isolated sciatic nerves of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice.

Fig. 4.

Alpelisib improves PIK3CA^MPZ-Cre+mice outcome depending on their ages. (A) Kaplan–Meier survival curves of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice treated with vehicle, alpelisib starting at either P21 or P60. (B) Whole-body T2-weighted MRI at P90 of PIK3CA^MPZ-Cre−, PIK3CA^MPZ-Cre+ vehicle-treated, PIK3CA^MPZ-Cre+-treated with either alpelisib started at P21 or P60. Neurolemma volume quantification. (C) Semithin section analysis of sciatic nerves at P90 of PIK3CA^MPZ-Cre− and PIK3CA^MPZ-Cre+ mice treated with vehicle, alpelisib starting at either P21 or P60. The graphs show the quantification of area, myelinated fibers, density (myelinated fibers on area), and percentage of aberrant fibers on myelinated fibers. (D) Western blot and quantification of p110, AKT phosphorylation on residues Ser⁴⁷³ and S6RP in sciatic nerves of PIK3CA^MPZ-Cre−, PIK3CA^MPZ-Cre+ vehicle-treated, PIK3CA^MPZ-Cre+-treated mice with either alpelisib started at P21 or P60. (E) Representative immunofluorescence costaining of S100β and P-AKT^Thr308 in sciatic nerves of PIK3CA^MPZ-Cre−, PIK3CA^MPZ-Cre+ vehicle-treated, PIK3CA^MPZ-Cre+-treated mice with either alpelisib started at P21 or P60 (Upper). Representative immunofluorescence costaining of S100β and P-S6RP in sciatic nerves of PIK3CA^MPZ-Cre−, PIK3CA^MPZ-Cre+ vehicle-treated, PIK3CA^MPZ-Cre+-treated mice with either alpelisib started at P21 or P60 (Lower). (F) Western blot and quantification of p110, AKT phosphorylation at Ser⁴⁷³ and S6RP in isolated sciatic nerves of PIK3CA^MPZ-Cre− or PIK3CA^MPZ-Cre+ mice that were then treated ex vivo with either vehicle or alpelisib for 8 h. Sciatic nerves were collected at P7, P21, and P60. (G) Alpelisib measurements in plasma and tissues of PIK3CA^MPZ-Cre− or PIK3CA^MPZ-Cre+ mice treated with alpelisib started either at P10 or P60.