Altered iPSC-derived neurons' sodium channel properties in subjects with Monge's disease

Abstract

Monge's disease, also known as chronic mountain sickness (CMS), is a disease that potentially threatens more than 140 million highlanders during extended time living at high altitudes (over 2500m). The prevalence of CMS in Andeans is about 15-20%, suggesting that the majority of highlanders (non-CMS) are rather healthy at high altitudes; however, CMS subjects experience severe hypoxemia, erythrocytosis and many neurologic manifestations including migraine, headache, mental fatigue, confusion, and memory loss. The underlying mechanisms of CMS neuropathology are not well understood and no ideal treatment is available to prevent or cure CMS, except for phlebotomy. In the current study, we reprogrammed fibroblast cells from both CMS and non-CMS subjects' skin biopsies into the induced pluripotent stem cells (iPSCs), then differentiated into neurons and compared their neuronal properties. We discovered that CMS neurons were much less excitable (higher rheobase) than non-CMS neurons. This decreased excitability was not caused by differences in passive neuronal properties, but instead by a significantly lowered Na(+) channel current density and by a shift of the voltage-conductance curve in the depolarization direction. Our findings provide, for the first time, evidence of a neuronal abnormality in CMS subjects as compared to non-CMS subjects, hoping that such studies can pave the way to a better understanding of the neuropathology in CMS.

Keywords: Na(+) channel; chronic mountain sickness; induced pluripotent stem cells; neurons.

Figure 1. Generation of iPSCs-derived neurons from fibroblasts

A) Morphology of human fibroblasts obtained from skin biopsies. B-D) Formation of iPSC colonies through reprogramming processes. E) Formation of EBs from iPSCs and F-G) rosettes were picked to generate NPCs. H) Morphology of induced neurons from NPCs. Scale bar = 200 μm unless otherwise stated.

Figure 2. Characterization of iPSCs, NPCs and neurons

A) Representative images of iPSCs expressing pluripotency markers including NANOG and TRA-1-81. B) Rosettes expressing neural precursor markers including PAX6 and SOX1. C) iPSCs-derived neurons expressing neuronal markers including MAP2 and TUJ1. Scale bar for A-C = 200 μm. D) Representative images of iPSCs-derived neurons from CMS subjects expressing glutamatergic neuronal markers including MAP2 and VGLUT1, Scale bar = 50 μm.

Figure 3. Characterization of iPSCs and neurons

A) No karyotypic abnormalities were observed in iPSC lines. B) Alkaline phosphatase staining of iPSCs. Scale bar = 200 μm. C) RT-PCR analysis of iPSC lines detected the transcripts of endogenous pluripotency embryonic stem cell markers. D) RT-PCR analysis of EBs detected the variety of differentiation markers for the three germ layers. E) Genomic RT-PCR revealed integration of all four retrovirus factors in iPSCs lines. F) The expression of transgenes was low or undetectable in the mRNA of iPSCs. Lane 1-6 was either iPSCs or EBs obtained from non-CMS (n = 3) and CMS (n = 3) subjects. Fibroblast cells (Fibro) were used as negative control, H9 cells and retrovirus-infected fibroblasts (F + OSKM) were used as positive control accordingly. G) Similar percentages of MAP2⁺/DAPI⁺ neurons and VGLUT1⁺/MAP2⁺ neurons observed in CMS and non-CMS neuronal populations. H) Similar neuronal differentiation and developmental stages between non-CMS and CMS neuronal cultures. Real time PCR results confirmed that neuronal cultures from both groups had a similar gene expression profile (p > 0.05).

Figure 4. Characterization of action potentials from 33.7 days old non-CMS neurons and 32.8 days old CMS neurons after differentiation

A) A representative image of neurons from CMS neurons for patch clamping. B) Representative traces of evoked action potential from non-CMS (left) and CMS (right) neurons. C) 2 μM TTX completely eliminates the rapid inward current in neurons. D) CMS neurons (42) have a higher rheobase than non-CMS neurons (89). * indicates p < 0.05.

Figure 5. Decreased Na⁺ currents, current densities and protein expression in CMS neurons

A) Representative Na⁺ current traces from non-CMS (left) and CMS (right) neurons. B) Decreased Na⁺ currents from non-CMS neurons (89) and CMS neurons (42), C) current densities and E) protein expression in CMS neurons. D) Representative immunoblots probed with anti-pan Na and -actin. * indicates p < 0.05.

Figure 6. Na⁺ current activation and inactivation

A) Activation of Na⁺ currents from non-CMS neurons (29) and CMS neurons (15). B) Representative steady state inactivation of Na⁺ currents from non-CMS (left) and CMS (right), data summarized in C). D) Recovery from inactivation of Na⁺ currents from non-CMS (left, 18) and CMS neurons (right, 10), data were summarized in E). * indicates p < 0.05.

Figure 7. Gene expression profiles of SENP1 and ANP32D in non-CMS and CMS neurons under normoxia and hypoxia

A) Real time PCR analysis confirmed that expression of SENP1 was not different between non-CMS and CMS neurons under normoxia (p > 0.05), but there was a significantly different response between the two groups to 1% hypoxia treatment for 24 hrs (p < 0.05). Hypoxia treatment significantly decreased SENP1 expression in non-CMS neurons as compared to that under normoxia (p < 0.05), but not in CMS neurons. B) Real time PCR analysis confirmed that expression of ANP32D was not different between non-CMS and CMS neurons under normoxia (p > 0.05), but there was a significantly different response between the two groups to 1% hypoxia treatment for 24 hrs (p < 0.05). Hypoxia treatment significantly decreased ANP32D expression in both non-CMS and CMS neurons as compared to that under normoxia (p < 0.05). * indicates p < 0.05.