Human neurons from Christianson syndrome iPSCs reveal mutation-specific responses to rescue strategies

Abstract

Christianson syndrome (CS), an X-linked neurological disorder characterized by postnatal attenuation of brain growth (postnatal microcephaly), is caused by mutations in SLC9A6, the gene encoding endosomal Na+/H+ exchanger 6 (NHE6). To hasten treatment development, we established induced pluripotent stem cell (iPSC) lines from patients with CS representing a mutational spectrum, as well as biologically related and isogenic control lines. We demonstrated that pathogenic mutations lead to loss of protein function by a variety of mechanisms: The majority of mutations caused loss of mRNA due to nonsense-mediated mRNA decay; however, a recurrent, missense mutation (the G383D mutation) had both loss-of-function and dominant-negative activities. Regardless of mutation, all patient-derived neurons demonstrated reduced neurite growth and arborization, likely underlying diminished postnatal brain growth in patients. Phenotype rescue strategies showed mutation-specific responses: A gene transfer strategy was effective in nonsense mutations, but not in the G383D mutation, wherein residual protein appeared to interfere with rescue. In contrast, application of exogenous trophic factors (BDNF or IGF-1) rescued arborization phenotypes across all mutations. These results may guide treatment development in CS, including gene therapy strategies wherein our data suggest that response to treatment may be dictated by the class of mutation.

Correction of a pathogenic mutation in iPSCs derived from a patient with Christianson syndrome using CRISPR/Cas9 genome editing

“…A family with one son affected with CS and carrying a c.1569G>A (p.(W523X)) mutation in NHE6 and an unaffected, biologically related son was recruited for this study as a part of our larger CS study (Lizarraga et al, 2021; Pescosolido et al, 2014). The NHE6 protein has three domains: 1) an N-terminal domain; 2) a 12-TM domain, which includes the Na+/H+ exchanger domain; and 3) a cytoplasmic domain.…”

“…The reduction in NHE6 mRNA and lack of NHE6 protein in CS iPSCs containing this mutation support that it is a loss-of-function mutation. Although not explored in detail here, based on our experimental results from other CS iPSC lines with nonsense mutations (Lizarraga et al, 2021), we anticipate that the c.1569G>A (p.(W523X)) mutation is subject to nonsense-mediated mRNA decay.…”

“…Following separation of proteins by electrophoresis, gels were transferred to nitrocellulose membranes (Novex #LC2000). Western blots were performed using standard procedures (Lizarraga et al, 2021; Ouyang et al, 2019; Xu et al, 2017) and were analyzed with the Li-CoR Odyssey Imaging System. Proteins were detected using rabbit anti-NHE6 antibody (1:1,000 working dilution; Covance #048) (Ouyang et al, 2013) and mouse anti-α-Tubulin (1:5,000 working dilution; Sigma #T6074).…”

“…The copyright holder for this preprint (which this version posted May 20, 2021. ; https://doi.org/10.1101/2021 …”

Human iPSC lines from a Christianson syndrome patient with NHE6 W523X mutation, a biologically-related control, and CRISPR/Cas9 gene-corrected isogenic controls

“…Following separation of proteins by electrophoresis, gels were transferred to nitrocellulose membranes (Novex #LC2000). Western blots were performed using standard procedures ( Lizarraga et al, 2021 ) and were analyzed with the Li-CoR Odyssey Imaging System. See Table 2 for antibodies and dilutions used.…”