Glutamate Stimulation Dysregulates AMPA Receptors-Induced Signal Transduction Pathway in Leber’s Inherited Optic Neuropathy Patient-Specific hiPSC-Derived Retinal Ganglion Cells

Abstract

The mitochondrial genetic disorder, Leber’s hereditary optic neuropathy (LHON), is caused by a mutation in MT-ND4 gene, encoding NADH dehydrogenase subunit 4. It leads to the progressive death of retinal ganglion cells (RGCs) and causes visual impairment or even blindness. However, the precise mechanisms of LHON disease penetrance and progression are not completely elucidated. Human-induced pluripotent stem cells (hiPSCs) offer unique opportunities to investigate disease-relevant phenotypes and regulatory mechanisms underlying LHON pathogenesis at the cellular level. In this study, we successfully generated RGCs by differentiation of LHON patient-specific hiPSCs. We modified the protocol of differentiation to obtain a more enriched population of single-cell RGCs for LHON study. Based on assessing morphology, expression of specific markers and electrophysiological activity, we found that LHON-specific hiPSC-derived were more defective in comparison with normal wild-type RGCs. Based on our previous study, whereby by using microarray analysis we identified that the components of glutamatergic synapse signaling pathway were significantly downregulated in LHON-specific RGCs, we focused our study on glutamate-associated α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. We found that the protein expression levels of the subunits of the AMPA receptor, GluR1 and GluR2, and their associated scaffold proteins were decreased in LHON-RGCs. By performing the co-immunoprecipitation assay, we found several differences in the efficiencies of interaction between AMPA subunits and scaffold proteins between normal and LHON-specific RGCs.

Modelling Mitochondrial Disease in Human Pluripotent Stem Cells: What Have We Learned?

“…Other approaches include microscopic visualization of key cellular features to determine a mutation’s impact on cell structure or function [ 100 ]. For cardiomyocytes and neurons, electrophysiology can provide highly sensitive data to identify even subtle functional changes [ 101 ]. Calcium imaging can be particularly informative in the context of mitochondrial diseases, since calcium handling is a key role of mitochondria [ 102 , 103 ].…”

“…Initial studies in a LHON iPSC model carrying homoplasmic double mtDNA mutations in MT-ND1 and MT-ND6 determined that their differentiation efficiency to retinal ganglion cells (iPSC-RGCs) was unaffected, but apoptosis was more prominent in these cells when compared to both a non-isogenic control and a cybrid corrected isogenic control [ 64 ]. Furthermore, in LHON patient iPSC-RGCs carrying a m.11778G > A MT-ND4 mutation, optic vesicles derived from the LHON iPSCs were smaller, with a notable difference in the appearance of the neuroblastic layer compared to controls [ 101 ]. Additionally, LHON iPSC-RGCs possessed shorter neurites, and formed fewer connections with neighbouring RGC bundles, while mitochondrial motility was also impaired [ 165 ].…”

“…However, it is unclear if the differentiation defects were a direct manifestation of mitochondrial dysfunction ( Table A4 ) or are linked to the increased apoptosis [ 64 , 166 ]. Additionally, the use of non-isogenic controls makes it difficult to assign phenotypes directly to the m.11778G > A MT-ND4 variant [ 101 , 165 ]. Nonetheless, glutamate uptake was compromised in m.11778G > A LHON iPSC-RGCs.…”

Hereditary Optic Neuropathies: Induced Pluripotent Stem Cell-Based 2D/3D Approaches

“…Additionally, they demonstrated in another study the existence of an impaired mitochondrial respiration in affected RGCs, which led to deficient neuronal function. The LHON-RGCs presented electrophysiological activity dysfunction, implying that the mutation could cause defects in the ionic channels [ 105 ].…”

Exploiting hiPSCs in Leber's Hereditary Optic Neuropathy (LHON): Present Achievements and Future Perspectives

“…Furthermore, affected RGCs displayed a significant increase of KIF5A, a member of the kinesin-1 family KIF5, involved in the transport of mitochondria along the axons. Another study carried out on hiPSC-derived RGCs by Yang et al ( 50 ) highlighted the possible role played by AMPA receptors and excitotoxicity in m.11778G>A LHON patients. They used a modified protocol of differentiation of hiPSCs to RGCs to obtain a highly homogeneous RGCs population.…”