Remodeling of Mitochondria-Endoplasmic Reticulum Contact Sites Accompanies LUHMES Differentiation

Abstract

Neural progenitor cells (NPCs) are often used to study the subcellular mechanisms underlying differentiation into neurons in vitro. Works published to date have focused on the pathways that distinguish undifferentiated NPCs from mature neurons, neglecting the earlier and intermediate stages of this process. Current evidence suggests that mitochondria interaction with the ER is fundamental to a wide range of intracellular processes. However, it is not clear whether and how the mitochondria-ER interactions differ between NPCs and their differentiated counterparts. Here we take advantage of the widely used NPC line LUHMES to provide hints on the mitochondrial dynamic trait changes that occur during the first stage of their maturation into dopaminergic-like neurons. We observed that the morphology of mitochondria, their interaction with the ER, and the expression of several mitochondria-ER contact site resident proteins change, which suggests the potential contribution of mitochondria dynamics to NPC differentiation. Further studies will be needed to explore in depth these changes, and their functional outcomes, which may be relevant to the scientific community focusing on embryonic neurogenesis and developmental neurotoxicity.

Keywords: LUHMES; MERCs; Neural precursor cells; differentially expressed genes; mitochondria; mitochondria–ER contact sites.

Figure 1

Differentiation of LUHMES cells. (A) Scheme of LUHMES differentiation: at day 0, cells were cultured in a medium supplemented with GDNF, dcAMP, and tetracycline, and cell samples were collected every 2 days, up to 8 days of differentiation. The orange arrow indicates the cell collection phase. (B) Western blot analysis of β-tubulin III expression. (C) Quantification of β-tubulin III expression using densitometry (n = 4 independent experiments; normalized to day 8). (D) Differentiation of LUHMES cells was visualized using bright-field microscopy; scale bars: 100 μm (up) and 15 μm (bottom). Statistical significance was determined using ordinary One-way ANOVA test. Statistical significance: * p-value < 0.05, **** p-value < 0.0001. Western blot original images are in the supplementary materials.

Figure 2

Analysis of MERC structure in differentiating LUHMES cells via TEM. (A) Representative TEM pictures of LUHMES cells at the indicated timepoint; scale bars: 2 μm (up), 1 μm (middle); 0.2 μm (bottom). Representative MERCs are indicated by red arrows; the ER membrane is marked by a magenta line, the outer mitochondrial membrane by a cyan line, and the MERC width by a yellow line. (B) Quantification of the MERC width. (C) Histogram of the distribution of the MERC width along differentiation. (D) Quantification of the MERC length. (E) Quantification of the perimeter of mitochondria in contact with the ER. (F) Quantification of the perimeter of mitochondria not engaged in contact sites with the ER. Error bars represent the standard error of the mean (SEM). Statistical significance was determined using the Kruskal–Wallis test. Statistical significance: ** p-value < 0.01, **** p-value < 0.0001.

Figure 3

Differentially expressed genes along differentiation of LUHMES cells. (A) Heatmap of the identified differentially expressed genes (DEGs). A total of 3083 DEGs were identified using a multiple comparison analysis with Bonferroni correction, p-value = 0.005. The Z-score was calculated based on log(cpm) values scaled by rows. (B) Multidimensional scaling (MDS) plot showing sample dissimilarity based on pairwise distances, with similar samples clustering together (C) Birch clustering showing the patterns of change in gene expression; 30 clusters were identified, of which 14 clusters, shown here, contained more than 50 DEGs. Birch threshold: 0.75.

Figure 4

MERC resident proteins and their protein interactors among DEGs. (A) Cluster 1 MERC resident proteins and their known protein interactors among DEGs using the BIOGRID database. (B) Western blot of the MERC resident proteins and their interactors, CIT and RECQL4. GAPDH was used as an internal control. (C) Densitometry analysis of Western blots. A small region of interest was drawn around each band, and the band intensity was acquired after background subtraction. Ordinary one-way ANOVA was used for statistical significance. Statistical significance: * p value < 0.05, ** p value < 0.01, *** p value < 0.001, **** p value < 0.0001. (D) Western blots of mouse brain subcellular fractions; 30 μg of protein were loaded. CANX: Calnexin, FACL4: Acyl-CoA Synthetase Long-Chain Family Member 4; PNS: post-nuclear supernatant fraction; MAMs: mitochondria-associated membranes fraction. Western blot original images are in the supplementary materials.