Mitochondria are unique double membrane organelles that form networks. This network is regulated by a dynamic process of position and fusion/fission. A balance between these processes ensures the health of mitochondria and the cell in general. Cell stress, including oxidative stress is accompanied by mitochondrial fission: an increase in short individual mitochondria, compared to a highly connected elongated network in resting cells. The fragmentation of mitochondrial network is detected in many pathological conditions associated with excitotoxicity and oxidative stress. There is evidence that the fragmentation protects cells against damage. How oxidative stress triggers fragmentation is unknown.
Cytosolic Dynamin-related Protein 1 (DRP1), and Mitochondrial fission protein 1 (Fis1) play vital roles in mitochondrial fission. DRP1 translocate from the cytoplasm to cleave the mitochondria. Mitochondrial fragmentation in response to oxidative stress is well documented. However, the specific protein(s) that triggers DRP1 recruitment in response to oxidative stress is unknown.
In addition to fusion/fission, mitochondria change their position in response to oxidative stress and move to center of the cell. Mitochondrial positioning is driven by Mitochondria Rho (Miro), in complex with Milton/Trafficking Kinesin-binding protein 1 (TRAK1), and Kinesin heavy chain (KHC/Kif5) complex. This complex controls the anterograde movement of mitochondria. Dynein and dynamin complex modulate the retrograde movement and to an extent, the anterograde movement. However, how Miro-1 responds to oxidative stress is unknown.
Further, Ganglioside-induced differentiation-associated protein 1 (GDAP1), appears to regulate mitochondrial network in response to oxidative stress: GDAP1 knockdown eliminates the fragmentation is response to oxidative stress. It has been shown that GDAP1 mutations in Charcot Marie Tooth disease cause mitochondria networks dysregulation. Although GDAP1 was theorized to protect cells from oxidative damage by promoting mitochondrial fragmentation, the mechanism through which GDAP1 regulates mitochondrial fragmentation remains unclear.
Therefore, to understand the mechanism with which GDAP1 regulates fragmentation and possibly positioning, we must determine how GDAP1 interacts with the mitochondrial transport machinery. I have found that GDAP1 overexpression is associated with the mitochondrial shape transition (MiST) phenotype indistinguishable from Miro-1 dysregulation. Miro-1 constitutively active mutant reverses the MiST phenotype caused by GDAP1 overexpression. Dynein and Miro-1 inhibition phenocopy GDAP1 overexpression. Therefore, I propose that GDAP1 affects mitochondrial network by inhibiting Miro-1; this promotes Miro-1 sensitivity to calcium. I will test this model using FRET, co-IP, PLA, CRISPR-Cas9