Birk, A. | bioRxiv Science

bioRxiv2023

High density aromatic peptides protect retinal ganglion cells in mouse retina following optic nerve crush injury.

Mitochondrial dysfunction is a critical early driver of retinal ganglion cell (RGC) loss in optic nerve injury. We evaluated whether HDAP2, a mitochondria-targeted aromatic peptide designed to support mitochondrial membrane integrity, could preserve neuronal structure after optic nerve crush (ONC). Systemically administered HDAP2 penetrated the blood-retinal barrier and localized to RGCs and mitochondrial-rich retinal layers. Daily treatment (IP, 3 mg/kg for 14 days) significantly improved RGC survival compared to saline-treated ONC animals. RGC densities increased across central, midperipheral, and peripheral regions, and surviving RGCs exhibited approximately two-fold higher RBPMS expression, suggesting improved cellular health. Transmission electron microscopy revealed that HDAP2 substantially reduced mitochondrial loss within crushed optic nerve axons. Mitochondrial density in HDAP2-treated nerves reached 70% of uninjured levels (0.129 {+/-} 0.029 vs. 0.185 {+/-} 0.017) and was 5.3-fold higher than untreated ONC nerves (0.025 {+/-} 0.012; Kruskal-Wallis p = 0.039; Cohens d = 2.77). Mitochondrial morphology was similar across groups, indicating that HDAP2 prevents mitochondrial loss rather than rescuing damaged organelles. HDAP2-treated nerves also contained a higher density of structurally intact axons, consistent with reduced ultrastructural degeneration following injury. These findings demonstrate that HDAP2 limits mitochondrial loss and attenuates neuronal degeneration after ONC. Together, the results support HDAP2 as a promising therapeutic candidate for protecting CNS projection neurons by maintaining mitochondrial stability after axonal injury.

neuroscience↗

bioRxiv2023

Targeting high-density aromatic peptides to cardiolipin optimizes the mitochondrial membrane potential.

The mitochondrial membrane potential ({Delta}{Psi}m) is created by the accumulation of protons on an outer leaflet of the inner mitochondrial membrane and drives the synthesis of most cellular ATP, which is essential for cellular bioenergetics and survival. The {Delta}{Psi}m also facilitates the electrogenic transport of cations, such as Ca2+, and regulates generation of reactive oxygen species, which serves as a powerful bioenergetic and stress-signaling regulator. Proton trapping on the outer leaflet of the inner mitochondrial membrane of mitochondrial cristae could be controlled by cardiolipin when the local pH is above 8. However, there is presently no technology that effectively targets strong bases to cardiolipin. We have developed a novel, high-density aromatic peptide (HDAP2) to preserve a proton gradient-driven potential in mitochondria by increasing proton trapping on cardiolipin (CL). HDAP2-induced formation of cardiolipin-HDAP2 complexes accumulated positive charges at the head of CL. The HDAP2-CL vesicles could accumulate the mitochondrial transmembrane potential probe, Tetramethylrhodamine (TMRM). This potential could be uncoupled with Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and Dinitrophenol (DNP), indicating that an interaction of HDAP2 with CL could support a proton gradient-driven transmembrane potential. We demonstrated that this novel, water-soluble peptide is cell-permeable, targets mitochondria without causing cell toxicity, and promotes cell survival during serum starvation. Importantly, the HDAP2-cardiolipin complex-mediated optimization of the proton gradient was supported by the ability of HDAP2 to prevent CCCP-mediated mitochondrial depolarization in ARPE-19 cells in a dose-dependent manner. Based on its mechanism of action, HDAP2 could promote cellular homeostasis, which would have broad clinical applicability for the prevention, recovery and reversal of many acute and chronic disease conditions, such as neurodegeneration, ischemia- reperfusion injury, and inflammation.

biochemistry↗