Retrograde and Anterograde Axonal Transport Impairments Are Early Indicators For OPA1 Haploinsufficiency

Abstract

Purpose : Dominant Optic Atrophy (DOA) is an inherited optic nerve disorder characterized by bilateral optic nerve degeneration. It severely impairs retinal ganglion cells (RGC) and their axons. The most prevalent cause of DOA is OPA1 mutations, which generally result in OPA1 haploinsufficiency. Although OPA1 is a multifunctional mitochondrial protein, the disease's specific mechanism remains unclear. Mitochondrial energy metabolism and apoptotic stimuli sensitivity are assumed to be implicated, and these deficiencies should impair axonal energy balance and transport. Therefore, we proposed that axonal transport defects should be one of the early markers of DOA, and showed the effects of OPA1 haploinsufficiency on RGC axonal transport.

Methods : The transport defects are evaluated in both anterograde and retrograde directions. For the anterograde transport, intravitreal injections of Alexa flour-488 and 594 conjugated cholera toxin subunit B (CTB) (1 mg/ml) was performed to Opa1^wt/del and Opa1^wt/wt and the RGC reporter, Pv ^Cre/Cre; Thy1-LSL-YFP; Opa1^wt/del and Pv ^Cre/Cre; Thy1-LSL-YFP; Opa1^wt/wt mice, respectively. Animals were perfused and brains were fixed in 4% PFA and dissected. Subsequently, the superior colliculus (SC) and lateral geniculate nucleus (LGN) were imaged. For the retrograde transport, CTB-488 was intracranially injected to SC of Opa1^wt/wt and Opa1^wt/del mice in the same age group. Fundus fluorescent imaging and flow cytometry analysis were used to evaluate the retrograde transport deficiency. DRAQ7 was used for live/dead selection. Ratio paired t-test was used for comparison.

Results : Intravitreal CTB injections allowed visualization of axonal transport after the RGC uptake. Anterograde transport was delayed in haploinsufficient mice in SC and LGN regions according to fluorescent images of coronal brain sections for both CTB-488 and 594 injections. Flow cytometry analysis showed 2.5 fold reduction in retrograde transport between haploinsufficient and wt mice in terms of CTB-488 positive RGCs.

Conclusions : The degeneration of RGC axons in Opa1^wt/del mice are almost undetectable at young ages. However, both retrograde and anterograde CTB transport rate was hampered. As a result, the observed transport delay is unrelated to axonal degeneration. We demonstrated an early marker for OPA1 haploinsufficiency, which could also be a valuable therapeutic target for DOA.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.