Dynactin 6 deficiency enhances tubular ER‐associated dystrophic neurites formation

Background The formation of dystrophic neurites (DNs) is a contributing factor to synaptic dysfunction in Alzheimer’s disease (AD) patients’ brains. We have shown that tubular endoplasmic reticulum (ER) shaping proteins reticulon 3 (RTN3), REEP2 and REEP5 are participated in the formation DNs, named as RTN3 immunoreactive DNs or RIDNs. RIDNs are appeared as clustered or dispersed forms in AD and aging mouse brain, respectively. Ultrastructural investigation showed that RIDNs are constitute with abnormal clustering of tubular ER. The over expression of RTN3 accelerates aging associated disperse RIDNs formation in Tg‐RTN3 mouse brain hippocampus. To determine a molecule that may cause RTN3 mediated abnormal tubular ER clustering, we aimed to identify RTN3 interacting proteins and to elucidate its functional roles in tabular ER accumulation and RIDNs formation in aging and AD brain. Methods Yeast two‐hybrid system, co‐immunoprecipitation, domain mapping and immune‐confocal studies to identify RTN3 interacting proteins. WB was performed to measure the protein level. Formation of RIDNs in mouse brains were detected using immuno‐confocal study. AAV9‐RTN3‐mCherry and AAV9‐DCTN‐GFP were injected at hippocampus of wild type mice to over expressed RTN3 and DCTN6. Results We have identified that N‐terminal domain of RTN3 interacts with dynactin 6 (DCTN6), a protein component that involved in dynein‐mediated retrograde transport of cargo vesicles. Our immune‐confocal studies on DCTN6 and RTN3 over expressed cells and primary cultured neuron indicate that RTN3‐DCTN6 interaction likely mediates the tubular ER trafficking. The DCTN6 protein level decreases during aging and it reduces in hippocampus compared to cortical samples of the same age mice. To understand the functional roles of DCTN6 in RIDNs formation during aging and beta‐amyloid plaque deposition, DCTN6 deficient Tg‐RTN3 and 5xFAD mice were generated. Tg‐RTN3; DCTN6+/‐ showed reduced DCTN6 protein level and enhanced hippocampus specific RINDs formation compare to Tg‐RTN3; DCTN6+/+ mouse. We are investigating whether RIDNs formation are increases in DCTN6 deficient 5xFAD mice and whether AAV9‐DCTN6 overexpression reduces RIDNs formation in AAV9‐RTN3 overexpressed mouse brain. Conclusion RTN3‐DCTN6 interaction likely mediates the tubular ER transport in axons and the DCTN6 deficiency is a contributing factor for tubular ER accumulation, RIDNs formation in AD and aging brain.