The Role Of Autophagy In The Metabolism Of Htt-552aa Fragments In Primary Neurons

Aim:To study the role of autophagy in the metabolism of Htt-552aa fragments in primary neurons, and to evaluate the autophagy on clearance of htt-552aa.Methods: Mouse cerebral cortial neurons were infected with adenovirus vector carrying htt-552aa fragments.Western blot analysis and immunohistochemistry were applied to identify expressed htt,Beclin1 and LC3 levels in neurons.The contribution of autophagy to htt-552aa catabolism was determined with treatment with 3-MA and rapamycin.Immunofluorescence were used to detect the distribution and expression of Beclin1,LC3,cathepsin B and cathepsin L.Results: 1. Wild-type Htt-552aa(htt-552aa) and mutant Htt-552aa (mhtt-552aa) were successfully expressed in primary neurons. 2. 3-MA inhibited the expression levels of Beclin1 and LC3, and increased the accumulation of htt in neurons;whereas rapamycin enhanced Beclin1,LC3 expression in neurons, and decreased htt accumulation. 3. Autophagy/lysosome related protein expression was changed in neurons expressing Htt-552aa. The distribution of Beclin1 and LC3 was altered. Htt-552aa changed cathepsin B distribution,while mhtt-552aa inhibited its expression. Cathepsin L expression in neurons expressing wild-type htt-552aa was higher than that expressing mhtt-552aa,suggesting that mhtt-552aa attenuate cathepsins expression. 4. Autophagy activity was higher 7 days after culture, then decreased thereafter;htt-552aa accumulation was higher in neurons cultured 13 days than those cultured 7 days.Conclusions: 1. Different from PC12, mhtt-552aa can form aggregates mainly in the neurites of primary neurons. 2. Autophagy may be one major way of htt metabolism. 3.Mhtt attenuated cathepsins expression. 4. Higher autophagy activity in young neurons have lower htt-552aa accumulation than that aged neurons with lower autophagy activity.These results suggest that autophagy may be one major way of htt metabolism.