Localization of α-, ß-, and γ-synuclein during neuronal development and alterations associated with the neuronal response to axonal trauma

Abstract

Genetic and protein studies have indicated abnormalities in α-synuclein in neurodegenerative diseases. However, the developmental localization and cellular role of synuclein isoforms is contentious. We investigated the cellular localization of α-, ß-, and γ-synuclein in developing cultured rat neurons and following axonal transection of relatively mature neurons, a model that disrupts the axonal cytoskeleton and results in regenerative sprouting. Cortical neurons were grown up to 21 days in vitro (DIV). Axon bundles at 21 DIV were transected and cellular changes examined at 4 and 24 h post-injury. Immunohistochemistry demonstrated that α- and ß-synuclein were localized to cellular cytosol and growth cones at 3DIV, with accumulating puncta-like labeling within axons and growth cones by 10-21DIV. In contrast, γ-synuclein immunoreactivity was limited at all time points. By 21DIV, α- and ß-synuclein were present in the same neurons but largely in separate subregions, only 26% of puncta contained both α- and ß-synuclein immunoreactivity. Less than 20% of α-, ß-, and pan-synuclein immunoreactive puncta directly colocalized to synaptophysin profiles at 10DIV, decreasing to 10% at 21DIV. Both α- and ß-synuclein accumulated substantially within damaged axons at 21DIV and were localized to cytoskeletal abnormalities. At latter time points post-injury, α- and ß-synuclein immunoreactive puncta were localized to growth cone-like structures in regenerating neurites. This study shows that α- and ß-synuclein have a precise localization within cortical neurons and are generally nonoverlapping in their distribution within individual neurons. In addition, synuclein proteins accumulate rapidly in damaged axons and may have a role in regenerative sprouting.