The tyrosine capsid mutations on retrograde adeno-associated virus accelerates gene transduction efficiency.

Adeno-associated virus (AAV) vector is a critical tool for gene delivery through its durable transgene expression and safety profile. Among many serotypes, AAV2-retro is typically utilized for dissecting neural circuits with its retrograde functionality. However, this vector requires a relatively long-term incubation period (over 2 weeks) to obtain enough gene expression levels presumably due to low efficiency in gene transduction. Here, we aimed to enhance transgene expression efficiency of AAV2-retro vectors by substituting multiple tyrosine residues with phenylalanines (YF mutations) in the virus capsid, which is previously reported to improve the transduction efficiency of AAV2-infected cells by evading host cell responses. We found that AAV2-retro with YF mutations (AAV2-retroYF)-mediated transgene expression was significantly enhanced in the primary culture of murine cortical neurons at 1 week after application, comparable to that of the conventional AAV2-retro at 2 week after application. Moreover, transgene expressions in the retrogradely labeled neurons mediated by AAV2-retroYF were significantly increased both in the cortico-cortical circuits and in the subcortical circuits in vivo, while the retrograde functionality of AAV2-retroYF was equally effective as that of AAV2-retro. Our data indicate that YF mutations boost AAV2-retro-mediated retrograde gene transduction in vivo and suggest that the AAV2-retroYF should be useful for efficient targeting of the projection-defined neurons, which is suited to applications for dissecting neural circuits during development as well as future clinical applications.

Adeno-Associated Virus-Mediated Single-Cell Labeling of Mitral Cells in the Mouse Olfactory Bulb: Insights into the Developmental Dynamics of Dendrite Remodeling.

Neurons typically remodel axons/dendrites for functional refinement of neural circuits in the developing brain. Mitral cells in the mammalian olfactory system remodel their dendritic arbors in the perinatal development, but the underlying molecular and cellular mechanisms remain elusive in part due to a lack of convenient methods to label mitral cells with single-cell resolution. Here we report a novel method for single-cell labeling of mouse mitral cells using adeno-associated virus (AAV)-mediated gene delivery. We first demonstrated that AAV injection into the olfactory ventricle of embryonic day 14.5 (E14.5) mice preferentially labels mitral cells in the olfactory bulb (OB). Birthdate labeling indicated that AAV can transduce mitral cells independently of their birthdates. Furthermore, in combination with the Cre-mediated gene expression system, AAV injection allows visualization of mitral cells at single-cell resolution. Using this AAV-mediated single-cell labeling method, we investigated dendrite development of mitral cells and found that ~50% of mitral cells exhibited mature apical dendrites with a single thick and tufted branch before birth, suggesting that a certain population of mitral cells completes dendrite remodeling during embryonic stages. We also found an atypical subtype of mitral cells that have multiple dendritic shafts innervating the same glomeruli. Our data thus demonstrate that the AAV-mediated labeling method that we reported here provides an efficient way to visualize mitral cells with single-cell resolution and could be utilized to study dynamic aspects as well as functions of mitral cells in the olfactory circuits.