Adeno-associated virus serotypes 1, 8, and 9 share conserved mechanisms for anterograde and retrograde axonal transport

Abstract

Adeno-associated virus (AAV) vectors often undergo long-distance axonal transport after brain injection. This leads to transduction of brain regions distal to the injection site, although the extent of axonal transport and distal transduction varies widely among AAV serotypes. The mechanisms driving this variability are poorly understood. This is a critical problem for applications that require focal gene expression within a specific brain region, and also impedes the utilization of vector transport for applications requiring widespread delivery of transgene to the brain. Here, we compared AAV serotypes 1 and 9, which frequently demonstrate distal transduction, with serotype 8, which rarely spreads beyond the injection site. To examine directional AAV transport in vitro, we used a microfluidic chamber to apply dye-labeled AAV to the axon termini or to the cell bodies of primary rat embryonic cortical neurons. All three serotypes were actively transported along axons, with transport characterized by high velocities and prolonged runs in both the anterograde and retrograde directions. Coinfection with pairs of serotypes indicated that AAV1, 8, and 9 share the same intracellular compartments for axonal transport. In vivo, both AAV8 and 9 demonstrated anterograde and retrograde transport within a nonreciprocal circuit after injection into adult mouse brain, with highly similar distributions of distal transduction. However, in mass-cultured neurons, we found that AAV1 was more frequently transported than AAV8 or 9, and that the frequency of AAV9 transport could be enhanced by increasing receptor availability. Thus, while these serotypes share conserved mechanisms for axonal transport both in vitro and in vivo, the frequency of transport can vary among serotypes, and axonal transport can be markedly increased by enhancing vector uptake. This suggests that variability in distal transduction in vivo likely results from differential uptake at the plasma membrane, rather than fundamental differences in transport mechanisms among AAV serotypes.

FIG. 1.

High-speed anterograde transport is highly conserved among AAV serotypes 1, 8, and 9. Anterograde-directed AAV puncta were tracked within the microfluidic groove from 1 to 4 hr after specific application to cell bodies. (A) Histograms of instantaneous velocity, with negative values representing steps in reverse of the overall direction of movement (e.g., a anterograde-directed punctum stepping backward in the retrograde direction). Histograms depict the range of velocities at which each population is capable of moving, as well as the relative frequency of each velocity. (B–F) Box plots portray the quantified axonal transport of anterograde-directed AAVs and GFP-Rab7-labeled late endosomes/lysosomes. The interquartile range and median are represented, with whiskers depicting the 5th–95th percentile and outliers omitted because of large sample sizes. No significant differences were detected among AAV serotypes 1, 8, and 9. All three AAV serotypes were found to move anterograde at significantly higher average velocity (B), make longer individual runs (C), progress further total distance (D), and spend less time paused (F) than GFP-Rab7-labeled late endosomes/lysosomes (all p<0.0001), although no difference was detected in the duration of individual pauses (E). See Supplementary Table S1 for sample sizes. AAV9 and GFP-Rab7 data were obtained previously using identical methods and are included for comparison (Castle et al., 2014). (G) Excerpted frames from live imaging sequences depict representative movement from each population (arrows). Images were inverted and thresholds adjusted for maximum visibility. Full image series are shown in Supplementary Movies S1–S4. AAV, adeno-associated virus.

FIG. 2.

AAV serotypes 1, 8, and 9 are targeted for high-speed retrograde transport. Retrograde-directed AAV puncta were tracked within the microfluidic groove from 1 to 4 hr after specific application to axon termini. (A) Histograms of instantaneous velocity, defined as the speed (μm/sec) of movement between two sequential images during a run, with negative values representing steps in reverse of the overall direction of movement (e.g., a retrograde-directed punctum stepping backward in the anterograde direction). Histograms depict the range of velocities at which each population is capable of moving, as well as the relative frequency of each velocity. (B–F) Box plots portray the quantified axonal transport of retrograde-directed AAVs and GFP-Rab7-labeled late endosomes/lysosomes. The interquartile range and median are represented, with whiskers depicting the 5th–95th percentile and outliers omitted because of large sample sizes. AAV8 was found to move retrograde at significantly higher average velocity (B), make longer individual runs (C), progress further total distance (D), and spend less time paused (F) than AAV9 (all p<0.01), although in each case the observed difference was small (see Supplementary Table S1). No significant differences were detected between AAVs 1 and 8 or between AAVs 1 and 9. All three AAV serotypes were found to move retrograde at significantly higher average velocity (B), make longer individual runs (C), progress further total distance (D), and spend less time paused (F) than GFP-Rab7-labeled late endosomes/lysosomes (all p<0.0001). No difference was detected in the duration of individual pauses (E). See Supplementary Table S1 for sample sizes. AAV9 and GFP-Rab7 data were obtained previously using identical methods and are included for comparison (Castle et al., 2014). (G) Excerpted frames from live imaging sequences depict representative movement from each population (arrows). Colors were inverted and thresholds adjusted for maximum visibility. Full image series are shown in Supplementary Movies S5–S8.

FIG. 3.

AAVs 1, 8, and 9 colocalize strongly and consistently during both retrograde and anterograde transport. From 1.5 to 4 hr after simultaneous application of 3×10⁹ GC bCy3-AAV and 3×10⁹ GC AF488-AAV to a mass culture of 100,000 rat E18 cortical neurons, all AF488-AAV puncta that progressed more than 50 μm retrograde or anterograde over 10 min were counted. Each was classified as either colocalized with a Cy3-AAV punctum or uncolocalized. Each AF488-labeled serotype 1, 8, or 9 was compared against the other two Cy3-labeled serotypes individually. Bars represent the mean among a total of n=3 cultures for each condition, with error bars indicating one standard deviation from the mean. An average n=83 and minimum n=51 AF488-AAV puncta were counted per condition. No significant differences among groups were detected via ANOVA (p=0.9733 and p=0.9527 for retrograde and anterograde, respectively). Representative colocalized movement is shown in Fig. 3; Supplementary Movie S9.

FIG. 4.

AAV1 exhibits more frequent axonal transport than AAV8 or 9 in vitro. 3×10⁹ GC AAV1, 8, or 9 was applied to mass cultures of 100,000 rat E18 cortical neurons, or 1×10⁹ GC AAV9 was applied after treatment of cultures with 30 mU NA for 2 hr. These NA data were obtained previously using identical methods (Castle et al., 2014). From 1.5 to 4 hr after application of vector, the number of AAV puncta progressing more than 50 μm retrograde or anterograde over 10 min of imaging was counted, and then divided by the total length of axon imaged. Stars depict Newman–Keuls posttest significance between groups following ANOVA. *p<0.05; **p<0.01; ***p<0.001. AAV1 and AAV9+NA both made significantly more retrograde and anterograde runs when compared against AAV8 or AAV9 alone (p=0.0150 and p=0.0002, respectively). No significant differences were observed between AAV1 and AAV9+NA, or between AAV8 and AAV9. Bars represent the mean among cultures, with error bars indicating one standard deviation from the mean. AAV1 contains data from n=5 cultures, n=160 retrograde puncta, and n=163 anterograde puncta; AAV8 from n=6, n=106, and n=101; AAV9 from n=6, n=103, and n=107; and AAV9+NA from n=4, n=139, and n=142. NA, neuraminidase.

FIG. 5.

Retrograde transport of AAV peaks rapidly and remains constant, while anterograde transport increases gradually. The number of AAV puncta that progressed more than 50 μm retrograde or anterograde was counted for each axon imaged in mass culture (A) or for each image series obtained from a microfluidic chamber (B and C), and these data were analyzed for changes over time. Data from serotypes 1, 8, and 9 were combined, as no difference among serotypes was observed. Stars represent the significance of Kruskal–Wallis tests (Dunn's posttest). *p<0.05; **p<0.01; ***p<0.001. Boxes represent the median and interquartile range among cultures (A) or image series (B and C), with whiskers indicating the 10th–90th percentile and outliers omitted. In (A), n=18 cultures were analyzed at each time point. In (B) and (C), an average of n=38 and minimum of n=32 image series were analyzed at each time point, with the same series used for both retrograde and anterograde counts. (A) After application of 3×10⁹ GC AAV to mass cultures, there was a significant increase in the frequency of anterograde AAV transport over time (p<0.0001). Retrograde transport peaked rapidly after application and remained constant, with no significant differences detected. The decrease observed from 2.5 to 3 hr after application was not significant (p=0.1692). (B) After application of AAV to the axon terminus side of the microfluidic chamber, retrograde transport of AAV peaked rapidly and remained constant over time, with no significant differences detected (p=0.6491). Anterograde transport of AAV was rare after application to the axon terminus and did not change significantly (p=0.9763). (C) After application of AAV to the cell body side of the microfluidic chamber, both anterograde and retrograde transport of AAV significantly increased over time (both p<0.0001).

FIG. 6.

AAV8 and AAV9 exhibit identical retrograde axonal transport after dentate gyrus injection. (A) Adult C57BL/6 mice were unilaterally injected with 1.8×10¹⁰ GC AAV8- or AAV9-CMV.eGFP in a volume of 0.5 μl into DG. Four weeks after injection, mice were sacrificed and cryosectioned at 20 μm thickness, and transduced cells were visualized via colorimetric ISH against the eGFP transgene mRNA sequence. Both AAV8- and AAV9-injected mice exhibited strong local transduction throughout HPC, as well as strong distal transduction of ipsilateral EC after retrograde transport of the vector. In addition, retrograde transduction of cells within contralateral HPC (primarily CA3) and contralateral EC was also observed (arrows), indicating that both serotypes can transduce retrogradely over long distances. No fundamental differences in axonal transport were observed between serotypes, although distal transduction of the contralateral hemisphere was stronger for AAV9. All four brain sections in each column are from the same animal. (B) A schematic details the relevant connections among hippocampal and entorhinal brain regions that underlie the axonal transport of AAV and the distal transduction observed here and in Fig. 7. DG, dentate gyrus; EC, entorhinal cortex; GC, genome copies; HPC, hippocampus; ISH, in situ hybridization.

FIG. 7.

AAV8 and AAV9 exhibit identical anterograde axonal transport after entorhinal cortex injection. Adult C57BL/6 mice were unilaterally injected with 9.0×10⁹ GC AAV8- or AAV9-CMV.eGFP in a volume of 0.5 μl into EC. An identical volume and titer of AAV9 was also coinjected with 2 mU NA. Four weeks after injection, mice were sacrificed and cryosectioned at 20 μm thickness, and transduced cells were visualized via colorimetric ISH against the eGFP transgene mRNA sequence. Local EC transduction was strong and similarly distributed in all conditions. Strong distal transduction of DG was observed with both AAV8 and 9, indicating that both serotypes undergo anterograde axonal transport and transduce second-order cell bodies within the granule layer of DG (arrows). Axonal projections from EC to DG are nonreciprocal, and thus DG transduction provides unambiguous evidence of anterograde transport. Distal transduction of CA1 and CA3 was also observed with both serotypes. Although CA3 transduction likely resulted from anterograde transport as well, CA1 transduction could reflect either retrograde or anterograde transport of the vector (Fig. 6B). No enhancement of local or distal transduction was observed after coinjection of AAV9 with NA, which appeared similar to both AAV8 and AAV9 without NA. This may be because of the low concentration of NA that was used. All three sections in each row are from the same animal.

FIG. 8.

AAV8 exhibits identical local and distal transduction among mouse strains C57BL/6, 129, BALB/c, and C3H. Adult mice from each strain were unilaterally injected with 1 μl AAV8 in four sites along two injection tracts: the HPC and thalamus, and the cortex and STR. Four weeks after injection, mice were sacrificed and cryosectioned at 20 μm thickness, and transduced cells were visualized via colorimetric ISH against the eGFP transgene mRNA sequence. No differences were observed among the four mouse strains. Transduction of the cortex and thalamus was consistently strong but focal. Transduction of the HPC and STR was more widespread, with evidence of axonal transport to connected brain regions. Neither the pattern nor the overall strength of transduction varied among mouse strains. Axonal transport of vector was also highly conserved among mouse strains, with similar levels of distal transduction observed within contralateral CA3 and ipsilateral SNr among all strains (arrows). Axonal transport of vector to the SNr could have occurred from either the STR or the thalamus infusion of AAV. Images of the SNr are −3.2 mm caudal from bregma.