Reprogramming axonal behavior by axon-specific viral transduction - PubMed (original) (raw)

Reprogramming axonal behavior by axon-specific viral transduction

B A Walker et al. Gene Ther. 2012 Sep.

Abstract

The treatment of axonal disorders, such as diseases associated with axonal injury and degeneration, is limited by the inability to directly target therapeutic protein expression to injured axons. Current gene therapy approaches rely on infection and transcription of viral genes in the cell body. Here, we describe an approach to target gene expression selectively to axons. Using a genetically engineered mouse containing epitope-labeled ribosomes, we find that neurons in adult animals contain ribosomes in distal axons. To use axonal ribosomes to alter local protein expression, we utilized a Sindbis virus containing an RNA genome that has been modified so that it can be directly used as a template for translation. Selective application of this virus to axons leads to local translation of heterologous proteins. Furthermore, we demonstrate that selective axonal protein expression can be used to modify axonal signaling in cultured neurons, enabling axons to grow over inhibitory substrates typically encountered following axonal injury. We also show that this viral approach also can be used to achieve heterologous expression in axons of living animals, indicating that this approach can be used to alter the axonal proteome in vivo. Together, these data identify a novel strategy to manipulate protein expression in axons, and provides a novel approach for using gene therapies for disorders of axonal function.

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Figures

Figure 1. Immunoelectron microscopy shows eGFP-labeled ribosomes localized to nodes of Ranvier in the corticospinal tract

(a) eGFP-L10a exhibits characteristic ribosomal localization in Glt25d2::eGFP-L10a mice. Immunoelectron microscopy was performed on cortical sections of 8-10 week old transgenic mice using anti-GFP antibodies and peroxidase labeling. Anti-eGFP immunoreactivity is detected in dendrite cross sections (boxes I and II, false-colored yellow) and rough endoplasmic reticulum (box III, false-colored blue) in the cortex of Glt25d2::eGFP-L10a mice. Enlarged images of regions I, II, and III are shown on the right. The localization of the eGFP-labeled ribosomes is consistent with previous descriptions of endogenous ribosomes. Scale bar, 1 μm. (b) Anti-eGFP immunoreactivity is not present in the dendrites (boxes I and II, false-colored yellow) or rough endoplasmic reticulum (box III, false-colored blue) of wild-type animals. Enlarged images of regions I, II, and III are shown on the right. Scale bar, 1 μm. (c) eGFP-labeled ribosomes are localized to nodes of Ranvier in the corticospinal tract. Corticospinal tracts from 8 – 10 week old Glt25d2::eGFP-L10a or wild-type mice were examined with anti-GFP immunoelectron microscopy and visualized with peroxidase. Specific eGFP signal in Glt25d2::eGFP-L10a mice is localized beneath the membrane in nodes of Ranvier, which are recognized by characteristic loops of myelin (false-colored green). Scale bar, 500 nm. (d) Quantification of percent of positive nodes in the corticospinal tract of Glt25d2::eGFP-L10a mice and wild-type controls. Nodes were considered positive if the average signal intensity in a 67 nm2 area below the nodal membrane was at least two standard deviations higher than the average signal intensity in wild-type controls. The number of positive nodes was significantly higher in Glt25d2::eGFP-L10a mice compared to wild-type controls (*P < 0.0001, unpaired two-tailed Student’s _t_-test, n = 30 nodes measured from each animal. N =3 per group). Data are expressed as mean ± s.e.m.

Figure 2. The Sindbis-IRES virus enables axon-specific protein expression

(a) Schematic representation of Sindbis-IRES virus expression cassette. The Sindbis-IRES-eGFP virus is a plus-strand, single-stranded RNA genome which contains an IRES for direct translation by axonal ribosomes. The open reading frame of the top schematic expresses a Myc epitope fused to the eGFP sequence. The 3′ untranslated region includes a string of 37 adenosines. The poly(A) tract is expected to confer stability to the RNA genome. Below is a schematic of a similar construct that expresses a sACt fusion protein. (b) Diagram of microfluidic chamber. The culture chamber consists of a PDMS mold suitable for cell culture. The chamber is comprised of a cell body and an axonal compartment connected by microgrooves that are 10μm wide and 450 μm long. When neurons (drawn in green in schematic on left) are cultured in the cell-body compartment, axons grow through the microgrooves into the fluidically isolated axonal compartment. Shown on the right is an immunofluorescence image of a microfluidic chamber. Rat DRG neurons with axons extending through the microgrooves are visualized by anti-GAP43 immunohistochemistry (green). This cell culture platform enables experimental treatments to be selectively applied to the axonal compartment, without introduction into the cell body compartment. Scale bar, 100 μm. (c) Application of Sindbis-IRES-eGFP to the axonal compartment does not result in detectable eGFP in cell bodies. E15 rat DRG neurons were cultured in microfluidic chambers and either Sindbis-IRES-eGFP or vehicle as indicated (above the line) was added to the indicated compartments (below the line). eGFP expression in cell bodies was detected by anti-eGFP immunofluorescence (green), and Tau expression (red) was used to label neuronal cell bodies and processes after 8 h. eGFP was readily detected in the cell bodies of neurons infected with Sindbis-IRES-eGFP in both the axonal and cell body compartments. No eGFP was detected in the cell bodies of neurons infected with Sindbis-IRES-eGFP in the axonal compartment alone. Taken together, these data indicate that infection of axons by Sindbis-IRES virus does not lead to retrograde transport and Sindbis expression in the cell body. Scale bar, 50 μm. (d) Axon-specific infection with Sindbis-IRES-eGFP leads to local translation of eGFP. Sindbis-IRES-eGFP was applied to the axonal compartment as indicated, and axonal expression of eGFP (green) was detected by immunofluorescence. Axons were counterstained with Tau (red). Application of Sindbis-IRES-eGFP to the axonal compartment induced the appearance of eGFP in axons. This effect was blocked by selective application of cycloheximide to the axonal compartment at the time of infection. Axonal eGFP was not blocked by selective application of cycloheximide to the cell body compartment, confirming that the expression of eGFP in axons does not involve translocation of the virus to the cell body and somatic translation, but is a consequence of axonal mRNA translation. Scale bar, 100 μm.

Figure 3. Axon-specific expression of soluble adenylyl cyclase alters signaling in the cell body

(a) Local translation of eGFP-sACt in axons upon axonal application of Sindbis-IRES-eGFP-sACt. Vehicle or Sindbis-IRES-eGFP-sACt was applied to the axonal compartment of microfluidic chambers containing P6 rat DRG neurons. eGFP-sACt was visualized by anti-eGFP immunofluorescence (green) in axons counterstained with Tau immunofluorescence (red). Axonal application of Sindbis-IRES-eGFP-sACt induced appearance of eGFP-sACt, confirming that postnatal DRG axons can translate proteins. Scale bar, 50 μm. (b) Axonal expression of sACt increases pCREB in neuronal nuclei. P6 DRG neurons were cultured in microfluidic chambers. Sindbis-IRES virus and experimental treatments were added to the axonal compartment as indicated. pCREB levels in neuronal nuclei were detected by anti-pCREB immunofluorescence (green). The cell bodies of neurons with distal axons extended into the axonal compartment were identified by retrograde DiI labeling (red). Few pCREB positive neurons were detected after vehicle or Sindbis-IRES-eGFP infection of axons. Application of 8-Br-cAMP (10 μM) to axons for 1 h increased the number of pCREB-positive neurons. Axonal application of Sindbis-IRES-eGFP-sACt for 8 h resulted in a similar increase in pCREB positive neurons. This effect was blocked by administration of cycloheximide (1 μM) to the axon at the time of infection, confirming that axonal translation is required for the induction of pCREB by axonally applied Sindbis-IRES-sACt. Scale bar, 50 μm. (c) Quantification of the increase in pCREB-positive nuclei in (b). Treatment of axons with either 10 μM 8-Br-cAMP or IRES-eGFP-sACt resulted in a statistically significant increase in the number of pCREB positive neurons relative to vehicle (PBS) treatment (*P < 0.0001, n = 176 neurons measured across three experiments, and *P < 0.0001, n = 93 neurons measured across three experiments, respectively). The number of pCREB-positive nuclei were not significantly increased following treatment with either IRES-eGFP (P = 0.62, n = 166 neurons measured across three experiments) or IRES-eGFP-sACt with CHX (P = 0.34, n = 179 nuclei measured across three experiments) as compared with vehicle control. Student’s _t_-test (unpaired, two-tailed) was applied. Data are expressed as mean ± s.e.m.

Figure 4. Axon-specific expression of soluble adenylyl cyclase alters axon behavior

(a) Axonal application of Sindbis-IRES-eGFP-sACt results in increased axonal growth on CSPG. P6 DRG neurons were cultured in microfluidic chambers in which the axonal compartment was coated with either laminin, or laminin containing CSPG as indicated. The axonal compartment was incubated with either vehicle or Sindbis-IRES-eGFP-sACt as indicated. Axonal growth was measured by imaging axon terminals over 3 h by phase contrast microscopy. Shown are phase contrast images at t = 3 h. Axon termini are marked with yellow arrowheads. The position of axon termini at t = 0 h are indicated with blue arrowheads. Axon growth was slowed in microfluidic chambers containing CSPG in the axonal compartment. However, axonal growth rates of axons expressing Sindbis-IRES-eGFP-sACt extending across CSPG were comparable to those of the laminin control. Scale bar, 50 μm. (b) Quantification of axonal outgrowth rate in (a). Axon growth was normalized to the rate of vehicle-treated axons grown on laminin. Axons grow significantly slower across laminin containing CSPG than laminin alone (*P < 0.0001, n = 34 axons measured across a minimum of three experiments). Treatment of axons with 10 μM 8-Br-cAMP significantly increased outgrowth rates across CSPG as compared to vehicle control (*P < 0.0001, n = 41 axons measured across a minimum of three experiments). Axonal expression of eGFP-sACt significantly increased outgrowth rates across CSPG compared to axonal expression of eGFP, which itself was not significantly different than vehicle treatment on CSPG (*P = 0.0002, n = 29 axons measured across three experiments and P = 0.657, n = 28 axons measured across three experiments, respectively). Comparisons were made using the unpaired, two-tailed Student’s _t_-test. Data are expressed as mean ± s.e.m. (c) In vivo expression of eGFP-sACt in axons in the corticospinal tract. Sindbis-IRES-eGFP-sACt was stereotaxically injected into the corticospinal tract of adult C57bl/6 mice and allowed to express for 4 d. eGFP-sACt levels in axons were detected by anti-eGFP immunofluorescence (green) and colocalized with Tuj1 staining (red). eGFP immunofluorescence (green) does not strongly colocalize with a marker of myelinating glial cells, GalC (red). Taken together, these data indicate that eGFP-sACt is expressed in myelinated axons in the corticospinal tract. Scale bar, 5 μm.

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Reprogramming axonal behavior by axon-specific viral transduction - PubMed (original) (raw)