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Comparative Study
. 2011 Sep 30;201(1):228-38.
doi: 10.1016/j.jneumeth.2011.08.003. Epub 2011 Aug 9.

Lentiviral Vectors Express Chondroitinase ABC in Cortical Projections and Promote Sprouting of Injured Corticospinal Axons

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Free PMC article
Comparative Study

Lentiviral Vectors Express Chondroitinase ABC in Cortical Projections and Promote Sprouting of Injured Corticospinal Axons

Rong-Rong Zhao et al. J Neurosci Methods. .
Free PMC article

Abstract

Several diseases and injuries of the central nervous system could potentially be treated by delivery of an enzyme, which might most effectively be achieved by gene therapy. In particular, the bacterial enzyme chondroitinase ABC is beneficial in animal models of spinal cord injury. We have adapted the chondroitinase gene so that it can direct secretion of active chondroitinase from mammalian cells, and inserted it into lentiviral vectors. When injected into adult rat brain, these vectors lead to extensive secretion of chondroitinase, both locally and from long-distance axon projections, with activity persisting for more than 4 weeks. In animals which received a simultaneous lesion of the corticospinal tract, the vector reduced axonal die-back and promoted sprouting and short-range regeneration of corticospinal axons. The same beneficial effects on damaged corticospinal axons were observed in animals which received the chondroitinase lentiviral vector directly into the vicinity of a spinal cord lesion.

Figures

Fig. 1
Fig. 1
Western blots showing that active chondroitinase is secreted following transduction of tissue culture cells with the lentiviral vectors. Neu7 conditioned medium (a source of CSPGs) was placed on transfected cells for 24 h, then analyzed by western blotting. (a) LV-C and LV-D. The first two lanes are positive and negative controls with Neu7 medium not exposed to transfected cells; lane 1 was digested in vitro with commercial chondroitinase (Sigma, 20 mU, 37°, 3 h). (Top panel) Probed for carbohydrate ‘stub’ epitope produced by chondroitinase action (antibody 1B5). Neu7 conditioned medium (lane 2) shows little immunoreactivity, but incubation with cells after transduction with LV-ChABCs generates extensive reactivity. (Middle) Probed for NG2. Undigested NG2 (lane 2) appears largely as a characteristic ‘smear’ above the core protein band due to the heterodispersed high-Mr GAG chains, and this is all converted to core protein by digestion with commercial chondroitinase (lane 1) or by incubation with cells after transduction with LV-ChABC. (Bottom) Probed for chondroitinase ABC. Commercial chondroitinase (lane 1) shows both full-length band (Ch) and a shorter band (Ch**) due to proteolytic activity during incubation with medium. LV-ChABC all generate a diffuse chondroitinase band (GlyCh: partially glycosylated): this migrates more slowly than bacterial chondroitinase, confirming that it has some glycosylation at sites which were not mutated. Some lanes are overexposed as the experiment was principally intended to detect digestion of CSPGs; also see (c). Black bar or star indicates intense bands which partially bleached during imaging. (b) LV-GFP (control) and LV-P. (Top) Probed for NG2; the high-Mr smear due to GAG chains (marked NG2:CSPG), whose distribution varies according to cell type, is all reduced to core protein following LV-P transduction. (Bottom) Probed for chondroitinase ABC. (c) Confirmation of chondroitinase secretion. (1) Commercial chondroitinase (Sigma); (2) medium from HEK cells transduced with LV-C; (3) the same after treatment with N-glycosidase to remove residual glycosylation.
Fig. 2
Fig. 2
Immunocytochemistry showing expression of the vectors in tissue culture cells. Cells were transduced with lentiviral vectors, fixed, reacted with antibody against chondroitinase, and visualized with peroxidase diaminobenzidine reaction. Scale bars, 100 μm. Top row: LV-D in HEK293T cells, Neu7 cells, and SCTM41 cells. Middle rowLV-P in HEK293T cells, Neu7 cells, and primary astrocytes. Bottom row: negative control cultures with no vector.
Fig. 3
Fig. 3
In vivo LV-ChABC injection into the rat cortex results in chondroitinase activity and the activity is also transported along axons. Injection was into deep layers of cortex on the left. Scale bar, 1 mm. (a–c) LV-C, 2 weeks: adjacent coronal sections from one brain stained for chondroitinase-produced stub (a), perineuronal net (b) and chondroitin sulfate (c). Chondroitinase digestion (area outlined with red arrows) is seen around the injection site and in the cortical white matter and adjacent hippocampus. On the original of (a), a faint column of reactivity can also be seen in the contralateral cortex. M, midline. (d–f) LV-C, 4 weeks: a similar series of sections, showing that chondroitinase digestion has extended along axon tracts and to the contralateral cortex. (g,h) LV-GFP, 4 weeks: similar sections from a brain injected with a lentivector encoding farnesyl-GFP; 4 weeks; stained with antibody against GFP. (g) Cortex near injection site, showing strong expression in astrocytes at surface (A), and oligodendrocytes in white matter/corpus callosum (O), and large numbers of neuronal fibers (N). (h) High-power views of contralateral side showing GFP-positive axons that have crossed in the corpus callosum and (inset) ascending in the cortex opposite the injection site. (i) Enzyme activity assay of functional chondroitinase level 4 weeks after LV injection into the cortex. Disks were loaded with equal amounts of chondroitin sulfate (CS) and each was incubated with extract of cortex from one brain to allow digestion by chondroitinase expressed in the sample. They were then stained for CS. Top row: incubated with extract of control brain (0), and with various amounts of commercial chondroitinase (12.5–100 mU). Bottom panels: incubated with extracts of LV-injected brains. All samples from the injected side gave some digestion, especially the 4-week samples, as did the 4-week samples from the opposite side. (j,k) 2B6 staining on spinal cord after LV-P cortical injection. (j) 2 weeks: horizontal section at level C3-C4. (k) 4 weeks: transverse section at level C1. M, midline. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4
Expression of chondroitinase in rat brain from different LVs at different times: coronal sections stained with Ab-2B6 as in Fig. 3. Scale bar, 1 mm. (a) LV-P, 2 weeks: there is widespread digestion on both sides (red arrows). White arrow marks the approximate position of the injection. (b) LV-D, 4 weeks: more circumscribed digestion within ∼0.5 mm of the injection track, but also strong digestion within the corpus callosum extending to the contralateral side. (c) LV-C, 8 weeks: patchy digestion in various regions, especially ipsilateral hippocampus (Hipp) and thalamus (Thal). (d) PBS-injected control, 4 weeks: no specific immunoreactivity. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Fig. 5
In animals given a spinal cord lesion, LV-ChABC injected into cerebral cortex enhances corticospinal axon growth towards the lesion and lateral axon sprouting. Horizontal sections of spinal cord at the level of the dorsal columns are shown, 4 weeks after lesion ± LV-P cortical injection, stained for BDA, which had been injected into cortex to trace the CST. Cranial at top; lesion cavity at bottom in (a–c). M, midline. Images are laterally inverted, so the right CST (labeled from the left cortex) is shown to the left. Scale is indicated in (a); scale bars 100 μm for other panels. (a) Methods to quantify retraction, regeneration and sprouting. Spinal cord at C3-C4 from a lesioned animal with no vector treatment. At bottom, grid for measuring longitudinal retraction/regeneration relative to the lesion front (the rostral edge of the lesion cavity). At top, grid for measuring lateral sprouting in gray matter (gm) relative to the edge of the white matter (wm). (b) Control spinal cord lesion without LV injection: axons have retracted from the cranial edge of the lesion cavity (retraction bulbs arrowed). (c) In animals which received LV-P cortical injection, axons grow along the lesion edge caudally (arrowed). (d) Spinal cord 1.0–1.5 mm above the lesion, just left of the midline, in a control lesioned animal; axons show little lateral sprouting. (e) Similar section in a LV-P-treated animal shows substantial lateral sprouting into the gray matter. (f) Measurements of longitudinal retraction/regeneration: percentage of axons which remain at various distances above and below the lesion front, for control and LV-treated CST (±SEM). Significance by t-test: *P < 0.05; **P < 0.01. (g) Measurements of lateral axon sprouting index (per section per 100 axons labeled, ±SD), for control and LV-treated CST. Significance, P < 0.05. (h) Diagram of the surgical procedures.
Fig. 6
Fig. 6
Intraspinal LV-ChABC injection promotes axon sprouting and growth in lesioned spinal cord. Procedures and illustration formats are as for Fig. 5, except that instead of LV-P injection into the brain, LV-C was injected into the spinal cord at the cranial and caudal edges of the lesion. (a) Diagram of the surgical procedures. (b–e) Horizontal sections of spinal cord, 4 weeks after lesion ± LV-C injection, stained for BDA, which had been injected into cortex to trace the CST. Cranial at top. M, midline; gm, gray matter; wm, white matter. Scale bars, 100 μm. (b) Control spinal cord lesion with saline injection: axons have retracted from the lesion front. (c) In a LV-C-treated animal, axons show little retraction, and some grow along the lesion edge. (d) Spinal cord 1.0–1.5 mm above the lesion in a control animal; axons show little lateral sprouting. (e) Robust axon lateral sprouting into the gray matter in a LV-C treated animal. (f) Measurements of longitudinal retraction/regeneration, at 4 weeks: percentage of axons which remain at various distances above and below the lesion front, for control and LV-treated CST (±SEM). Significance by t-test: *P < 0.05; **P < 0.01. (g) Measurements of lateral axon sprouting index (per section per 100 axons labeled, ±SD), for control and LV-treated CST, at 4 weeks (top) and in a separate group of animals at 2 weeks (bottom). Significance, P < 0.01.

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