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. 2000 Nov 7;97(23):12804-9.
doi: 10.1073/pnas.210350097.

Sparing of neuronal function postseizure with gene therapy

J McLaughlin  1 , B Roozendaal, T Dumas, A Gupta, O Ajilore, J Hsieh, D Ho, M Lawrence, J L McGaugh, R Sapolsky
Affiliations

Sparing of neuronal function postseizure with gene therapy

J McLaughlin et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A 2001 Jan 30;98(3):1317

Abstract

Numerous studies have demonstrated that gene therapy interventions can protect neurons from death after neurological insults. In nearly all such studies, however, "protection" consists of reduced neurotoxicity, with no demonstrated preservation of neuronal function. We used a herpes simplex virus-1 system to overexpress either the Glut-1 glucose transporter (GT) (to buffer energetics), or the apoptosis inhibitor Bcl-2. Both decreased hippocampal neuron loss to similar extents during excitotoxic insults in vitro and in vivo. However, the mediating mechanisms and consequences of the two interventions differed. GT overexpression attenuated early, energy-dependent facets of cell death, blocking oxygen radical accumulation. Bcl-2 expression, in contrast, blocked components of death downstream from the energetic and oxidative facets. Most importantly, GT- but not Bcl-2-mediated protection preserved hippocampal function as assessed spatial maze performance. Thus, gene therapeutic sparing of neurons from insult-induced death does not necessarily translate into sparing of function.

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Figures

Figure 1
Figure 1
(A) Significant reduction in KA-induced neurotoxicity in cultured hippocampal neurons by overexpression of GT (Left) or Bcl-2 (Right). n = 20–30/group. (B) Significant reduction in KA-induced hippocampal neurotoxicity by GT or Bcl-2. n = 10–15/group. *, P < 0.05; **, P < 0.02; ***, P < 0.001, by t test.
Figure 2
Figure 2
(A) Effects of GT (Left) or Bcl-2 (Right) on ROS accumulation post-KA. Values are expressed as percentage above that in mock-infected control cultures without KA. In control vector-treated wells, KA significantly increased ROS accumulation (P < 0.05 and 0.01 in GT and Bcl-2 studies, respectively, post hoc test after two-way ANOVA). In GT-infected wells, KA did not significantly increase accumulation, and in either condition (±KA), there was significantly less accumulation than in cognate control wells (P < 0.01 for both). In contrast, KA significantly increased accumulation in Bcl-2-treated wells (P < 0.05), and values did not differ significantly from the cognate control wells. (B) Effects of GT or Bcl-2 on metabolism in primary hippocampal cultures under hypoglycemic conditions as assessed by proton efflux rates, measured by microphysiometry. vIE1GT (Left) attenuated the drop of metabolism posthypoglycemia (***, P < 0.001 by post hoc test after two-way ANOVA, comparing experimental vector versus control vector at the same time point), whereas vα22βgalα4bcl-2 (Right) had no effect. Data on the left previously published (7), making use of a related HSV vector expressing either GT (vIE1GT) or β-Gal as a reporter gene (vIE1βGal).
Figure 3
Figure 3
Effects of GT (Center) and Bcl-2 (Right) on retention impairment in a submerged (A) and visible platform test (B) in a water maze produced by KA-induced lesions. Data were from the same rats used for the lesion data in Fig. 1B. *, P < 0.05; **, P < 0.01, by post hoc test after ANOVA.
Figure 4
Figure 4
(A) Immunoblot of immunoprecipitated cell lysates from cultures infected with Bcl-2 vector (bcl-2 +) or β-gal control vector (Bgal + ). The arrow indicates the bcl-2 protein band at approximately 30 kDa. (B and C) Cells double labeled for Bcl-2 (B) and β-gal (IC). ×200 magnification.
Figure 5
Figure 5
Equivalent patterns of expression and of decreasing lesion size after treatment with GT and Bcl-2. (A) X-Gal-stained neurons (×100) in representative sections (counterstained with Cresyl violet) from a rat receiving GT (Upper) or Bcl-2 (Lower). Cell bodies and large diameter dendrites of granule cells in the superior blade of the dentate gyrus are visibly stained. (B) Percentage of total X-Gal-positive cells in 1-mm regions along the anterior/posterior (A/P) axis of the dentate gyrus (n = 9/group). Mean percentage of total lesion differed between areas [F(3,64) = 27.23, P < 0.001] but not between groups. Zero on the x axis refers to the injection site. (C) Percentage of X-Gal-positive cells on the superior blade of the dentate gyrus grouped into three equal regions along the medial/lateral (M/L) axis (n = 9/group). Mean percentage of total lesion differed between areas [F(2,48) = 10.01, P < 0.001] but not between groups. The number “1” on the x axis refers to the most medial third. (D) Percentage of total lesion in 1-mm regions along the A/P axis for hippocampi treated with KA and Glut-1 or Bcl-2 (n = 5/group). Mean percentage of total lesion differed between areas [F(3,32) = 18.22, P < 0.001], but not between groups. (E) Percentage of total lesion grouped into three equal regions along the M/L axis (n = 5/group). Mean percentage of total lesion differed between areas [F(2,24) = 51.93, P < 0.001], but not between groups. The number “1” on the x axis refers to the most medial third.
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