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. 2011 Jan;19(1):46-52.
doi: 10.1038/mt.2010.217. Epub 2010 Oct 19.

Delayed dominant-negative TNF gene therapy halts progressive loss of nigral dopaminergic neurons in a rat model of Parkinson's disease

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Delayed dominant-negative TNF gene therapy halts progressive loss of nigral dopaminergic neurons in a rat model of Parkinson's disease

Ashley S Harms et al. Mol Ther. 2011 Jan.

Abstract

Parkinson's disease (PD) is a progressive neurodegenerative disorder typified by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Recent evidence indicates that neuroinflammation may play a critical role in the pathogenesis of PD, particularly tumor necrosis factor (TNF). We have previously shown that soluble TNF (solTNF) is required to mediate robust degeneration induced by 6-hydroxydopamine (6-OHDA) or lipopolysaccharide. What remains unknown is whether TNF inhibition can attenuate the delayed and progressive phase of neurodegeneration. To test this, rats were injected in the SNpc with lentivirus encoding dominant-negative TNF (lenti-DN-TNF) 2 weeks after receiving a 6-OHDA lesion. Remarkably, when examined 5 weeks after the initial 6-OHDA lesion, no further loss of nigral DA neurons was observed. Lenti-DN-TNF also attenuated microglial activation. Together, these data suggest that TNF is likely a critical mediator of nigral DA neuron death during the delayed and progressive phase of neurodegeneration, and that microglia may be the principal cell type involved. These promising findings provide compelling reasons to perform DN-TNF gene transfer studies in nonhuman primates with the long-term goal of using it in the clinic to prevent the delayed and progressive degeneration of DA neurons that gives rise to motor symptoms in PD.

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Figures

Figure 1
Figure 1
Experimental design and outcome measures. Rat striata were injected unilaterally with saline or 6-OHDA at week 0. Before surgery and again at weeks 1, 3, and 5, motor asymmetry was assessed using the cylinder test. A subset of rats (delayed inhibition) received a second injection of either lenti-GFP or lenti-DN-TNF 2 weeks after the striatal 6-OHDA lesion. Unbiased stereology was used to estimate DA neuron number within the substantia nigra pars compacta at 2 or 5 weeks after the 6-OHDA lesion. IHC, immunohistochemistry; DN-TNF, dominant-negative tumor necrosis factor; GFP, green fluorescent protein; 6-OHDA, 6-hydroxydopamine.
Figure 2
Figure 2
Effects of delayed DN-TNF gene transfer on nigral DA neuron survival. Total numbers of TH and NeuN double-positive cells at 2 and 5 weeks after 6-OHDA lesion were estimated by unbiased stereology and analyzed using a one-way analysis of variance. Data are reported as the mean ± SEM. Fisher's post hoc test was used to assess significance. Bars with different letters are statistically significantly different from each other (P < 0.05). Group N's: saline/GFP–5 weeks (n = 6); 6-OHDA– 2 weeks (n = 5); 6-OHDA/GFP–5 weeks (n = 7); 6-OHDA/DN-TNF–5 weeks (n = 9). DA, dopaminergic; DN-TNF, dominant-negative tumor necrosis factor; GFP, green fluorescent protein; NeuN, neuronal nuclei; 6-OHDA, 6-hydroxydopamine; TH, tyrosine hydroxylase.
Figure 3
Figure 3
Impact of delayed DN-TNF gene transfer on motor asymmetry in 6-OHDA lesioned rats. At weeks 0, 1, 3, and 5, 6-OHDA-lesioned animals were tested for deficits in forelimb asymmetry using the cylinder test. A repeated measures analysis of variance was used to examine potential differences between groups. Data are plotted as contralateral forelimb use as a percentage of total and expressed as mean ± SEM. Group N's: saline/GFP–5 weeks (n = 6); 6-OHDA–2 weeks (n = 5); 6-OHDA/GFP–5 weeks (n = 7); 6-OHDA/DN-TNF–5 weeks (n = 9). DN-TNF, dominant-negative tumor necrosis factor; GFP, green fluorescent protein; 6-OHDA, 6-hydroxydopamine.
Figure 4
Figure 4
Immunofluorescent localization of GFP+ cells, Iba1+ microglia, and DN-TNF protein in rats receiving DN-TNF gene transfer. (a,b) Anti-GFP immunofluorescence staining (green) in cells of glial morphology transduced in substantia nigra pars compacta (SNpc; white box). (c) Confocal microscope Z-stack of cells immunopositive for the microglial marker Iba1 (Alexa-546, red) and for hDN-TNF (Alexa-488, green) in SNpc. a, bar = 400 µm; b, bar = 200 µm; c, bar = 20 µm. DN-TNF, dominant-negative tumor necrosis factor; GFP, green fluorescent protein.
Figure 5
Figure 5
Effects of nigral DN-TNF gene transfer on microgliosis and astrogliosis. (a) Activated astrocytes were identified by GFAP immunostaining (green) in the striatum. The area lesioned by the 6-OHDA neurotoxic is highlighted with a white circle. (b,c) Microglia were identified by Iba1 immunostaining (green) in the substantia nigra pars compacta (SNpc) area identified by tyrosine hydroxylase immunostaining (red). a, bar = 400 µm; b, bar = 100 µm; in c, bar = 200 µm. (d) Quantification of microglia in SNpc of 6-OHDA/lenti-GFP and 6-OHDA/lenti-DN-TNF-injected rats. Values represent the mean ± SEM of Iba1+ microglia within SNpc calculated from three separate brain sections (12 random fields/section) using threshold analysis in three animals per treatment group. Two-way analysis of variance for comparison of 6-OHDA/lenti-GFP and 6-OHDA/lenti-DN-TNF with Fisher's protected least significant difference post hoc test (###P < 0.001). DN-TNF, dominant-negative tumor necrosis factor; GFP, green fluorescent protein; 6-OHDA, 6-hydroxydopamine.

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References

    1. Frank-Cannon TC, Alto LT, McAlpine FE., and, Tansey MG. Does neuroinflammation fan the flame in neurodegenerative diseases. Mol Neurodegener. 2009;4:47. - PMC - PubMed
    1. McGeer PL., and, McGeer EG. Glial reactions in Parkinson's disease. Mov Disord. 2008;23:474–483. - PubMed
    1. Aggarwal BB, Samanta A, Feldmann M.2000. TNFα. In: Oppenheim JJ, Feldman M (eds) Cytokine Reference. Academic: New York. 414–434.
    1. Boka G, Anglade P, Wallach D, Javoy-Agid F, Agid Y., and, Hirsch EC. Immunocytochemical analysis of tumor necrosis factor and its receptors in Parkinson's disease. Neurosci Lett. 1994;172:151–154. - PubMed
    1. Mogi M, Harada M, Riederer P, Narabayashi H, Fujita K., and, Nagatsu T. Tumor necrosis factor-α (TNF-α) increases both in the brain and in the cerebrospinal fluid from parkinsonian patients. Neurosci Lett. 1994;165:208–210. - PubMed

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