Volume 17, Issue 6, Pages 980-991 (June 2009) Nigrostriatal rAAV-mediated GDNF Overexpression Induces Robust Weight Loss in a Rat Model of Age-related Obesity  Fredric P Manfredsson, Nihal Tumer, Benedek Erdos, Tessa Landa, Christopher S Broxson, Layla F Sullivan, Aaron C Rising, Kevin D Foust, Yi Zhang, Nicholas Muzyczka, Oleg S Gorbatyuk, Philip J Scarpace, Ronald J Mandel  Molecular Therapy  Volume 17, Issue 6, Pages 980-991 (June 2009) DOI: 10.1038/mt.2009.45 Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 1 GDNF distribution and viral transduction pattern. (a) Sections from an animal injected in the SNc with the CBA-GDNF/HSV-TK-GFP virus were stained for GDNF to show the level of transduction of the entire striatum using bright field microscopy, and fluorescence to show the transduction pattern with precise anatomical tracing of the tract as assessed by GFP (green) and the distribution of GDNF (red) relative to the GFP+ fibers. (b) At the level of the SN, GDNF immunoreactivity was observed throughout the SN both intra- and extracellularly, with transgene observed relatively distal to the transduced cells, arrows point to the substantia nigra pars compacta (SNc). (c) Higher magnification of area outlined in b. (d–f) GDNF immunoreactivity was observed in a wide area adjacent to the MFB throughout the nigrostriatal tract, including the posterior portions of the lateral hypothalamus. (e,f) Increased magnifications of area outlined in d. (g–i) GDNF and GFP expression at the level of the anterior hypothalamus. (i) Higher magnification of area outlined in h. Conversely, no GDNF or GFP expression was observed in the contralateral nigrostriatal tract. (j) In the terminal region of the striatum, GDNF immunoreactivity was seen throughout the striatum while a majority of GFP+ fibers were observed in the ventral portions of the striatum. (k) Higher magnification of area outlined in j. (l) Again, no GDNF or GFP expression was observed in the contralateral hemisphere. Bars in a–c = 1 mm, d, g, h, j, and l = 0.5 mm, in c, e, i, and k = 0.1 mm, and in e = 25 µm. AC, anterior commissure; fx, fornix; GDNF, glial cell line–derived neurotrophic factor; GFP, green fluorescent protein; HSV-TK, herpes simplex virus thymidine kinase; ic, internal capsule; LV, lateral ventricle; MFB, medial forebrain bundle; mt, mammillothalamic tract; STR, striatum; SNr, substantia nigra pars reticulata; VTA, ventral tegmental area; 3v, third ventricle. Molecular Therapy 2009 17, 980-991DOI: (10.1038/mt.2009.45) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 2 Animal measures. (a) Weekly change in body mass and weekly FI (b) before and after rAAV2/5-administration (week 0). Open circles represent the hyp-GFP group (n = 7), open triangles SN-GFP (N = 8), closed circles hyp-GDNF (n = 6), and closed triangles SN-GDNF (n = 7). The SN-GDNF group displayed a significant decrease in body weight as compared to the GFP control from week 2 (*) and when compared to the hyp-GDNF (#) from week 3. The hyp-GDNF group displayed a significant weight loss as compared to the GFP control from week 6 (%) (P < 0.01). There was also a transient decrease in FI (b) in the SN-GDNF animals when compared to the GFP control (25%) 2–5 weeks following the rAAV2/5 injection (*) and the hyp-GDNF group (11%) (weeks 2,3) (#). The hyp-GDNF-injected animals consumed 11% less food 3–4 weeks following the injections as compared to the GFP control (%) (P < 0.001). Cumulative FI postinjection (b-inset) was significantly lower in the SN-GDNF group when compared to the GFP control and the hyp-GDNF group (P < 0.001). Activity measures (c) done at ∼45 days postsurgery indicate no significant differences in activity among the groups. (d) ELISA measurements specific for the transgene indicate that there was no difference due to injection site, however, hypothalamic GDNF levels were >50-fold in measurements taken from the striatum and NAcc. ELISA, enzyme-linked immunosorbent assay; FI, food intake; GDNF, glial cell line–derived neurotrophic factor; GFP, green fluorescent protein. Molecular Therapy 2009 17, 980-991DOI: (10.1038/mt.2009.45) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 3 GDNF expression in the hyp-GDNF group. Sections were stained for GDNF expression using a transgene specific antibody. Images were taken throughout the entire axis of transgene expression (a) stretching from the striatum, (b) to the midbrain, (c–e) throughout the hypothalamus. (a) GDNF expression in the area of the striatum at approximately the level of bregma. (b) GDNF expression at the level of the midbrain. (Bregma −4.8 mm). (c) GDNF staining at the anterior (bregma −2.8 mm), (d) medial (bregma −3.6 mm) and posterior (bregma −4.2 mm), (e) portions of the hypothalamus. AC, anterior commissure; BedNu, bed nucleus of the stria terminalis; GDNF, glial cell line–derived neurotrophic factor; MFB, medial forebrain bundle; mm, medial mammilary nucleus; mp, mammillary peduncle; MS, medial septum; Str, striatum, SN, substantia nigra; Bar = 1 mm. Molecular Therapy 2009 17, 980-991DOI: (10.1038/mt.2009.45) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 4 Phosphorylated-ERK immunoreactivity as a result of GDNF overexpression. Sections were stained using a p-ERK specific antibody. (a) Hypothalamic rAAV2/5-GDNF injections resulted in a significant increase in ERK phosphorylation in the hypothalamus. (b) Higher magnification of area indicated in a. (c,d) However, hypothalamic injections with rAAV2/5-GFP yielded no detectable p-ERK in the hypothalamus. (d) Higher magnification of area outlined in c. (e,f) Likewise, injections in the SN with rAAV2/5-GDNF resulted in p-ERK immunoreactivity in the SNc as well as (g,h) in the hypothalamus (g,h). Panels f and h are high magnification images of areas indicated in e and g, respectively. Bars in a and c = 1 mm, b, d, f, and h = 50 µm, e, g = 0.5 mm. GDNF, glial cell line–derived neurotrophic factor; p-ERK, phosphorylated extracellular signal-regulated kinase. Molecular Therapy 2009 17, 980-991DOI: (10.1038/mt.2009.45) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 5 Anatomical distribution of p-ERK immunoreactivity. (a–f) Animal injected in the SNc with rAAV-GDNF was analyzed for oxytocin and p-ERK immunoreactivity to characterize the subset of activated paraventricular neurons. The area of p-ERK immunoreactivity (red) was largely distinct from that of oxytocin (blue). (a) Mapping adapted from Swanson.49 (b,c) Individual channels of a. (d–f) Increased magnification of a–c. Yellow arrow identifies the same oxytocin expressing cell in all panels. Green arrow identifies the same p-ERK expressing cell in all panels. Abbreviations are all for subdivisions of the PVH: dp, dorsal parvocellular; pml, posterior magnicellular; mpv, medial parvocellular ventral zone; mpd, medial parvocellular dorsal zone. (g,h) Injections of rAAV2/5-GFP in the SN did not result in hypothalamic p-ERK immunoreactivity. (h) Higher magnification of area outlined in g. Bars in a–f = 0.1 mm and g, h = 0.5 mm. GDNF, glial cell line–derived neurotrophic factor; PVH, paraventricular hypothalamic; rAAV, recombinant adeno-associated virus. Molecular Therapy 2009 17, 980-991DOI: (10.1038/mt.2009.45) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 6 Catecholamine analysis. (a) DA analysis of the striatum indicated a slightly lower DA content in the SN-GFP (n = 7) injected animals as compared to the hyp-GFP group (n = 6) (P < 0.05 by ANOVA) and the SN-GDNF group (n = 6) (P = 0.07). (b) No differences were observed in hypothalamic DA content in-between the various groups. (c) DA content in the NAcc was significantly elevated in the hyp-GDNF group when compared to the SN-GDNF group, in addition, the hyp-GFP group contained slightly higher DA levels when compared to the SN-GFP control (P < 0.05 by ANOVA). (d) Evaluation of serum norepinephrine content indicated a significant increase in the SN-GDNF group when compared to the GFP control (P < 0.05 by ANOVA). (e,f) Low power photomicrographs showing no GDNF-induced alteration of nigral TH expression. TH immunoreactivity in animal injected in the (e) SN with rAAV2/5-GDNF, and (f) rAAV2/5-GFP f. Bars in e,f = 1 mm. ANOVA, analysis of variance; DA, dopamine; GDNF, glial cell line–derived neurotrophic factor; GFP, green fluorescent protein. Molecular Therapy 2009 17, 980-991DOI: (10.1038/mt.2009.45) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 7 Protein and mRNA analysis. Measurement of (a) TH mRNA levels and (b) protein levels in the adrenal gland indicated no significant difference among the various groups. (c) Evaluation of hypothalamic TH protein content indicated a slight but significant (P = 0.03 by ANOVA) increase in the hyp-GFP (n = 6) group as compared to the SN-GFP (n = 6) group. (d) Levels of AT1 protein in the hypothalamus was roughly equal among all groups except from the hyp-GDNF group, this result was not significant, however (P = 0.1 by ANOVA). (e) The SN-GDNF (n = 6) group displayed dramatically elevated levels (twofold increase) of NPY mRNA in the adrenal gland when compared to the GFP control (n = 6) and the hyp-GFP group (n = 6) (P < 0.0001 by ANOVA). ANOVA, analysis of variance; GDNF, glial cell line–derived neurotrophic factor; GFP, green fluorescent protein; mRNA, messenger RNA; NPY, neuropeptide Y; TH, tyrosine hydroxylase. Molecular Therapy 2009 17, 980-991DOI: (10.1038/mt.2009.45) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions