Figure 1.
Sequence origin and homology of DNSP-11.
(A) DNSP-11 (filled) is an 11 amino acid sequence present in the proprotein region of the 211 amino acid human pre-proGDNF sequence. After cleavage of the pre-signal sequence (gray), DNSP-11 is predicted to be cleaved from the proprotein at flanking dibasic cleavage sites by endopeptidases. Further predicted processing yields the C-terminal amidated peptide. The N-terminal (striped) and C-terminal (checkered) proprotein fragments and mature GDNF (open) protein are shown. The sequence figure is not drawn to scale to highlight the processing of DNSP-11. (B) DNSP-11 shows high sequence homology to the rat and mouse proGDNF sequences suggesting a conserved function. (C) In vivo expression of the DNSP-11 sequence in the substantia nigra of the ventral mesencephalon from rat pups at PN10. Rows indicate the type of stain: the top panel is DNSP-11 (green); the middle panel is a dopaminergic neuron marker, tyrosine hydroxylase (TH+, red); the bottom panel represents a merged image of the previous stains (yellow). The bottom panel demonstrates co-localization of DNSP-11 sequence within dopaminergic cell bodies at PN10. The scale bar represents 30 µm.
Figure 2.
Uptake of DNSP-11 into the rat substantia nigra 30 minutes after a single injection.
The fluorescent immunostaining for DNSP-11 (A), TH (B) and the two photomicrographs merged (C) show that DNSP-11 is taken up by neurons in both the substantia nigra, pars reticulata (SNr) and substantia nigra, pars compacta (SNc). TH-positive dopamine neurons (B) populate the SNc and the ventral tegmental area (VTA). Figures D-F are higher power micrographs from the SNc. Immunostaining (D) revealed uptake of DNSP-11 into the perikaryon (large arrow), nucleus (small arrow), and neurites of TH+ cells (E), which appear yellow in the merged imaged (F), including the two cells denoted by arrowheads. The injection site is indicated (star). Scale bar = 200 µm in A-C; 15 µm in D-F.
Figure 3.
Neurotrophic effects of DNSP-11 and GDNF in Primary Dopaminergic Neurons.
(A) E14 rat embryo primary dopaminergic neurons from the ventral mesencephalon were grown for 5 days in vitro and neurotrophic molecules were added at each media change, including initial plating and day 2. GDNF (open bars) and DNSP-11 (blue bars) were added at various concentrations (0.03, 0.1, 1.0 and 10 ng/ml; 10 mM citrate buffer +150 mM NaCl, pH 5) and were seen to significantly increase TH+ neuron counts (+ SE; one-way ANOVA with Newman-Keuls post hoc analysis, *p<0.05 and **p<0.01) (B) Photographs of treated E14 primary dopaminergic neurons demonstrating that both GDNF and DNSP-11 treated cells (0.1 ng/ml) displayed enhanced cell survival, neurite length, and total number of branches.
Table 1.
E14 Primary mesencephalic neuron survival and morphological data following treatment with GDNF and DNSP-11.
Figure 4.
Neurotrophic effects of DNSP-11 in vivo.
(A) 28 days after DNSP-11 (30 µg) or citrate buffer vehicle was delivered to the nigral cell bodies, neurochemical studies were carried out in the ipsilateral striatum with in vivo microdialysis and levels of DA and its metabolites, DOPAC and HVA were determined. The DNSP-11 treatment group showed significantly higher basal neurochemical concentrations of DA, DOPAC and HVA. Basal DA increased from 26.0±2.7 nM in the vehicle treatment group to 45.8±7.7 nM in the DNSP-11 treatment group (t(31) = 2.255, p = 0.0314). Basal concentrations of DOPAC increased from 3355±338 nM in the vehicle group to 6544±836 nM in the DNSP-11 group (t(31) = 3.293, p = 0.0025), and HVA, increased from 2419±251 nM with vehicle treatment to 4516±502 nM with DNSP-11 treatment (t(30) = 3.588, p = 0.0012). All data were analyzed using a two-tailed unpaired t-test *p<0.05. (B) Apomorphine (0.05 mg/kg) induced rotational behavior was assessed prior to infusion treatment (Pre) and once weekly for 4 weeks after DNSP-11 (100 µg) or vehicle treatment. Drug-induced rotational behavior is expressed as a percentage of vehicle treatment and showed a significant decrease in rotational behavior beginning one week after DNSP-11 treatment that lasted for all 4 weeks post DNSP-11. The data were analyzed using a one-way ANOVA for repeated measures (F(4,39) = 4.807, p = 0.0005) with Bonferroni's multiple comparison test *p<0.05, **p<0.01, ***p<0.001. (C) DNSP-11 treatment significantly increased levels of DA, (74%) and DOPAC (132%) in the substantia nigra of unilateral 6-OHDA-lesioned rats. DA content was determined to be 34.7±6.4 ng/g in the vehicle treatment group and 59.1±7.3 ng/g in the DNSP-11 treatment group (t(13) = 2.521, p = 0.0265). DOPAC tissue content was determined to be 7.10±1.40 ng/g in the vehicle treatment group and 16.48±4.01 ng/g (t(13) = 2.33, p = 0.0364) in the DNSP-11 treatment group. All data were analyzed using a two-tailed unpaired t-test, * p<0.05.
Figure 5.
DNSP-11 functions differently than GDNF.
Both DNSP-11 and GDNF protect against 6-OHDA toxicity as demonstrated by reductions in TUNEL staining at 24 h (A) and caspase-3 (B) activity at 3 h after 6-OHDA exposure. MN9D dopaminergic cells were incubated for 1 hour with either citrate buffer (control), 1 ng/mL of DNSP-11 or GDNF prior to 100 µM 6-OHDA exposure for 15 min. Data are + SD, one-way ANOVA with Tukey's post hoc analysis, *p<0.05, **p<0.01, ***p<0.001 vs. control; #p<0.05, ##p<0.01, ###p<0.001 vs. 6-OHDA. (C) DNSP-11 reduces staurosporine- induced cytotoxicity (1 µM) by ∼125% as measured by LIVE/DEAD® assay in dopaminergic B65 cells, whereas GDNF offers no protection at 20 hours after treatment. Control was citrate buffer alone; GDNF and DNSP-11 were added at 1 ng/mL. STS-staurosporine. (D) Similarly, DNSP-11 reduces gramicidin-induced cytotoxicity (1 µM) by ∼60%, whereas GDNF offers no protection at 20 hours after treatment. One-way ANOVA used to test for significance among groups, followed by Tukey's post hoc analysis (*p<0.05, **p<0.01, ***p<0.001 vs control; #p<0.05, ##p<0.01, ###p<0.001 vs toxin). Gram-gramicidin.
Table 2.
Identified cytosolic proteins pulled-down by DNSP-11 from substantia nigra of Fischer 344 rats.
Figure 6.
DNSP-11 prevents staurosporine-induced cytochrome c release from mitochondria.
Confocal microscopy images demonstrate that DNSP-11 prevents cytochrome c (green) release from mitochondria (red) of B65 cells, supporting its protection from staurosporine-induced cytotoxicity (Figure 5C). GDNF does not prevent cytochrome c diffusion from the mitochondria when exposed to 1 µM staurosporine. The control was citrate buffer alone. The nucleus is stained blue in all images.