Evaluation of the physical and in vitro protective activity of three synthetic peptides derived from the pro- and mature GDNF sequence.

Recently, a small 11-amino acid amidated peptide, dopamine neuron stimulating peptide-11 (DNSP-11), was shown to exert neurotrophic-like actions on primary dopaminergic neurons and in parkinsonian rat models. This suggests smaller neurotrophic-like molecules may be deliverable and modifiable for therapeutic use. Here we evaluate the molecular and cellular protection properties of DNSP-11 and two other amidated-peptides, a 5-mer (DNSP-5) and a 17-mer (DNSP-17), hypothesized to be endoproteolytically processed from the pro- and mature glial cell line-derived neurotrophic factor (GDNF) protein sequence, respectively. Far-UV circular dichroism spectra show that the three DNSPs are soluble and act independently in vitro. Reverse phase HPLC and mass spectrometry analysis show that the three peptides are stable for one month at a variety of storage and experimental conditions. To gain insight into their biodistribution properties in the brain, we used affinity chromatography to show that DNSP-17 binds heparin equally as tight as GDNF, whereas DNSP-5 and DNSP-11 do not bind heparin, which should facilitate their delivery in vivo. Finally, we present data showing that DNSP-11 provides dose-dependent protection of HEK-293 cells from staurosporine and 3-nitropropionate (3-NP) cytotoxicity, thereby supporting its broad mitochondrial-protective properties.

Dopamine neuron stimulating actions of a GDNF propeptide.

BACKGROUND: Neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), have shown great promise for protection and restoration of damaged or dying dopamine neurons in animal models and in some Parkinson's disease (PD) clinical trials. However, the delivery of neurotrophic factors to the brain is difficult due to their large size and poor bio-distribution. In addition, developing more efficacious trophic factors is hampered by the difficulty of synthesis and structural modification. Small molecules with neurotrophic actions that are easy to synthesize and modify to improve bioavailability are needed. METHODS AND FINDINGS: Here we present the neurobiological actions of dopamine neuron stimulating peptide-11 (DNSP-11), an 11-mer peptide from the proGDNF domain. In vitro, DNSP-11 supports the survival of fetal mesencephalic neurons, increasing both the number of surviving cells and neuritic outgrowth. In MN9D cells, DNSP-11 protects against dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA)-induced cell death, significantly decreasing TUNEL-positive cells and levels of caspase-3 activity. In vivo, a single injection of DNSP-11 into the normal adult rat substantia nigra is taken up rapidly into neurons and increases resting levels of dopamine and its metabolites for up to 28 days. Of particular note, DNSP-11 significantly improves apomorphine-induced rotational behavior, and increases dopamine and dopamine metabolite tissue levels in the substantia nigra in a rat model of PD. Unlike GDNF, DNSP-11 was found to block staurosporine- and gramicidin-induced cytotoxicity in nutrient-deprived dopaminergic B65 cells, and its neuroprotective effects included preventing the release of cytochrome c from mitochondria. CONCLUSIONS: Collectively, these data support that DNSP-11 exhibits potent neurotrophic actions analogous to GDNF, making it a viable candidate for a PD therapeutic. However, it likely signals through pathways that do not directly involve the GFRalpha1 receptor.