BackgroundNeurotrophic 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 FindingsHere 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.ConclusionsCollectively, 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 GFRα1 receptor.

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 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 the DNSPs 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.

A major challenge of systems biology is explaining complex traits, such as the biological clock, in terms of the kinetics of macromolecules. The clock poses at least four challenges for systems biology: (i) identifying the genetic network to explain the clock mechanism quantitatively; (ii) specifying the clock's functional connection to a thousand or more genes and their products in the genome; (iii) explaining the clock's response to light and other environmental cues; and (iv) explaining how the clock's genetic network evolves. Here, the authors illustrate an approach to these problems by fitting an ensemble of genetic networks to microarray data derived from oligonucleotide arrays with approximately all 11 000 Neurospora crassa genes represented. A promising genetic network for the clock mechanism is identified.

Dopamine neuron stimulating peptide‐11 (DNSP‐11), an eleven amino acid amidated peptide derived from the glial cell‐line derived neurotrophic factor (GDNF) prosequence, has been shown recently to provide neuroprotective and neurorestorative effects in dopaminergic cell culture and parkinsonian rat model systems. However, unlike GDNF, DNSP‐11 was found to prevent staurosporine‐induced toxicity and cytochrome c release from mitochondria in nutrient‐deprived dopaminergic B65 cells. The objective of this study is to investigate the neuroprotective effects of DNSP‐11 on the mitochondria. We treated dopaminergic cell lines with mitochondrial‐specific toxins and evaluated DNSP‐11’s protective effects by measuring mitochondrial potential, caspase‐3 activity, and TUNEL staining assays. In addition, proteomic profile analyses were performed to determine changes in the membrane, cytosolic and mitochondrial proteins involved in DNSP‐11 protection. These data support a mitochondrial neuroprotective hypothesis and further suggest that DNSP‐11 is a promising candidate for further evaluation as a downstream therapeutic for age‐related neurogenerative diseases, like Parkinson's disease. Support provided by: NIH COBRE Pilot (P20RR20171), NIA (T32 AG000242), NIDA (T32 DA022738), NINDS (NS039787), PhRMA Foundation, and University of Kentucky College of Medicine Start‐up Funds.