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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 20, 2001 as doi:10.1096/fj.00-0603fje. |
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Department of Neurosciences, School of Medicine, Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0634, USA
2Correspondence: Department of Neurosciences, School of Medicine, Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0634, USA. E-mail: jsobrien{at}ucsd.edu
SPECIFIC AIMS
Our aim was to evaluate the efficacy of ProsaptideTM D5, a bioactive protease-resistant 11-mer retro-inverso trophic peptidomimetic of prosaposion, in a model of Parkinson’s disease (PD). Experiments were devised to test the efficacy of ProsaptideTM D5 both in vitro and in vivo in the rescue of dopaminergic (DA) neurons from MPP+/MPTP toxicity, a specific toxin for DA neurons.
PRINCIPAL FINDINGS
1. ProsaptideTM D5 promoted neurite outgrowth and
rescued DA neuron from MPP+ toxicity in the primary culture
of DA neurons
The addition of MPP+ (48 h incubation at 20
µM) significantly inhibited neurite outgrowth of primary DA neurons;
after addition of MPP+, the average number of
neurites per neuron decreased to 55% (primary neurites) and 34%
(secondary neurites) of controls, respectively, and the neurites were
also shorter in length (Fig. 1B
). ProsaptideTM D5 treatment (1 ng/ml)
promoted neurite outgrowth in the presence of
MPP+ (Fig. 1C
, D
); the
numbers of both primary and secondary neurites were normalized after
the addition of ProsaptideTM D5 (P >
0.05 vs. controls). At the highest dose of
ProsaptideTM D5 tested (10 ng/ml), an abundant
neurite network was apparent (Fig. 1D
) and the number of
secondary neurites was further increased (P < 0.05 vs.
controls). The survival of DA neurons after
MPP+ addition was reduced to 56% of control
wells (P < 0.001 vs. controls).
ProsaptideTM D5 dose-dependently increased the number
of surviving DA neurons in the presence of added
MPP+; after ProsaptideTM D5
treatment, the number of surviving DA neurons exceeded those of the
controls. At the highest dose tested (10 ng/ml), treatment with
ProsaptideTM D5 increased the survival of DA
neurons by 284% in the MPP+ wells and by 158%
in the control wells. Compared with GDNF (1 ng/ml), a well-known
neurotrophic factor for DA neurons, ProsaptideTM
D5 had the same bioactivity to rescue DA neurons from
MPP+ toxicity in a dose-dependent manner. An
inert all-D linear peptide D1 was not effective in these experiments.
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The addition of PD98059 (25 µM), an inhibitor of the MAP kinase
pathway (Fig. 1E
, F
, G
, H
), blocked the effect of
ProsaptideTM D5 on DA neuron survival and neurite
sprouting, suggesting that it is a crucial pathway involved in neurite
sprouting and survival of DA neurons.
In DA high-affinity uptake experiments, addition of MPP+ decreased DA uptake by 37% compared to controls after 48 h of incubation (P < 0.01 vs. controls). After ProsaptideTM D5 treatment, uptake activity was increased in a dose-dependent manner and normalized at the highest dose of ProsaptideTM D5 (10 ng/ml) (P > 0.05 vs. controls).
2. ProsaptideTM D5 rescued DA neurons dose-dependently
in MPTP-treated C57BL/6 mice
We then studied MPTP-treated mice to investigate the effect
of ProsaptideTM D5 in vivo.
There was a significant loss of the DA neurons in the substantia nigra
2 wk after MPTP treatment (Fig. 2B
), i.e., about 30% of the control group.
ProsaptideTM D5 injected subcutaneously (every
other day for 2 wk; 7 injections) reduced the loss of DA neurons from
MPTP toxicity in a dose-dependent manner; the number of DA neurons in
the substantia nigra was restored to the control level at a high dose
of ProsaptideTM D5 (200 µg/kg). An all-D linear
peptide D1 was ineffective in the same model.
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CONCLUSIONS
Various growth factors have been identified to support injured DA neurons, such as basic fibroblast growth factor, insulin growth factor, epithelial growth factor, transforming growth factor, brain-derived neurotrophic factor, ciliary neurotrophic factor, and neurotrophin (NT 4/5). GDNF was reported to promote the survival and differentiation of DA neurons that result in extensive neurite outgrowth and increased high-affinity DA uptake. One major therapeutic drawback is the delivery of GDNF into the brain; the blood–brain barrier excludes GDNF from crossing into the central nervous system. We report here that ProsaptideTM D5 was as bioactive as GDNF in rescue of DA neurons in vitro and in vivo. ProsaptideTM D5 is a unique peptidomimetic trophic factor that has been shown to be effective on a PD model when given subcutaneously.
Our strategy was to initially focus on protective treatment; ProsaptideTM D5 is a small (1.13 kDa), stable, bioactive retro-inverso peptidomimetic that entered the brain intact when given systemically. This strategy was successful in rescuing DA neurons in the MPTP model of Parkinson’s disease. At the optimal dose used, ProsaptideTMD5 treatment rescued 93% of the DA neurons in the substantia nigra from MPTP-induced death over 2 wk. In our experiments, after 2 wk the number of DA neurons that were reduced in the MPTP mouse model averaged 70%. In human studies, positron emission tomography measuring the DA terminal transporter has shown that such terminals were reduced by 40% in the putamen of PD patients as compared with normal individuals. Thus, the toxic effect of MPP+ on the DA neurons in our experiments was similar to the damage seen in humans with PD.
In conclusion, this report focused on a short 15-day treatment regimen using a well-known PD model. For potential therapeutic application for PD patients, further experiments are planned to assess the efficacy of ProsaptideTM D5 treatment in higher animals and in other models of PD.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0603fje ; to cite this
article, use FASEB J. (February 20, 2001) 10.1096/fj.00-0603fje 
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