benzyloxycarbonyl-isoleucyl-glutamyl(o-tert-butyl)-alanyl-leucinal and Nerve-Degeneration

Repeated subcutaneous administration of proteasome inhibitor 1 [PSI, Z-Ile-Glu(OtBu)-Ala-Leu-CHO] to rats causes progressive motor deficits and nigral dopaminergic cell loss in our laboratories, but this is controversial since others have not reproduced these findings. For this reason, we have investigated the role that the dose of PSI and its route of administration have on motor activity and neuronal loss in rat brain. PSI (8, 12 or 16 mg/kg, s.c.) was administered to female Wistar rats on 6 occasions on alternative days over 2 weeks. Subsequently PSI (8 mg/kg) was administered by oral, s.c. and i.p. routes on alternate days to separate groups of animals. Rats were assessed for motor function on a weekly basis up to 5 months after the end of PSI treatment. Locomotor activity was decreased following s.c. administration of 8 and 12 mg/kg PSI but not following 16 mg/kg. In subsequent experiments PSI (8 mg/kg) decreased motor activity after p.o. but not i.p. administration. PSI 8 mg/kg s.c. or p.o., but not i.p., caused neuronal loss in the substantia nigra, raphe nuclei, locus coeruleus, nucleus basalis of Meynert and dorsal motor nucleus of vagus. These data confirm that systemic administration of PSI reduces locomotor activity in rats and induces widespread neuronal degeneration in brain. However, the effects of PSI and its time course of action are dose and route dependent.

Topics: Administration, Oral; Analysis of Variance; Animals; Brain; Cell Count; Dose-Response Relationship, Drug; Female; Immunohistochemistry; Injections, Intraperitoneal; Injections, Subcutaneous; Motor Activity; Nerve Degeneration; Neurons; Oligopeptides; Rats; Rats, Wistar; Tyrosine 3-Monooxygenase

Systemic administration of the proteasomal inhibitor I (PSI) to rats was reported to cause progressive nigral dopaminergic neuronal loss but this is disputed. A major controversy centres over the use of manual counting of tyrosine hydroxylase (TH) positive neurons at the level of third cranial nerve as opposed to employing systematic stereological analysis of cell loss in the entire substantia nigra (SN). To provide a method of marking SN neurones independent of protein expression, fluorogold (FG) was stereotaxically injected bilaterally into the striatum of male Wistar rats to retrogradely label nigral dopaminergic neurons. After 1 week, animals were treated with six doses of PSI (8 mg/kg, s.c.) or its vehicle (dimethyl sulphoxide) on alternate days over a 2-week period. Five weeks after the last treatment, PSI-treated animals showed decreased spontaneous locomotor activity and reduced TH positive SN cell number at the level of the third cranial nerve compared to control rats. Manual cell counting showed loss of FG-labelled SN neurones at this level, with a subpopulation of surviving neurons displaying abnormal morphology. Manual counting of all FG-labelled cells in the entire SN also showed regional PSI-induced loss of neurones with both normal and compromised morphology. Stereological optical fractionator estimates of total FG-labelled cell number confirmed the manual cell counting data both at the level of the third cranial nerve and throughout the entire SN. These findings confirm that PSI does cause a persistent nigral dopaminergic neuronal loss. The reason for the lack of reproducibility between laboratories requires further investigation. We suggest that a failure to distinguish between TH-positive neurones with normal and abnormal morphology following PSI administration contributes to equivocal results.

Topics: Animals; Immunohistochemistry; Male; Motor Activity; Nerve Degeneration; Oligopeptides; Rats; Rats, Wistar; Stereotaxic Techniques; Substantia Nigra

We investigated to determine whether acute administration of proteasome inhibitor can cause dopaminergic cell loss in mice, in comparison with that of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The four intraperitoneally administrations of MPTP at 1-h intervals to mice decreased significantly the concentration of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum after 5 days, in comparison with vehicle-treated animals. In contrast, the three subcutaneously administrations of carbobenzoxy-L-gamma-t-butyl-L-glutamyl-L-alanyl-L-leucinal (PSI) did not show significant changes in the concentration of dopamine, DOPAC and HVA in the striatum after 5 days, in comparison with vehicle-treated animals. Our Western blot analysis also showed that the four administrations of MPTP at 1-h intervals to mice produced a significant reduction of anti-tyrosine hydroxylase antibody (TH) protein levels in the striatum after 5 days after. In PSI-treated mice. In contrast, no significant change of TH protein levels was observed in the striatum 5 days after the final treatment with PSI. Furthermore, a significant decrease of TH protein levels was observed in the striatum of MPTP-treated mice, as compared with PSI-treated animals. The present study demonstrates that the acute treatment with proteasome inhibitor PSI did not cause the dopaminergic neurotoxicity in mice, as compared with acute treatment with MPTP. Thus, our findings suggest that acute proteasome inhibition is not a reliable model for Parkinson's disease.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 3,4-Dihydroxyphenylacetic Acid; Animals; Corpus Striatum; Cysteine Proteinase Inhibitors; Disease Models, Animal; Dopamine; Drug Administration Schedule; Homovanillic Acid; Male; Mice; Mice, Inbred C57BL; Nerve Degeneration; Neurons; Neurotoxins; Oligopeptides; Parkinsonian Disorders; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Reproducibility of Results; Time Factors; Tyrosine 3-Monooxygenase

Proteasomal dysfunction has been suggested to contribute to the degeneration of nigrostriatal dopamine neurons in Parkinson's disease. A recent study reported that systemic treatment of rats with the proteasome inhibitor Z-lle-Glu(OtBu)-Ala-Leu-al (PSI) causes a slowly progressive degeneration of nigrostriatal dopamine neurons, the presence of inclusion bodies in dopamine neurons, and motor impairment. We examined in vitro and in vivo the effects of PSI on nigrostriatal dopamine neurons. Mass spectrometric analysis was employed to verify the authenticity of the PSI compound. PSI was non-specifically toxic to neurons in ventral mesencephalic organotypic slice cultures, indicating that impairment of proteasome function in vitro is toxic. Moreover, systemic administration of PSI transiently decreased brain proteasome activity. Systemic treatment of rats with PSI did not, however, result in any biochemical or anatomical evidence of lesions of nigrostriatal dopamine neurons, nor were any changes in locomotor activity observed. These data suggest that systemic administration of proteasome inhibitors to normal adult rats does not reliably cause an animal model of parkinsonism.

Topics: Animals; Animals, Newborn; Corpus Striatum; Cysteine Proteinase Inhibitors; Dopamine; Male; Motor Activity; Nerve Degeneration; Oligopeptides; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Substantia Nigra