Cerebellin: a quantifiable marker for Purkinje cell maturation.

Abstract

The cerebellum-specific hexadecapeptide cerebellin has been localized by immunocytochemical means to the perikarya and dendrites of cerebellar Purkinje cells. Biochemical analysis using ion-pairing HPLC shows cerebellin to first appear 5 days after birth, whereafter levels rise to a maximum at 25 days postpartum, and then decline to stable adult values. This same pattern of development occurs with a lag of approximately 5 days for the major metabolite of cerebellin, des-Serl-cerebellin. The immunocytochemical picture of cerebellin in developing Purkinje cells mirrors the biochemical data. These results show that cerebellins represent unique quantifiable markers for the investigation of Purkinje cell maturation and lend support to the feasibility of using unique endogenous peptides to chart neurodevelopment.We have proposed that by systematically identifying naturally occurring unique peptides of neural structures these could be used as quantifiable molecular markers with which to investigate neurodevelopment (1). In a prototype study two cerebellum-specific peptides were isolated and sequenced and shown to be a hexadecamer termed cerebellin (NH2-Ser-Gly-Ser-Ala-Lys-Val-Ala-Phe-Ser-Ala-Ile-ArgSer-Thr-Asn-His-OH) and an apparent metabolite des-Serlcerebellin (1). It thus remained to establish the utility of the cerebellins as molecular markers for neurodevelopment and to determine their cellular location. Since both cerebellins have been synthesized (1), we have been able to develop antibodies to the peptides that can be used for their localization at the level of the light microscope. Furthermore, as sensitive biochemical analysis of the peptides is possible (1), immunocytochemical evidence could be corroborated and quantitated in adult and developing cerebellum. In this study the immunocytochemical localization of cerebellin in Purkinje cells is reported as well as the characteristics of cerebellin expression during ontogeny. The demonstration of these parameters lends support to the feasibility of using native endogenous peptides to map development in the brain. MATERIALS AND METHODSPreparation of Immunogen. Cerebellin immunogen was synthesized by incubating together 2 mg of cerebellin and 30 mg of bovine thyroglobulin (Sigma) in 2.8 ml of 50 mM sodium phosphate (pH 7.8) containing a final concentration of 0.5% glutaraldehyde for 2 hr at room temperature. The complex was dialyzed against incubation buffer, and then 1-mg aliquots were used to immunize rabbits. Details of the immunization procedures are as previously reported (2).Affinity Purification of Rabbit Antiserum to Cerebeflin. One milliliter of rabbit antiserum (titered by using a cerebellin radioimmunoassay; unpublished data) was passed over a 1-ml protein A-Sepharose column (Pharmacia) under the conditions recommended by the suppliers. The IgG fraction was neutralized with 0.2 vol of 1 M sodium phosphate (pH 7.6) and passed over a 1-ml cerebellin-agarose affinity column at a flow rate of 10 ml/hr. After loading, the column was washed with 20 ml...

Isolation, sequence, and developmental profile of a brain-specific polypeptide, PEP-19.

“…Another Purkinje cell marker peptide, cerebellin, shows a similar time course of appearance but decreases in concentration after day 25 postpartum until a stable adult level is attained (11). PEP-19 does not exhibit this characteristic decline in concentration (Fig.…”

“…Precise details of the production and characterization of the anti-PEP-19 antisera will be described elsewhere. Briefly, a peptide was synthesized corresponding to residues 43-55 of PEP-19 and coupled to bovine thyroglobulin as described (11). This region of PEP-19 has no tTo whom reprint requests should be addressed.…”

“…Indeed, the fact that a number of neural proteins were shown to have differential expression during development was only realized some time after their initial discovery. Because we have shown (10,11) that HPLC mapping techniques combined with rapid isolation methods can be used to identify brain region-specific peptides, we felt that the same approach would be amenable to the identification of developmentally regulated polypeptides. Thus a database was constructed of the retention times of polypeptides present in neonatal and adult rat cerebellum using the HPLC systems described (10,11).…”

Cbln1 Is Essential for Interaction-Dependent Secretion of Cbln3

“…Cbln1⌬21N and Cbln3⌬24N are the secreted mature forms of Cbln1 and Cbln3, respectively. Cbln1⌬54N is the product generated by cleavage at a putative tumor necrosis factor (TNF) alpha-cleaving enzyme-like site in Cbln1 responsible for liberating the peptide, cerebellin (1,33,34,40), and Cbln3⌬57N is the equivalent fragment in Cbln3. Cbln1⌬61N and Cbln3⌬63N constitute just the C1q domains of the two family members.…”

Precerebellin-related genes and precerebellin 1 peptide in the adrenal gland of the rat: Expression pattern, localization, developmental regulation and effects on corticosteroidogenesis

“…Initially, it was suggested that the N-terminal truncated version of CER, des-Ser1-CER, is an apparent metabolite of CER. However, recent data failed to confirm this contention (1,34,36,37). In regards to the adrenal gland, both CER and desCER exert a modulatory effect on corticosteroid secretion (25,(38)(39)(40)(41).…”