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Expression of creatine kinase isoenzyme genes during postnatal development of rat brain cerebellum: evidence for transcriptional regulation.

ABSTRACT: Transcription and accumulation of brain-type creatine kinase (CKB) mRNA and its protein was examined during postnatal development of rat brain cerebellum, the brain region containing highest CKB mRNA in the adult. CKB protein was extremely low at day 1, increased about 10-fold until week 4 and remained constant until week 10. This time course was paralleled by cerebellar CKB mRNA, which was also extremely low at day 1 and increased 5-fold during the first 3 weeks and then remained constant. High levels of CKB protein were also detected in cultured primary cerebellar granular neurons. Nuclear run-on assays directly showed that CKB mRNA accumulation during postnatal cerebellar development was due to increased transcription. When compared with cerebrum and whole brain, cerebellar CKB mRNA accumulation during postnatal development was temporally delayed. Analysis of myocyte enhancer factor (MEF)-2 and Sp1, factors known to initiate or sustain CKB transcription in tissues other than brain, revealed that MEF-2 in cerebellum was low at week 1 but increased 3.5-fold by week 7, while Sp1 remained unchanged. The increase in CKB protein during cerebellar postnatal development was coincident with that of the ubiquitous mitochondrial CK protein and mRNA, indicating that a functional phosphocreatine energy shuttle probably exists for efficient ATP regeneration in the cerebellum. This should be beneficial for the many energy-demanding requirements during cerebellar development, as indicated by the observed temporal co-expression of CKB with myelin basic protein, which is involved in axon myelination by oligodendrocytes.

SUBMITTER: Shen W

PROVIDER: S-EPMC1222886 | biostudies-other | 2002 Oct

REPOSITORIES: biostudies-other

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Project description:Creatine kinase (CK) is an energy storage enzyme that plays an important role in energy metabolism. CK/phosphocreatine functions as an energy buffer and links ATP production sites with ATP utilization sites. Several key mutations in the ?A-crystallin (cryaa) and ?B-crystallin (cryab) genes have been linked with autosomal-dominant, hereditary human cataracts. The cryaa-R49C mutation was identified in a four-generation Caucasian family. We previously identified an increase in the quantity of CK complexed with ?-crystallin in the lenses of knock-in mice expressing the cryaa-R49C mutation using proteomic analyses. Increased levels of CK in postnatal cataractous lenses may indicate increased ATP requirements during early cataract development. To gain a further understanding of the relationship between CK and ?-crystallin, we investigated whether ?-crystallin interacts with and forms complexes with CK, in vitro. Isothermal titration calorimetry (ITC) showed that each CK dimer bound to 28 ?-crystallin subunits, with a Kd of 3.3 × 10-7 M, and that the interaction between ?-crystallin and CK was endothermic, thermodynamically favorable, and entropy-driven. High-salt concentrations did not affect the interaction between CK and ?-crystallin, suggesting that the interaction between CK and ?-crystallin is primarily hydrophobic. Gel permeation chromatography (GPC) detected water-soluble ?-crystallin and CK complexes, as determined by increased light scattering after complex formation. In addition, CK and ?-crystallin formed partially-water-insoluble, high-molecular-mass complexes. Enzyme-linked immunosorbent assay (ELISA)-based enzymatic activity analyses of lens homogenates showed a 17-fold increase in CK activity in the postnatal lenses of cryaa-R49C knock-in mice. These studies indicate that the interaction between ?-crystallin and CK is functionally important and that increased CK levels may be necessary to meet the increased ATP demands of ATP-dependent functions in cataractous lenses.

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Expression of creatine kinase isoenzyme genes during postnatal development of rat brain cerebellum: evidence for transcriptional regulation.

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