Alterations in the cell cycle in the cerebellum of hyperbilirubinemic Gunn rat: a possible link with apoptosis?

Abstract

Severe hyperbilirubinemia causes neurological damage both in humans and rodents. The hyperbilirubinemic Gunn rat shows a marked cerebellar hypoplasia. More recently bilirubin ability to arrest the cell cycle progression in vascular smooth muscle, tumour cells, and, more importantly, cultured neurons has been demonstrated. However, the involvement of cell cycle perturbation in the development of cerebellar hypoplasia was never investigated before. We explored the effect of sustained spontaneous hyperbilirubinemia on cell cycle progression and apoptosis in whole cerebella dissected from 9 day old Gunn rat by Real Time PCR, Western blot and FACS analysis. The cerebellum of the hyperbilirubinemic Gunn rats exhibits an increased cell cycle arrest in the late G0/G1 phase (p < 0.001), characterized by a decrease in the protein expression of cyclin D1 (15%, p < 0.05), cyclin A/A1 (20 and 30%, p < 0.05 and 0.01, respectively) and cyclin dependent kinases2 (25%, p < 0.001). This was associated with a marked increase in the 18 kDa fragment of cyclin E (67%, p < 0.001) which amplifies the apoptotic pathway. In line with this was the increase of the cleaved form of Poly (ADP-ribose) polymerase (54%, p < 0.01) and active Caspase3 (two fold, p < 0.01). These data indicate that the characteristic cerebellar alteration in this developing brain structure of the hyperbilirubinemic Gunn rat may be partly due to cell cycle perturbation and apoptosis related to the high bilirubin concentration in cerebellar tissue mainly affecting granular cells. These two phenomena might be intimately connected.

Figure 1. Plasma total unconjugated bilirubin amounts, and tissue cerebellar unconjugated bilirubin concentration.

A) plasma total unconjugated bilirubin amounts (Tbil) , and B) tissue unconjugated bilirubin concentrations (TUCB). □ Normal homozygous JJ and ■ Hyperbilirubinemic homozygous jj Gunn rat. Data are expressed as mean ± SD. 6 animals of both genotypes were used. Statistical significance: *** p < 0.001.

Figure 2. mRNA relative expression of Cyclin D1, Cyclin E1, Cyclin A, A1 and Cdk2.

□ Normal homozygous JJ and ■ Hyperbilirubinemic homozygous jj Gunn rat. Data are expressed as mean ± SD. Statistical significance: ** p < 0.01.

Figure 3. Representative Western blot and protein relative expression of Cyclin D1, Cyclin E, Cyclin A, A1 and Cdk2.

A) Representative Western blot, and B) protein relative quantification. FL: full length Cyclin E (50 kDa), LWM: low molecular weight Cyclin E forms (49-34 kDa), Total: FL plus LMW Cyclin E forms. □ Normal homozygous JJ and ■ Hyperbilirubinemic homozygous jj Gunn rat. Data are expressed as mean ± SD. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4. Representative Western blot and protein relative expression of p18-Cyclin E, Parp-1, cParp-1 and cCasp3.

A) Representative Western blot, and B) protein relative quantification. cParp-1: cleaved Parp-1. cCasp3: active caspase3 □ Normal homozygous JJ and ■ Hyperbilirubinemic homozygous jj Gunn rat. Data are expressed as mean ± SD. Statistical significance: * p < 0.05, ** p < 0.01.

Figure 5. Cell Cycle profile in JJ and jj P9 Gunn rats.

Cell cycle was analysed on cells from whole cerebella (A), and in population A and B, identified by forward- scattering (B). □ Normal homozygous JJ and ■ Hyperbilirubinemic homozygous jj Gunn rat. Data are expressed as mean ± SD. Statistical significance: * p < 0.05, ** p < 0.01.

Figure 6. Effects of bilirubin on cell cycle and apoptosis in cerebellar granular and astrocytes primary cell cultures.

A) Celll cycle analysis by FACS on cerebellar primary cell cultures exposed to 126 nM of Bf (black bars) and vehicle: 0.5% DMSO (white bars). B) Western blot analysis of cCasp3 expression in astrocytes (grey bars) and granular cells (black bars) primary cell cultures exposed to 126 nM of Bf vs. vehicle (0.5% DMSO, white bars). Data are expressed as mean ± SD. Statistical significance: * p < 0.05, ** p < 0.01.