Increased Cerebral Serum Amyloid A2 and Parameters of Oxidation in Arylsulfatase B (N-Acetylgalactosamine-4-Sulfatase)-Null Mice

Abstract

Introduction: Chondroitin sulfate and chondroitin sulfate proteoglycans have been associated with Alzheimer's Disease (AD), and the impact of modified chondroitin sulfates is being investigated in several animal and cell-based models of AD. Published reports have shown the role of accumulation of chondroitin 4-sulfate and decline in Arylsulfatase B (ARSB; B-acetylgalactosamine-4-sulfatase) in other pathology, including nerve injury, traumatic brain injury, and spinal cord injury. However, the impact of ARSB deficiency on AD pathobiology has not been reported, although changes in ARSB were associated with AD in two prior reports. The enzyme ARSB removes 4-sulfate groups from the non-reducing end of chondroitin 4-sulfate and dermatan sulfate and is required for their degradation. When ARSB activity declines, these sulfated glycosaminoglycans accumulate, as in the inherited disorder Mucopolysaccharidosis VI.

Methods: Reports about chondroitin sulfate, chondroitin sulfate proteoglycans and chondroitin sulfatases in Alzheimer's Disease were reviewed. Measurements of SAA2, iNOS, lipid peroxidation, chondroitin sulfate proteoglycan 4, and other parameters were performed in cortex and hippocampus from ARSB-null mice and controls by QRT-PCR, ELISA, and other standard assays.

Results: SAA2 mRNA expression and protein, CSPG4 mRNA, chondroitin 4-sulfate and i-NOS were increased significantly in ARSB-null mice. Measures of lipid peroxidation and redox state were significantly modified.

Discussion: Findings indicate that decline in ARSB leads to changes in expression of parameters associated with AD in the hippocampus and cortex of the ARSB-deficient mouse.

Conclusions: Further investigation of the impact of decline in ARSB on the development of AD may provide a new approach to prevent and treat AD.

Fig.1.. Arylsulfatase B (ARSB) and chondroitin 4-sulfate (C4S) in cortex and hippocampus of ARSB null mice and controls.

A. ARSB activity was significantly reduced in the brain tissue of the ARSB-null mice, as expected. Level in the control mice was 113.1 ± 10.8 nmol/mg protein/h and 2.1 ± 0.2 nmol/mg protein/h (n=6 in each group) in hippocampal tissue and similar in cortical tissue. B. Consistent with the ARSB activity, the C4S in the hippocampus of the ARSB-null mice was 12.0 ± 0.7 μg/g protein and 7.1 ± 0.6 μg/g protein in the control mice, with similar values in the cortical tissue (n=6 in each group). C. mRNA expression of chondroitin sulfate proteoglycan 4 (CSPG4) increased to 2.8 times the control level in the cortex of the ARSB-null mice (n=6 in each group). *** for p<0.001 by unpaired t-test, two-tailed, unequal variance; p<0.01 by Mann-Whitney U test for non-parametric data. [con=control; null=ARSB-null]

Fig.2.. SAA in cortex, hippocampus, and serum of ARSB null mice and controls.

A. SAA was measured by ELISA in the cortex and hippocampus of control and ARSB null mice. In cortex, control value was 1.30 ± 0.09 μg/mg protein compared to 6.78 ± 0.63 μg/mg protein in the ARSB-null mouse cortex. Values in hippocampus were similar (1.06 ± 0.12 vs. 5.13 ± 0.47 μg/mg protein; n=6 in all groups). P-values are <0.001 for all comparisons (unpaired t-test, two-tailed). B. QRT-PCR data indicate 1.89-times higher expression in the ARSB-null mice than the control mice (n=6; p<0.001, unpaired t-test, two-tailed). Values were similar for male and female mice. C. Serum levels of SAA were similar in control and ARSB-null mice, and higher in the male mice than the female mice (n=4 in each group). [con=control; null=ARSB-null]

Fig.3.. Inducible nitric oxide synthase, total thiols, and products of lipid peroxidation are increased in the cortex of ARSB-null mice.

A. Inducible nitric oxide synthase (i-NOS) expression was markedly increased in the ARSB-null mice (5.68 ± 0.39 vs. 1.04 ± 0.08, n=6; p<0.001, unpaired t-test, two-tailed, unequal variance). B. Total thiols, including protein-thiol groups and inorganic SH groups were lower in the ARSB-null mice than the controls (71.6 ± 4.9 vs. 44.1 ± 1.0; n=6, p<0.001), consistent with lower reducing capacity in the ARSB-null mice. C. The lipid peroxidation products, MDA and HAE, were measured in the cortical tissue of the ARSB-null and control mice. In the ARSB-null mouse tissue, levels are several-fold higher (n=6, p<0.001). [con=control; HAE=4-hydroxyalkenals; MDA=malondialdehyde; null=ARSB-null; SH=sulfhydryl]