Minocycline prevents early age-related cognitive decline in a mouse model of intellectual disability caused by ZBTB18/RP58 haploinsufficiency

Abstract

Haploinsufficiency of the transcriptional repressor ZBTB18/RP58 is associated with intellectual disability. However, the mechanisms causing this disability are unknown, and preventative measures and treatments are not available. Here, we assessed multiple behaviors in Zbtb18/Rp58 heterozygous-knockout mice, and examined local field potentials, DNA fragmentation, mitochondrial morphology, and performed histochemical and transcriptome analyses in the hippocampus to evaluate chronic inflammation. In wild-type mice, object location memory was present at a similar level at 2 and 4-5 months of age, and became impaired at 12-18 months. In contrast, Zbtb18/Rp58 heterozygous-knockout mice displayed early onset impairments in object location memory by 4-5 months of age. These mice also exhibited earlier accumulation of DNA and mitochondrial damage, and activated microglia in the dentate gyrus, which are associated with defective DNA repair. Notably, chronic minocycline therapy, which has neuroprotective and anti-inflammatory effects, attenuated age-related phenotypes, including accumulation of DNA damage, increased microglial activation, and impairment of object location memory. Our results suggest that Zbtb18/Rp58 activity is required for DNA repair and its reduction results in DNA and mitochondrial damage, increased activation of microglia, and inflammation, leading to accelerated declines in cognitive functions. Minocycline has potential as a therapeutic agent for the treatment of ZBTB18/RP58 haploinsufficiency-associated cognitive dysfunction.

Keywords: Cognitive decline; DNA damage; DNA repair; Inflammation; Intellectual disability; ZBTB18/RP58.

Fig. 1

Spatial memory and hippocampal function in Rp58 hetero-KO mice. A Experimental schedule. B Object location memory in wild-type and Rp58 hetero-KO mice at different ages. One of two familiar objects was moved on day 3 and change in time spent exploring the object in the new location (∆ discrimination index) was measured (see Materials and Methods for details). *p < 0.05, **p<0.01; n = 12 for both genotypes at 2- or 4–5-months of age, n = 10 for 12–18-month-old wild-type mice, and n = 16 for 12–18-month-old Rp58 hetero-KO mice. C Theta power in the hippocampal CA1 region of 4–5-month-old wild-type- and age-matched Rp58 hetero-KO mice on day 2 and day 3 of the object location test. *p < 0.05, **p < 0.01; n = 5 for each genotype

Fig. 2

Cellular mechanisms of DNA damage in Rp58 hetero-KO mice. A Localization of RP58 protein in the hippocampal formation in adult mice. Triple staining for RP58, GluR2, and PV indicates that RP58 is expressed in pyramidal cells of hippocampal CA1-CA3, granular neurons, and hilar mossy cells (indicated by arrowheads) of the dentate gyrus, but not in PV-positive GABAergic neurons (indicated by arrows). Scale bar (left) = 200 µm; scale bar (right) = 50 µm. B Accumulation of ssDNA (indicated by arrows) as a marker of DNA damage in neurons in the dentate gyrus of wild-type- and Rp58 hetero-KO mice. *p < 0.05; n = 3 for both genotypes at 2- or 12–18 months of age, and n = 4 for both genotypes at 4–5 months of age. scale bar = 20 µm. C Accumulation of gamma-H2AX protein (indicated by arrows) as a marker of DNA damage in neurons in the dentate gyrus of wild-type and Rp58 hetero-KO mice. n = 3 per group. scale bar = 20 µm. D Microglia, detected using anti-Iba1 antibody (magenta), and activated microglia, detected using anti-CD68 antibody (cyan), in the dentate gyrus of wild-type- and Rp58 hetero-KO mice. *p < 0.05; n = 3 for both genotypes at 2- or 12–18-months of age, and n = 4 for both genotypes at 4–5-months of age. scale bar in top = 20 µm, scale bar in bottom = 10 µm. E Morphological abnormalities of neuronal mitochondria in the dentate gyrus of wild-type- and Rp58 hetero-KO mice. Mitochondria were classified according to their ultrastructural appearance into one of four categories as previously described [27]. Granule cell layer: chi-square = 284.837, degrees of freedom (df) = 3, p < 0.001; hilus: chi-square = 359.525, df = 3, ***p < 0.001; n = 3 per group. scale bar in right = 0.2 µm

Fig. 3

DNA repair dysfunction in Rp58 hetero-KO mice. A Sampling schedule after irradiation. B Accumulation of irradiation-induced ssDNA as a marker of DNA damage (magenta) in neurons in the dentate gyrus of wild-type and Rp58 hetero-KO mice. *p < 0.05; n = 3 per group. scale bar = 20 µm. C Irradiation-induced accumulation of gamma-H2AX protein (gray) as a marker of DNA damage in neurons in the dentate gyrus of wild-type- and Rp58 hetero-KO mice. **p < 0.01; n = 3 per group. scale bar = 20 µm. D Microglial activation detected by labeling microglia with Iba1 and CD68 in the dentate gyrus of wild-type- and Rp58 hetero-KO mice. *p < 0.05; n = 3 per group. scale bar in left = 20 µm, scale bar in right = 10 µm

Fig. 4

Minocycline for the treatment of age-related phenotypes in Rp58 hetero-KO mice. A Experimental schedule of drug intervention. B The effect of minocycline on ssDNA accumulation (a marker of DNA damage) in neurons in the dentate gyrus of Rp58 hetero-KO mice. *p < 0.05, **p < 0.01; n = 4 for both genotypes with normal water, and n = 3 for both genotypes with minocycline. scale bar = 20 µm. C The effect of minocycline on gamma-H2AX protein accumulation in neurons in the dentate gyrus of Rp58 hetero-KO mice. *p < 0.05; n = 3 per group. scale bar = 20 µm. D The effect of minocycline on microglial activation in the dentate gyrus of Rp58 hetero-KO mice. *p < 0.05, **p < 0.01; n = 4 for both genotypes with normal water, and n = 3 for both genotypes with minocycline. scale bar in left = 20 µm, scale bar in right = 10 µm. E The effect of minocycline on the impairment of spatial memory in Rp58 hetero-KO mice. *p < 0.05; n = 8 for 4–5-month-old wild-type mice with minocycline, and n = 13 for 4–5-month-old Rp58 hetero-KO mice with minocycline. In this figure, the data from Figs. 1 and 2 has been reposted to show the control conditions (normal water)