Data_Sheet_1_Cystatin B deficiency results in sustained histone H3 tail cleavage in postnatal mouse brain mediated by increased chromatin-associated cathepsin L activity.docx

Cystatin B (CSTB) is a cysteine cathepsin inhibitor whose biallelic loss-of-function mutations in human result in defects in brain development and in neurodegeneration. The physiological function of CSTB is largely unknown, and the mechanisms underlying the human brain diseases remain poorly understood. We previously showed that CSTB modulates the proteolysis of the N-terminal tail of histone H3 (H3cs1) during in vitro neurogenesis. Here we investigated the significance of this mechanism in postnatal mouse brain. Spatiotemporal analysis of H3cs1 intensity showed that while H3cs1 in wild-type (wt) mice was found at varying levels during the first postnatal month, it was virtually absent in adult brain. We further showed that the high level of H3cs1 coincides with chromatin association of de novo synthesized cathepsin L suggesting a role for nuclear cathepsin L in brain development and maturation. On the contrary, the brains of Cstb–/– mice showed sustained H3cs1 proteolysis to adulthood with increased chromatin-associated cathepsin L activity, implying that CSTB regulates chromatin-associated cathepsin L activity in the postnatal mouse brain. As H3 tail proteolysis has been linked to cellular senescence in vitro, we explored the presence of several cellular senescence markers in the maturing Cstb–/– cerebellum, where we see increased levels of H3cs1. While several markers showed alterations in Cstb–/– mice, the results remained inconclusive regarding the association of deficient CSTB function with H3cs1-induced senescence. Together, we identify a molecular role for CSTB in brain with implications for brain development and disease.</p

Sequence of pathological events in <i>Cstb</i>^-/- mice.

<p>The figure summarises the sequence of the pathological events in the <i>Cstb</i><b>^-/-</b> mice as detected in the current study. Emergence of key symptoms and previous findings reported by Pennacchio et al. (1998) and Tegelberg et al. (2012) have also been included in the figure.</p

ROI based quantification of FA values in different anatomical locations in <i>Cstb^-/-</i> and control mice.

<p>Values have been averaged from hemisphere-mirrored FA-skeletons. Brain regions showing significant (statistical comparison was done using one-way ANOVA with Bonferroni post hoc correction and with p<0.05 considered significant) differences in FA values are in bold. All values are shown with the standard error of mean (SEM).</p

TBSS analysis of DTI data showed WM-changes emerging at the thalamus and cerebellum.

<p>A) The TBSS comparison of FA between <i>Cstb</i>^-/- mice and controls shown in three orientations: sagittal (left), coronal (middle) and horizontal (right). Areas of significant (p<0.05, multiple comparison corrected, with threshold-free cluster enhancement) FA decrease are shown in blue at 2 months of age (first row), yellow at 4 months of age (second row) and red at 6 months of age (third row), and a composite image overlaying all timepoints (fourth row). B) Longitudinal TBSS analysis of the FA between 2 months and 6 months of age show areas of significantly lower FA at 2 months compared to 6 months of age. The green color represents areas of significant FA change in <i>Cstb^-/-</i> mice and pink shows control mice, with the <i>Cstb^-/-</i> group results on top. Results are shown layered on top of the mean FA map calculated from all brains in the study. The FA changes are shown in three orientations: sagittal (top), coronal (middle) and horizontal (bottom).</p

<i>In vivo</i> MRI-volumetry of <i>Cstb^-/-</i> mice revealed early and severe brain atrophy.

<p><i>In vivo</i> MRI-volumetry of <i>Cstb^-/-</i> mice from 1 to 6 months showed differences in the volumes of A) the whole brain (excluding the medulla), B) cerebellum, C) cerebral cortex, D) striatum and E) hippocampus. Time points showing a significant difference in volume are marked with asterisks (statistical comparison was done using one-way ANOVA with post hoc correction and with p<0.05 considered significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).</p

The expression of synapsin 1, VGAT and gephyrin positive synaptic puncta.

<p><b>(A)</b> The number of synapsin 1 positive puncta in the molecular layer of <i>Cstb^−/−</i> cerebellum was significantly lower compared to controls at P14 (p = 0.0035) and P20 (p = 0.0447). P7 and P30 animals did not show significant difference. <b>(B)</b> Molecular layer of P14 and P20 <i>Cstb^−/−</i> cerebellum shows less synapsin 1 positive puncta compared to control. <b>(C)</b> The number of VGAT positive puncta in the molecular layer of <i>Cstb^−/−</i> cerebellum was significantly lower compared to controls at P14 (p = 0.0115). P7, P20 and P30 animals did not show significant difference. <b>(D)</b> Molecular layer of P14 <i>Cstb^−/−</i> cerebellum shows less VGAT positive puncta compared to control. <b>(E)</b> The number of gephyrin positive puncta in the molecular layer of <i>Cstb^−/−</i> cerebellum was significantly lower compared to controls at P20 (p = 0.0448) and P30 (p = 0.0131). P7 and P14 animals did not show significant difference. <b>(F)</b> Molecular layer of P20 and P30 <i>Cstb^−/−</i> cerebellum shows less gephyrin positive puncta compared to control. The data are expressed as mean amount of positive puncta relative to control ± SE. *, p<0.05; **, p<0.01.</p

Differential expression of <i>Gabra6</i> and <i>Gabrd</i>.

<p>qPCR shows increased expression of <i>Gabra6</i> and <i>Gabrd</i> in P7 <i>Cstb^−/−</i> cerebellum compared to the control mice. The data are expressed as a fold change relative to controls ± SE. *, p<0.05.</p

The GO terms of biological processes, molecular functions and cellular components in P30 <i>Cstb^−/−</i> cerebellum.

<p>The GO terms of biological processes, molecular functions and cellular components in P30 <i>Cstb^−/−</i> cerebellum.</p

Autoradiography of GABA_A receptors.

<p><b>(A)</b> Binding of [³H]muscimol was significantly reduced in P30 <i>Cstb^−/−</i> cerebellum (p = 0.037). No change was seen at P7. <b>(B)</b> Binding of [³H]Ro15-4513 did not show significant changes at P7 or at P30. However, when diazepam (DZ) was added to reveal the diazepam-insensitive α6 subunit-dependent GABA_A receptor subtype, decreased binding for [³H]Ro15-4513 (p = 0.012) was seen. The data are expressed as mean radioactivity levels (nCi/mg) ± SE. *, p<0.05. <b>(C)</b> Representative images of [³H]muscimol binding to P30 <i>Cstb^−/−</i> and control brain. <b>(D)</b> Representative images of [³H]Ro15-4513 binding to P30 <i>Cstb^−/−</i> and control brain without and <b>(E)</b> in presence of diazepam.</p

The GO terms of biological processes, molecular functions and cellular components in P7 <i>Cstb^−/−</i> cerebellum.

<p>The GO terms of biological processes, molecular functions and cellular components in P7 <i>Cstb^−/−</i> cerebellum.</p