Developmental Role of Adenosine Kinase in the Cerebellum

Characterization of the expression profile of ADK-L and ADK-S proteins in the developing and adult brain. A, Expression profile of ADK-L and ADK-S proteins in the embryonic brain as well as the developing cerebrum. Western blot analysis shows ADK expression changes during prenatal and postnatal development of the cerebrum in the mouse. Representative blots show ADK (L and S) isoform expression at different embryonic (E) and postnatal (P) stages with ADK-L shown as the upper band and ADK-S as the lower band. In embryonic brain, the nuclear long form ADK-L dominates and shifts toward ADK-S dominance in the adult cerebrum. Control includes recombinant protein ADK-S (Rec). B, Western blot analysis of the expression profile of ADK-L and ADK-S proteins in the developing and adult CB. The postnatal as well as adult CB exhibited strong expression of ADK-L while ADK-S expression increased progressively from P3 into adulthood. C, Quantitative analysis of Western blot analyses using ImageJ V. 1.52 software and expressed as the ratio of optical densities of ADK-L/ADK-S bands. All values are presented as mean ± SEM (n = 3–10). One-way ANOVA with Tukey’s multiple comparison post hoc test (ns, no significance, and ****p < 0.0001 for significance). D, Line graph of normalized optical density of ADK-L bands at different developmental time points of cerebrum and CB.

ADK IR in prenatal and postnatal mouse cerebrum. ADK IR in the mouse cerebrum at different developmental stages; E16, and postnatal days (P2, P5, and P21) as shown by peroxidase staining. A, E, At E16, nuclear expression of ADK presented as dark punctate staining in cells of the ventricular zone (VZ), sub-ventricular zone (SVZ), cortical plate (CP) in the neocortex. B, F, At P2, most of nuclear ADK IR is observed in Layers II/III/IV of the neocortex. C, G, At P5, nuclear ADK IR is only observed in Layers III/IV. D–H, At P21, nuclear ADK IR reduced while diffuse IR consistent with cytoplasmic ADK-S is widespread. I, NeuN (green) IR illustrating cortical layers as shown by immunofluorescence at developmental stages P2, P5, P21. J, Single-cell image of ADK (red)-positive, GAFP (green) astrocytes in P21 cerebrum. Scale bars: 200 μm (A–D) 100 μm (H, I), 50 μm (E–G), and 15 μm (J).

ADK IR in postnatal mouse CB. ADK IR in mouse CB at different developmental stages (P0, P2, P5, P9, and P21) as shown by peroxidase staining. A, At P0, ADK IR is widespread in the external granular layer (EGL), Purkinje layer (PL), and internal granular layer (IGL). B, At P2, ADK IR is present in the distinct layers of the cerebellar cortex EGL, PL, IGL, whereas fewer ADK-positive cells are observed in the molecular layer (ML). C, D, At P5 and P9, ADK IR declines in EGL, PL, and ML. The expression pattern of ADK IR appeared as dark punctate staining in the cells of all layers, which suggests the dominance of ADK-L at these developmental stages as seen in high magnification images in the right panel. E, At P21, no ADK is detected in the outermost layer of the cerebellar cortex. The dark punctate staining consistent with ADK-L is scattered in ML, PL, and IGL, whereas the diffuse staining of ADK-S is extensively observed in ML and IGL. Scale bars: 100 μm (left panel) and 50 μm (right panel).

Effect of ADK on DNA synthesis in the developing CB. A, ADK (red) and Ki67 (green) IR in the mouse CB at different developmental stages (P0, P2, P5, and P9) as revealed by double immunofluorescence. A–C, At P0, ADK/Ki67 double-labeled cells are widespread in the entire developing cerebellar cortex with robust expression at the outermost EGL. D–F, At P2, the layers of cerebellar cortex become visible where ADK/Ki67-positive cells are observed in the external granular layer (EGL), the developing Purkinje layer (PL), and the internal granular layer (IGL). G–L, At P5 and P9, ADK/Ki67-labeled cells initially increase then decrease in the EGL, which period coincides with the radial migration of cerebellar granular neurons. G, In the internal white matter (WM) region at P5, a population of ADK/Ki67-positive cells was observed, which declined in P9 (H). Scale bars: 100 μm (A–C, G–L), 200 μm (D–F), 25 μm (D, G, insets). M, The number of Ki67/ADK double labeled cells in the EGL at P0, P2, P5, and P9 per mm² (n = 3/group). One-way ANOVA with Tukey’s multiple comparisons post hoc test (*p < 0.05 for P5 vs P0, ^#p < 0.05 for P9 vs P0, and ***p < 0.001 for significance). N, Western blot analysis of immature granular neuronal precursors (GNPs). Representative blot of protein extracts from control (Ctl) glioblastoma U373 cells and immature GNPs shows that the precursors express ADK-L exclusively whereas control cells express both isoforms. O, Inhibition of ADK with antagonist 5-iodotubercidin (5-ITU) reduces DNA synthesis of GNPs. Concentration-dependent reduction of cell proliferation in response to different concentrations (0.3, 0.6, 1 μm) of the ADK inhibitor 5-ITU as compared with vehicle (DMSO ≤ 0.002%)-treated cells. All values are presented as mean ± SEM (n = 6–9). One-way ANOVA with Tukey’s multiple comparisons post hoc test (*p < 0.05, ***p < 0.001, and ****p < 0.0001, for significance).

Association of ADK with the development and maintenance of cerebellar cortex cells. A, B, ADK (red) and Cal (green) IR in the developing cerebellum (CB) at different developmental stages (P5, P9, and P21) as shown by immunofluorescence. A–C, Purkinje cells that are ADK (red)/Cal (green)-positive are observed in the Purkinje layer (PL) where the punctate stain of nuclear ADK-L is prominent at P5 and P9. By P21, ADK-L IR is less prominent in Purkinje cells and only maintained in a few cells, whereas the rest of ADK-L cells are found in clusters between Purkinje cells in the PL. White arrows in A point at ADK-L cells between Purkinje cells in P21. B, The number of Cal/ADK double labeled cells in the PL at P5, P9, and P21 per mm² (n = 3–4/group). D, IR of ADK (red)/GFAP (green) in astrocytes of the cerebellar cortex at different developmental stages (P5, P21, and adult) as shown by immunofluorescence. E, Bergmann glial cells in the PL show punctate staining of nuclear ADK-L at all developmental stages (P5, P21, and adult). GFAP-positive astrocytes that are ADK-L-positive are prominent in the white matter at all developmental stages while astrocytes in the inner granular layer are observed at P21 and in adulthood. F, Magnified field of glial cells and ADK-positive cells in white matter of P5 and PL of P21. White arrows showed ADK-L-positive Bergman glial cells between Purkinje neurons. Scale bars: 50 μm (A, C, E), 200 μm (D), and 15 μm (inset in A, and F). One-way ANOVA with Tukey’s multiple comparisons post hoc test (*p < 0.05 for P5 vs P21, and ^#p < 0.05 for P9 vs P21 for significance).