Inositol Hexakisphosphate Kinase-3 Regulates the Morphology and Synapse Formation of Cerebellar Purkinje Cells via Spectrin/Adducin

Abstract

The inositol hexakisphosphate kinases (IP6Ks) are the principal enzymes that generate inositol pyrophosphates. There are three IP6Ks (IP6K1, 2, and 3). Functions of IP6K1 and IP6K2 have been substantially delineated, but little is known of IP6K3's role in normal physiology, especially in the brain. To elucidate functions of IP6K3, we generated mice with targeted deletion of IP6K3. We demonstrate that IP6K3 is highly concentrated in the brain in cerebellar Purkinje cells. IP6K3 physiologically binds to the cytoskeletal proteins adducin and spectrin, whose mutual interactions are perturbed in IP6K3-null mutants. Consequently, IP6K3 knock-out cerebella manifest abnormalities in Purkinje cell structure and synapse number, and the mutant mice display deficits in motor learning and coordination. Thus, IP6K3 is a major determinant of cytoskeletal disposition and function of cerebellar Purkinje cells.

Significance statement: We identified and cloned a family of three inositol hexakisphosphate kinases (IP6Ks) that generate the inositol pyrophosphates, most notably 5-diphosphoinositol pentakisphosphate (IP7). Of these, IP6K3 has been least characterized. In the present study we generated IP6K3 knock-out mice and show that IP6K3 is highly expressed in cerebellar Purkinje cells. IP6K3-deleted mice display defects of motor learning and coordination. IP6K3-null mice manifest aberrations of Purkinje cells with a diminished number of synapses. IP6K3 interacts with the cytoskeletal proteins spectrin and adducin whose altered disposition in IP6K3 knock-out mice may mediate phenotypic features of the mutant mice. These findings afford molecular/cytoskeletal mechanisms by which the inositol polyphosphate system impacts brain function.

Keywords: IP6K; Purkinje cell; adducin; cerebellar synapse; kinase independent; spectrin.

Figure 1.

IP6K3 is highly expressed in cerebellar Purkinje cells, defects of motor learning, and coordination in IP6K3 knock-outs. A, Schematic representation of the mouse IP6K3 gene with coding regions shaded black, noncoding regions in white, and the exon number indicated on top. A close-up view of exons 5 and 6 in the wild-type, targeted, and knock-out alleles is shown. The knock-out allele loses the splice site, coding region, and the 3UTR of exon 6. B, Immunostaining of IP6K3 in sagittal sections of wild-type and IP6K3 KO mice brain (top) reveals abundant levels of IP6K3 in the cortex and cerebellar molecular layer. C–E, H&E staining on wild-type and IP6K3 KO mice brain sagittal section. C, Whole brain. D, Hippocampus. E, Cerebellum. F, Open field behavioral analysis reveals negligible alterations in IP6K3 KO mice; y-axis, total activity (beams breaks); x-axis, 5 min intervals. G, Rotarod tests reveal reduced motor learning and coordination in IP6K3 KO mice; y-axis, the latency to falling in second; x-axis, days of testing. H, Gait analysis shows significantly decreased overlap of hindpaws and forepaws in IP6K3 KO mice; y-axis, the percentage of forepaw-hindpaw overlap, with the averages taken across trials on testing day. The stride length and step width do not display notable differences. I, Immunostaining of IP6K3 and calbindin in cerebellar molecular layer from wild-type and IP6K3 KO mice. IP6K3 is expressed mainly in Purkinje cells. Scale bar, 20 μm. J, Immunostaining of IP6K3 and calbindin in wild-type cerebellar molecular layer. IP6K3 is most highly expressed in Purkinje cells (arrow) with substantial levels as well in interneurons (top arrowhead, basket cell; bottom arrowhead, stellate cell). Data are represented as mean ± SEM, *p < 0.05, ***p < 0.001, 8-week-old male mice. B–E, I, J, n = 5; F–H, n = 12.

Figure 2.

IP6K3 binds spectrins and adducins in the cerebellum. A, Immunoprecipitation (IP) of IP6K3 from wild-type mice cerebellum with rabbit IgG as a control. Silver staining and mass spectrometric analysis identify spectrins and adducins pulled down by IP6K3. B, Immunoprecipitation of IP6K3, α-adducin, and β-adducin from wild-type mice cerebellum with mouse IgG and rabbit IgG as controls. Western blot indicates that IP6K3 binds to α-adducin and β-adducin. C, Immunoprecipitation of IP6K3, α2-spectrin, and β2-spectrin from wild-type mice cerebellum with mouse IgG and rabbit IgG as controls. Western blot shows IP6K3 binding to α2-spectrin and β2-spectrin. D, Immunoprecipitation of IP6K1, IP6K2, and IP6K3 from wild-type mice cerebellum. Silver staining and mass spectrometric analysis indicates pull-down of spectrins and adducins by IP6K3 but not IP6K1 or IP6K2. E, Immunoprecipitation of IP6K1, IP6K2, and IP6K3 from wild-type mice cerebellum. Western blot shows that IP6K3 but not IP6K1 or IP6K2 binds to α-adducin, β-adducin, α2-spectrin, and β2-spectrin.

Figure 3.

Impaired adducin/spectrin interaction in IP6K3-deleted cells. A, IP6K3 is deleted in HEK293 cells by shRNA lentiviral transduction. B, Knockdown of IP6K3 in HEK293 cells reduces adducin/spectrin binding. C, Immunostaining in IP6K3-deleted HEK293 cells of β-adducin and β2-spectrin (top two lanes) as well as F-actin and β2-spectrin (bottom two lanes). Colocalization of β-adducin with β2-spectrin is significantly reduced in IP6K3 knockdown cells, as is colocalization of F-actin with β2-spectrin. Data are presented as mean ± SEM, ***p < 0.001. IP, immunoprecipitation; Ctrl, control.

Figure 4.

Adducin/spectrin binding is substantially reduced in IP6K3-null mice cerebellum. A, Expression of α-adducin, β-adducin, α2-spectrin, and β2-spectrin is normal in IP6K3 KO mice cerebellum. B, C, Immunoprecipitation (IP) verifies IP6K3 binding to α-adducin, β-adducin, α2-spectrin, and β2-spectrin in mouse cerebellum. D, Immunoprecipitation of α-adducin, β-adducin, α2-spectrin, and β2-spectrin from cerebellum of both wild-type and IP6K3 KO mice. Binding of α-adducin and β-adducin with β2-spectrin is reduced in IP6K3 KO mice cerebellum.

Figure 5.

IP6K3 facilitates the binding of adducins with spectrins; kinase activity of IP6K3 is not required. A, In vitro expression and purification of proteins. Coomassie blue staining of the control GST, β2-spectrin CH domain (1–275 aa), β2-spectrin repeat domain (303–525 aa), β2-spectrin PH domain (2197–2307 aa), β-adducin (437–726 aa), wild-type IP6K3, and kinase-dead mutant IP6K3 (K217A). GST, GST control; CH, β2-spectrin CH domain (1–275 aa); RE, β2-spectrin repeat domain (303–525 aa); PH, β2-spectrin PH domain (2197–2307 aa). B, In vitro binding of IP6K3 with β2-spectrin domains and β-adducin (437–726 aa). Immunoprecipitation (IP) of IP6K3, which binds to β2-spectrin CH domain and β-adducin (437–726 aa). The upper band for RE is the full-length band. C, In vitro binding of β2-spectrin domains and β-adducin (437–726 aa) with IP6K3. Immunoprecipitation of β2-spectrin domains (pull-down GST) and β-adducin (437–726 aa, pull-down flag tag). Western blot shows that IP6K3 binds to β2-spectrin CH domain and β-adducin (437–726 aa). D, In vitro binding of β2-spectrin domains with β-adducin (437–726 aa). Immunoprecipitation of β2-spectrin domains (pull-down GST) reveals that β-adducin (437–726 aa) binds only to β2-spectrin CH domain, and the binding is significantly increased by IP6K3. E, In vitro binding of wild-type IP6K3 and kinase-dead mutant IP6K3 (K217A) with β2-spectrin CH domain and β-adducin (437–726 aa). Immunoprecipitation of β2-spectrin CH domain (pull-down GST) and β-adducin (437–726 aa, pull-down flag-tag); Western blot shows kinase-dead mutant IP6K3 (K217A) binds with β-adducin (437–726 aa) and β2-spectrin CH domain similarly to wild-type IP6K3. F, In vitro binding of kinase-dead mutant IP6K3 (K217A) with β-adducin (437–726 aa) and β2-spectrin CH domain. Immunoprecipitation of β2-spectrin CH domain (pull-down GST) indicates that kinase-dead mutant IP6K3 (K217A) binding to β2-spectrin CH domain facilitates the binding of β2-spectrin CH domain with β-adducin (437–726 aa). G, In vitro binding of β-adducin (437–726 aa) and β2-spectrin CH domain with kinase-dead mutant IP6K3 (K217A). Immunoprecipitation of β-adducin (437–726 aa, pull-down flag tag). Western blot shows that binding of kinase-dead mutant IP6K3 (K217A) to β-adducin (437–726 aa) facilitates the binding of β2-spectrin CH domain with β-adducin (437–726 aa). H, In vitro binding of β-adducin (437–726 aa) with β2-spectrin domains. Immunoprecipitation of β2-spectrin domains (pull-down GST); Western blot shows that physiological concentrations of IP6 (20 μm) or IP7 (5 μm) do not affect the binding of β-adducin with β2-spectrin.

Figure 6.

IP6K3 KO mutant mice manifest altered Purkinje cell structure and reduced synapses. A, H&E staining in cerebellum from wild-type and IP6K3 KO mice (8 weeks old). Width of the molecular layer is decreased in IP6K3 KO mice. The Purkinje cell number and the width of granule layer do not show any significant difference. B, Golgi staining reveals withered dendritic trees of Purkinje cells from 8-week-old IP6K3 KO mice cerebellum. Cell volume is significantly decreased in IP6K3 KO Purkinje cells. C, Golgi staining for the Purkinje cell dendrites from wild-type and IP6K3 KO mice cerebellum (8 weeks old). Spine number is significantly decreased in IP6K3 KO Purkinje cells. D, Golgi staining reveals withered dendritic trees of Purkinje cells from 12-month-old IP6K3 KO mice cerebellum. E, Golgi staining of granule cells, basket cells, Golgi cells, and stellate cells reveals shorter dendritic length (22 ± 5% less for basket cell, 15 ± 4% less for Golgi cell, and 25 ± 8% less for stellate cell) but no significant morphological change in the IP6K3 KOs (8 weeks old). We quantified the length of an average of five dendrites per cell, six cells per mouse. F, Electron microscopy of the cerebellar molecular layer from wild-type and IP6K3 KO mice (8 weeks old). Densities of both symmetric and asymmetric synapses are markedly decreased in IP6K3 KOs. The ultrastructure of synapses is relatively normal in the KOs. Data are represented as mean ± SEM, three mice per group, **p < 0.01, ***p < 0.001.

Figure 7.

IP6K3 KO mice display significantly fewer synapses on cerebellar Purkinje cells. A, Immunostaining of GAD65 and calbindin in cerebellar Purkinje cells from wild-type and IP6K3 KO mice. The density of GAD65 is substantially less in IP6K3 KOs. B, Immunostaining of VGlut1 and calbindin in cerebellar Purkinje cells from wild-type and IP6K3 KO mice. The density of VGlut1 is markedly reduced in IP6K3 KOs. C, Immunostaining of VGlut2 and calbindin in cerebellar Purkinje cells from wild-type and IP6K3 KO mice. The density of VGlut2 is notably diminished in IP6K3 KOs. D, Immunostaining of gephyrin and VGAT in cerebellar Purkinje cells from wild-type and IP6K3 KO mice. The density of double-stained GABAergic synapses is significantly reduced in IP6K3 KOs. E, Immunostaining of VGlut1 and Homer1 in cerebellar Purkinje cells from wild-type and IP6K3 KO mice. The density of double-stained parallel fiber synapses is significantly reduced in IP6K3 KOs. F, Immunostaining of VGlut2 and Homer1 in cerebellar Purkinje cells from wild-type and IP6K3 KO mice. The density of double-stained climbing fiber synapses is much less in IP6K3 KOs. G, Statistical analysis from D–F indicates that GABAergic, parallel fiber, and climbing fiber synapses are significantly reduced in IP6K3 KOs. Scale bar, 20 μm. Data are presented as means ± SEM, ***p < 0.001. Mice were 8-week-old males, n = 5.