DGKγ Knock-Out Mice Show Impairments in Cerebellar Motor Coordination, LTD, and the Dendritic Development of Purkinje Cells through the Activation of PKCγ

Abstract

Diacylglycerol kinase g (DGKg) regulates protein kinase C (PKC) activity by converting DG to phosphatidic acid (PA). DGKg directly interacts with PKCg and is phosphorylated by PKCg , resulting in the upregulation of lipid kinase activity. PKC dysfunction impairs motor coordination, indicating that the regulation of PKC activity is important for motor coordination. DGKg and PKC are abundantly expressed in cerebellar Purkinje cells. However, the physiological role of DGKg has not been elucidated. Therefore, we developed DGKg knockout (KO) mice and tested their cerebellar motor coordination. In DGKg KO mice, cerebellar motor coordination and long-term depression (LTD) were impaired, and the dendrites of Purkinje cells from DGKg KO mice were significantly retracted. Interestingly, treatment with the cPKC inhibitor Gö6976 (Gö) rescued the dendritic retraction of primary cultured Purkinje cells from DGKg KO mice. In contrast, treatment with the PKC activator 12-o-tetradecanoylphorbol 13-acetate (TPA) reduced morphologic alterations in the dendrites of Purkinje cells from wild-type (WT) mice. In addition, we confirmed the upregulation of PKCg activity in the cerebellum of DGKg KO mice and rescued impaired LTD in DGKg KO mice with a PKCg-specific inhibitor. Furthermore, impairment of motor coordination observed in DGKg KO mice was rescued in tm1c mice with DGKg reexpression induced by the FLP-flippase recognition target (FRT) recombination system. These results indicate that DGKg is involved in cerebellar LTD and the dendritic development of Purkinje cells through the regulation of PKCg activity, and thus contributes to cerebellar motor coordination.

Precise Regulation of the Basal PKCγ Activity by DGKγ Is Crucial for Motor Coordination

“…However, the basal phosphorylation level of PKCα in tm1d mice was equivalent to that in tm1c mice ( Figure 3 a: tm1c, 1.00; tm1d, 1.00 ± 0.070). These results suggested that the upregulation of basal PKCγ activity in tm1d mice causes the impairment of motor coordination, similarly seen in the conventional DGKγ KO mice [ 15 ].…”

“…We examined the DGKγ expression levels in the brain of tm1d mice using western blotting and found that the DGKγ expression was significantly decreased at 16 weeks old (about 50%) in the cerebellum but not in the cerebrum ( Figure 1 e). The reason why the expression did not disappear completely was due to DGKγ expression in the granule cells [ 15 ]. Thus, we tested the motor coordination of tm1d mice by the rotarod and beam tests.…”

“…We previously reported that PKCγ activity is regulated by DGKγ and that the upregulation of basal PKCγ causes motor dyscoordination [ 15 , 16 ]. To investigate the molecular mechanism of motor dyscoordination in tm1d mice, we examined the autophosphorylation level of PKCγ as a hallmark of PKC activity in the cerebellum of tm1d mice.…”

“…DGKγ floxed (tm1c) mice were used as previously reported [ 15 ]. L7/Pcp2 -specific Cre recombinase transgenic mice were kindly provided by Dr. Suzuki [ 47 ].…”

“…Among DGK subtypes, the γ isoform of DGK (DGKγ) is abundantly expressed in cerebellar Purkinje cells [ 14 ], and we recently reported that the conventional DGKγ KO mice showed impaired cerebellar LTD and motor dysfunction [ 15 ]. We also found abnormal upregulation of the basal PKCγ activity in DGKγ KO mice and that the PKCγ inhibitor normalized the impairment of LTD in DGKγ KO mice [ 15 ]. These results suggested that the functional correlation between DGKγ and PKCγ is responsible for LTD induction.…”