Distribution and neuronal expression of phosphatidylinositol phosphate kinase IIgamma in the mouse brain

Abstract

The role of cellular phosphatidylinositol 5-phosphate (PtdIns5P), as a signalling molecule or as a substrate for the production of small, compartmentalized pools of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)], may be dependent on cell type and subcellular localization. PtdIns5P levels are primarily regulated by the PtdIns5P 4-kinases (type II PIP kinases or PIP4Ks), and we have investigated the expression and localization in the brain of the least-studied PIP4K isoform, PIP4Kgamma. In situ hybridization and immunohistochemistry, using antisense oligonucleotide probes and a PIP4Kgamma-specific antibody, revealed that this isoform has a restricted CNS expression profile. The use of antibodies to different cell markers showed that this expression is limited to neurons, particularly the cerebellar Purkinje cells, pyramidal cells of the hippocampus, large neuronal cell types in the cerebral cortex including pyramidal cells, and mitral cells in the olfactory bulb and is not expressed in cerebellar, hippocampal formation, or olfactory bulb granule cells. In neurons expressing this enzyme, PIP4Kgamma has a vesicular distribution and shows partial colocalization with markers of cellular compartments of the endomembrane trafficking pathway. The PIP4Kgamma isoform expression is established after day 7 of postnatal development. Overall, our data suggest that PIP4Kgamma may have a role in neuron function, specifically in the regulation of vesicular transport, in specific regions of the developed brain.

Figure 1

PIP5K2 gene expression in adult mouse tissue cDNA libraries. Comparative levels of PIP5K2A, PIP5K2B, PIP5K2C, and β-actin control mRNA in different tissues as determined by RT-PCR (representative of results from cDNA libraries generated from three different animals).

Figure 2

PIP5K2C gene expression in adult mouse tissues by in situ hybridization. With PIP5K2C-specific probes on 20-μm mouse tissue sections, positive signal is spatially distributed in brain (A) but absent from heart tissue (C; not to scale). Control sections of brain (B) and heart (D) were incubated with excess of unlabeled probe, and identical results were obtained with three sections from two animals. Autoradiographic emulsion staining of hybridization slides (counterstained with methyl blue), shows PIP5K2C mRNA labeled with silver grains (E–J). Positive cells (arrows) are seen in the cerebellum (E, inset is Purkinje cell layer), hippocampal field CA1 (F, inset is stratum pyramidale), cerebral cortex (G, inset is layer V), and olfactory bulb (H, inset is mitral cell layer). Controls incubated with excess cold probe (I,J) were negative. au, Heart auricle; ve, heart ventricle; cm, molecular layer; cp, Purkinje layer; cg, granular layer (all cerebellar); ho, stratum oriens; hp, stratum pyramidale; hr, stratum radiatum (all hippocampal); op, outer plexiform layer; om, mitral cell layer; ip, inner plexiform layer; og, granule cell layer (all olfactory bulb). Scale bars = 40 μm in E–I; 10 μm in J and insets.

Figure 3

Specificity of antiphosphatidylinositol 5-phosphate 4-kinase γ (PIP4Kγ) antibody to PIP4Kγ in mouse brain. A: SDS-PAGE of total proteins from 50 μg whole mouse brain lysate (Coomassie stained) and 50 ng of purified recombinant (rec) PIP4Kγ (silver stained) showing mature protein of 47 kDa and degradation products at 26 kDa. B: Western blot of these proteins using anti-PIP4Kγ antibody, showing single band specificity in brain lysate. C: Control Western blot using anti-PIP4Kγ antibody preincubated with antigenic peptide. Results are representative of at least three experiments.

Figure 4

Endogenous PIP4Kγ expression in adult mouse brain. Quantification of PIP4Kγ levels in lysates (30 μg) from 10 different adult mouse CNS regions probed with PIP4Kγ-specific antibody (n = 3). Integrated pixel intensities for each band are shown (±SEM), normalized to the loading control (α-tubulin). Recombinant (rec) PIP4Kγ was included as a positive size control.

Figure 5

Multiple forms of PIP4Kγ are differentially expressed in brain regions. A: PIP4Kγ was detected as three different bands (47, 48, and 49 kDa) by 8% SDS-PAGE and Western blotting. The 49-kDa band was differentially expressed in cerebral cortex, hippocampus, spinal cord, cerebellum, and brainstem and was not present in the olfactory bulb. B: Only the 47-kDa band remained after lysates were pretreated with calf intestinal alkaline phosphatase (CIAP) and Western blotted with anti-PIP4Kγ antibody. Results are representative of at least three experiments.

Figure 6

PIP4Kγ expression in different regions of the adult mouse brain. PIP4Kγ was detected in whole saggital brain sections (approximately lateral 0.48 mm) by immunofluorescence with anti-PIP4Kγ antibody (A). Staining indicated differential expression levels of PIP4Kγ in different brain regions. A similar, fluorescent Nissl-stained section is included for morphological reference (B). Images are representative of a minimum of six stained sections from three different mice. Scale bars = 1 mm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 7

A–P: Localization of PIP4Kγ in adult mouse brain regions. Positive PIP4Kγ signal (arrows) was observed in the cerebral cortex (A–C), CA1–CA3 of the hippocampus (E–G) and cerebellum (M–O). No significant signal was observed in the inferior colliculus (I–K). Saggital brain sections were stained by immunofluorescent (A,B,E,F,I,J,M,N) and also immunochemical (C,G,K,O) methods. Fluorescent Nissl-stained images are included for reference (D,H,L,P). Images are representative of a minimum of six stained sections from three different mice. I, II/III, V, cerebral cortex layers; dg, dentate gyrus; py, hippocampal pyramidal cells; ec, external cortex of the inferior collicus; cn, central nucleus of the inferior collicus; ig, intermediate gray layer of the superior collicus; pu, cerebellar Purkinje cells; g, cerebellar granular layer; m, cerebellar molecular layer. Scale bars = 100 μm in A,E,I,M; 50 μm in B,F,J,N; 20 μm in C,G,K,O; 500 μm in D,H,L,P. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 8

A–T: Localization of PIP4Kγ in adult mouse brain regions. Positive PIP4Kγ signal (arrows) was observed in the cervical spinal cord (longitudinal section, A–C) and olfactory bulb (M–O). No significant signal was observed in other areas such as the medulla (E–G) or thalamus (I–K). Saggital brain sections were stained by immunofluorescent (A,B,E,F,I,J,M,N,S,T) and also immunochemical (C,G,K,O,Q,R) methods. Fluorescent Nissl-stained images are included for reference (D,H,L,P). Controls for immunochemical and immunofluorescent labeling (incubated with secondary antibody only or using preadsorbed anti-PIP4Kγ) are also included (Q–T). Images are representative of a minimum of six stained sections from three different mice. dw, Dorsal white matter of the spine; vw, ventral white matter of the spine; gr, spinal gray matter; ir, intermediate reticular nucleus of the medulla; mr, medullary reticular nucleus; md, mediodorsal/paracentral thalamic nuclei; ad, anterodorsal thalamic nucleus; gl, olfactory glomerular layer; op, olfactory outer plexiform layer; gr, olfactory granule cell layer; mi, olfactory mitral cells; g, cerebellar granular layer; m, cerebellar molecular layer. Scale bars = 100 μm in A,E,I,M; 50 μm in B,F,J,N; 20 μm in C,G,K,O,Q–T; 500 μm in D,H,L,P. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 9

Identification of cell types expressing PIP4Kγ in the adult mouse cerebral cortex and hippocampus. Endogenous PIP4Kγ was detected in cerebral cortex (A–L) and hippocampus (M–X) by using anti-PIP4Kγ antibody (green). Counterstaining for Nissl substance (B,E,N,Q) indicated that PIP4Kγ was expressed in neuronal cells (C,F,O,R). PIP4Kγ was excluded from glial cells stained with anti-glial fibrillary acidic protein (GFAP; G–I,S–U). PIP4Kγ was not expressed in cells that were positive for the neuronal cell marker calbindin D-28k in these regions (J–L,V–X). Images are representative of three stained sections. py, Stratum pyramidale of hippocampus; dg, dentate gyrus. Scale bars = 50 μm in C (applies to A–C); 20 μm in F (applies to D–F); 50 μm in I (applies to G–I); 50 μm in L (applies to J–L); 50 μm in O (applies to M–O); 20 μm in R (applies to P–R); 50 μm in U (applies to S–U); 50 μm in X (applies to V–X).

Figure 10

Identification of cell types expressing PIP4Kγ in the adult mouse cerebellum and olfactory bulb. PIP4Kγ (green) was detected in the cerebellum (A–I) and olfactory bulb (J–R). Signal was coincident with fluorescent Nissl stain (A–C,J–L) and was excluded from glial cells stained with anti-GFAP (D–F,M–O), indicating that PIP4Kγ was expressed in neuronal cells. PIP4Kγ was expressed in cells that were positive for the neuronal cell marker calbindin D-28k in the cerebellum (G–I) but not in the olfactory bulb (P–R). Images are representative of three stained sections. gr, Cerebellar granular layer; pu, cerebellar Purkinje layer; mi, mitral cell layer of olfactory bulb. Scale bars = 50 μm in C (applies to A–C); 50 μm in F (applies to D–F); 20 μm in I (applies to G–I); 50 μm in L (applies to J–L); 20 μm in O (applies to M–O); 50 μm in R (applies to P–R).

Figure 11

Identification of cell types expressing PIP4Kγ in the adult mouse spinal cord and in primary neuronal cultures. Endogenous PIP4Kγ (green) was detected in neurons in the spinal cord (A–F) by fluorescent costaining for Nissl substance (A–C) and was also seen in the subpopulation of neurons positive for calbindin D-28k (D–F). Hippocampal primary cell cultures enriched for pyramidal cells expressed PIP4Kγ (G–I), whereas cultures enriched for cerebellar granule cells did not (J–L). Images for (A–F) are representative of three stained sections. dw, Dorsal white matter of the spine; gr, spinal gray matter. Scale bars = 50 μm in C (applies to A–C); 50 μm in F (applies to D–F); 20 μm in I (applies to G–I); 20 μm in L (applies to J–L).

Figure 12

Subcellular localization of PIP4Kγ in neurons. Endogenous PIP4Kγ was detected in the cell body and dendritic trees of cerebellar Purkinje cells (A), olfactory bulb mitral cells (B), and large neurons in dorsal root ganglion (drg) preparations (C). PIP4Kγ was mostly excluded from the nucleus and was present in a vesicular compartment in the cytoplasm. This compartment seemed to have a stronger, but partial, colocalization with the cis-Golgi structural marker GM130 (J–L) than with the ER resident calnexin (D–F) or the -ergic marker p115 (G–I), although this could not be fully characterized in brain tissue. Images are representative of results from at least three stained sections. Scale bars = 20 μm in A–C; 20 μm in F (applies to D–F); 20 μm in I (applies to G–I); 20 μm in L (applies to J–L).

Figure 13

PIP4Kγ associates with a vesicular compartment that partially colocalizes with components of the endomembrane system. Expression of GFP-tagged PIP4Kγ in HeLa cells and mild permeabilization with digitonin (A–L) indicated a vesicular cytoplasmic pool of PIP4Kγ that partially colocalized with the Golgi marker GM130 (D–F) and the endosomal markers EEA1 (G–I) and mannose-6-phosphate receptor (M6PR; J–L) but not with the -ergic marker p115 (A–C). Results observed after permeabilization with Triton X-100 (M–X) suggested that the main pool of PIP4Kγ did not remain localized to any of these markers (O,R,U,X). Scale bars = 10 μm in C (applies to A–C); 10 μm in F (applies to D–F); 10 μm in I (applies to G–I); 10 μm in L (applies to J–L); 10 μm in O (applies to M–O); 10 μm in R (applies to P–R); 10 μm in U (applies to S–U); 10 μm in X (applies to V–X).

Figure 14

Expression of PIP4Kγ in the developing mouse brain. Immunohistochemical staining of mouse brain sections at different postnatal development stages (P1–P28 days after birth) indicated the levels of PIP4Kγ (green) detected in three different regions; cerebellum (A–E), hippocampal field CA3 (F–J), and olfactory bulb (K–O). Brain regions are pictured in the same orientation, and images are representative of two animals at each developmental stage. mo, Molecular layer; pu, Purkinje layer; gr, granular layer (cerebellum); so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; sl, stratum lacunosum-moleculare (hippocampal formation); pl, plexiform layer; mi, mitral cell layer; gr, granule layer (olfactory bulb). Scale bars = 50 μm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]