Acidic (leucine-rich) nuclear phosphoprotein 32 family, member A (ANP32A), has multiple functions involved in neuritogenesis, transcriptional regulation, and apoptosis. However, whether ANP32A has an effect on the mammalian developing brain is still in question. In this study, it was shown that brain was the organ that expressed the most abundant ANP32A by human multiple tissue expression (MTE) array. The distribution of ANP32A in the different adult brain areas was diverse dramatically, with high expression in cerebellum, temporal lobe, and cerebral cortex and with low expression in pons, medulla oblongata, and spinal cord. The expression of ANP32A was higher in the adult brain than in the fetal brain of not only humans but also mice in a time-dependent manner. ANP32A signals were dispersed accordantly in embryonic mouse brain. However, ANP32A was abundant in the granular layer of the cerebellum and the cerebral cortex when the mice were growing up, as well as in the Purkinje cells of the cerebellum. The variation of expression levels and distribution of ANP32A in the developing brain would imply that ANP32A may play an important role in mammalian brain development, especially in the differentiation and function of neurons in the cerebellum and the cerebral cortex.
ANP32A is a member of acidic nuclear phosphoprotein 32 kDa (ANP32) family [
All ANP32 proteins share two highly conserved regions: the N-terminal leucine-rich repeats (LRRs) sequence and the C-terminal acidic tail [
More studies have focused on ANP32A, the founding member of the ANP32 family. ANP32A (PP32) was originally found as a tumor suppressor [
ANP32A is known to be a key component of the inhibitor of acetyltransferase (INHAT) complex in the nucleus, involved in regulating chromatin remodeling or transcription initiation [
ANP32A plays essential roles in a variety of neural pathophysiology processes. The level of ANP32A (I1PP2A) is increased in Alzheimer’s disease (AD) and may be involved in regulatory mechanism of affecting Tau phosphorylation and impairing the microtubule network and neurite outgrowth [
The expression characteristic of ANP32A in the developing brain, especially details on the expression and distributions of ANP32A in the human brain, had rarely been reported [
Adult C57 BL/6 mice were kept with free accesses to food and water. The day of insemination was designated as embryonic day 0 (E0). The day of birth was designated as postnatal day 0 (P0). Brains from different embryonic period (E12 and E16), early time points after birth (P0, P5, and P12), pubescent male mice (approximately 5-6 weeks old), and adult male mice (approximately 8–10 weeks old) were frozen quickly and stored at −80°C until required for experiments. E12 and E16 brains were collected under the dissection microscope and the mesenchymal tissues were removed with fine forceps as much as possible. For immunohistochemistry preparation, brains were fixed in formalin (4% formaldehyde in 1× PBS, pH7.4) for 24 hours and then embedded in paraffin. Serial sections (4
The human MTE array (BD, America) was a positively charged nylon membrane to which poly A+ RNAs from different human tissues had been normalized and immobilized in separate dots, along with several controls. The MTE array made it possible to determine the relative expression levels of a target mRNA in different tissues and developmental stages [
A 750 bp
Mouse whole brain tissues were homogenized in RIPA buffer (Sigma, America) on ice, lysates were centrifuged at 12,000 ×g, 4°C for 15 minutes, and supernatants were collected for western blot analysis. Protein concentration of samples was determined by the Bio-Rad protein Assay (Bio-Rad, America). Protein extracts (20
Total RNA was extracted from mouse whole brains using a Trizol (Invitrogen, America). 1
Slides were deparaffinized in xylene and rehydrated in different concentrations of ethanol (100%, 95%, 90%, 80%, and 70%), and antigen retrieval performed using a citrate buffer. Slides were blocked (37°C, 2.5 h) with 10% fetal bovine serum and then incubated with anti-ANP32A antibody (2
The differences of the levels of ANP32A mRNA between various areas of human brain were tested by the spot hybridized with the human MTE assay. The MTE array provides a fast way to simultaneously compare the relative abundance of ANP32A mRNA in a wide array of tissues, normalized and immobilized in separate dots, along with several controls. It was shown that brain was the organ that expressed the most abundant ANP32A, followed with heart, liver, and kidney. As shown with blue column in Figure
Distribution of human ANP32A transcripts in brain. Human MTE array was probed with a 750 bp human ANP32A radiolabeled probe as described under “experimental procedures.” mRNA levels were determined by densitometric scanning of autoradiographs. Whole brain and fetal brain are shown as red column, and different anatomical region is shown as blue column.
The difference between the levels of ANP32A mRNA in adult human brain and fetal human brain was also analyzed by MTE array. Brain in embryonic stage was still the organ with most abundant ANP32A, compared with 7 important human fetal organs including heart, liver, and kidney. The level of ANP32A mRNA in the adult whole brain was about 1.5-fold that in the fetal brain (as shown with red column in Figure
Expression of ANP32A gene in mouse developing brain. (a). Total proteins from the developing C57 BL/6 whole brains were analyzed by western blotting; (b). Total RNAs from the developing C57 BL/6 whole brains were analyzed by qPCR. E12 and E16, embryonic days 12 and 16, respectively; P0, P5, and P12, postnatal days 0, 5, and 12, respectively; 5-6 W and 8–10 W, adult brain from 5-6 weeks and 8–10 weeks old C57 BL/6.
Compared with the embryonic mouse, ANP32A protein was expressed along with the differential cerebral cortex’s layer, more and more significantly, after birth. The changes of morphology of the positive stained cells were also coincidental to the development of the neuron in the nervous system. In the embryonic period, the layer of cerebral cortex is not clear. All positive staining cells are small and numerous, and the expression of ANP32A seemed to be nothing special. When the mice were birthed and growing, the positive staining cells were bigger and more strongly stained in external granular layer of the cerebral cortex (Figure
Immunohistochemical study of ANP32A in mouse cerebral cortex. ANP32A signal was visualized with DAB in C57 BL/6 brain. E12 and E16, embryonic days 12 and 16, respectively; P0, P5, and P12, postnatal days 0, 5, and 12, respectively; 5-6 W, adult brain from 5-6 weeks old C57 BL/6. Scale bar = 200
In detail, from the day of birth (P0), the molecular layer of cerebral cortex appeared, and the staining of the ANP32A in the molecular layer was initially decreased than other partitions of cerebral cortex. On the P5 and P12, the positive stain cells were stratification and conversely in the molecular layer and external granular layer. The difference of staining between the molecular layer and external granular layer in 5-6 weeks old mouse brain was much more significant. The expression of ANP32A was fairly abundant in the external granular layer, localized in the nucleus of the neurons, while being apparently lower in molecular layer.
The distribution of ANP32A changed with the migration of external granule cells in the developing mouse cerebellum. In the embryonic period, the ANP32A was expressed moderately in the nucleus of the internal granule cells and strongly in the external granule cells. In the postnatal day 12, the signals in internal granular layer became stronger, while in the 5-6 W mouse brain the ANP32A’s expression in the internal granule cells became much stronger which may be attributed in large part to migration of the external granule cells to internal granular layer.
The amount and cellular localization of the expression of ANP32A in Purkinje cells seemed to be associated with the mice age and the position in the adult mouse gyrus cerebelli. In the cerebellum P12, ANP32A was expressed moderately in only the nucleus of the Purkinje cells, which scattered evenly between the molecular layer and the granular layer. In the cerebellum 5-6W, it was absorbing that ANP32A was localized in both the nucleus and the cytoplasm, as well as dendrites arborization of Purkinje cells (Figure
Immunohistochemical study of ANP32A in mouse cerebellum. ANP32A signal was visualized with DAB in C57 BL/6 brain. E16, embryonic day 16; P12, postnatal day 12; 5-6 W, adult brain from 5-6 weeks old C57 BL/6. In each image, the upper rectangle inset is magnified and displayed in the corresponding lower image. Purkinje cells were marked by red arrow. Scale size = 100
ANP32 family members had been thought all functionally redundant in vivo. Because loss-of-function mutants for ANP32 family members include two independently targeted ANP32A-deficient mice [
In view of a broad array of physiological activities [
MTE array showed that the expression of ANP32A was higher in the human adult brain than the fetal brain. The similar evidences have been collected by the analysis of ANP32A abundance in a series of different time point mouse brain from embryonic stage to adult stage. Both ANP32A mRNA and proteins were elevated in a time-dependent manner in the developing mouse brain. Mutai et al. had reported that expression of PAL31/ANP32B mRNA and protein in the rat brain was high during the fetal period and decreased after birth [
In the embryonic mouse brain, all positive staining cells were distributed homogeneously, small and numerous. The expression of ANP32A seemed to be nothing special except that the staining in the external granule cells of cerebellum was a little stronger. However, in postnatal day 12, the signals in internal granular layer became stronger. ANP32A was significantly abundant in the granular layer of the cerebellum, and the cerebral cortex when the mice were 5-6 weeks old, as well as in the Purkinje cells of the cerebellum. It may be attributed in large part of migration of the external granule cells to internal granular layer [
The amount and cellular localization of the expression of ANP32A in Purkinje cells seemed to be associated with the mice age and the position. In the cerebellum P12, ANP32A was expressed moderately in the nucleus of the Purkinje cells, similar to some ANP32 proteins [
The distribution of ANP32A in the different adult brain areas was dramatically diverse. Strongly stained nuclei were observed in the external granular layer of cerebral cortex and the granule cells in the cerebellum. MTE array showed that ANP32A was abundant in the human nervous system with high expression in cerebellum, temporal lobe, nucleus accumbens, substantia nigra, and cerebral cortex.
The cerebellum is a region of the brain that plays an important role in motor control. It may also be involved in some cognitive functions such as attention and language, and in regulating fear and pleasure responses. Its movement-related functions are the most solidly established. Learning how to ride a bicycle is an example of a type of neural plasticity that may take place largely within the cerebellum [
On the other hand, ANP32A was lowly expressed in pons, medulla oblongata, and spinal cord. These areas of nervous system function primarily in vital activity, such as control sleep, respiration, swallowing, and motor organization. ANP32A should have tiny effects on these functional areas. In this context, it is not surprising that loss-of-function mutants for ANP32A are not fatal.
In conclusion, ANP32A was abundant in the central nervous system. The expression of ANP32A in the developing brain was raised in a time-dependent manner. And the distribution of ANP32A changed dramatically in different brain areas and layer of cerebellum or cerebral cortex, which implied the roles of ANP32A involved in differentiation and specific functional regulation of neurons. Potential mechanisms of ANP32A in the development and differentiation of nervous system may be involved in neuritogenesis modulating, apoptosis regulating, and transcription control, according to the protein localization in and out of the neurons [
All experiments were reviewed by the Ethics Committee of the Navy General Hospital of Chinese PLA.
There is no conflict of interests for any authors.
Shanshan Wang and Yunliang Wang contributed equally to this study.
This study was supported in part by the National Natural Science Foundation of China (nos. 31071256, 81272700, and 81472350) and the Innovation Fund of the Navy General Hospital of Chinese PLA (no. CX200904).