Novel Alzheimer risk factor IQ motif containing protein K is abundantly expressed in the brain and is markedly increased in patients with Alzheimer's disease

Abstract

Alzheimer's disease (AD) is complex and highly heterogeneous. Less than 10% of AD cases are early-onset (EOAD) caused by autosomal dominantly inherited mutations in amyloid precursor protein (APP), presenilin 1 (PS1), or presenilin 2 (PS2), each of which can increase Aβ generation and, thus, amyloid plaques. The remaining 90% of cases of AD are late-onset (LOAD) or sporadic. Intense research efforts have led to identification of many genes that increase the risk of AD. An IQ motif containing protein K (IQCK) was recently identified by several investigators as an Alzheimer's disease risk gene. However, how IQCK increases AD risk is completely unknown. Since IQCK is a novel gene, there is limited information on its physiological characterization. To understand its role in AD, it is first important to determine its subcellular localization, whether and where it is expressed in the brain, and what type of brain cells express the IQCK protein. Therefore, in this study, we show by immunocytochemical (ICC) staining that IQCK is expressed in both the nucleus and the cytoplasm of SH-SY5Y neuroblastoma cells as well as HeLa cells but not in either HMC3 microglial or CHO cells. By immunohistochemistry (IHC), we also show that IQCK is expressed in both mouse and human neurons, including neuronal processes in vivo in the mouse brain. IHC data also show that the IQCK protein is widely expressed throughout the mouse brain, although regional differences were noted. IQCK expression was highest in the brainstem (BS), followed by the cerebellum (CB) and the cortex (CX), and it was lowest in the hippocampus (HP). This finding was consistent with data from an immunoblot analysis of brain tissue homogenates. Interestingly, we found IQCK expression in neurons, astrocytes, and oligodendrocytes using cell-specific antibodies, but IQCK was not detected in microglial cells, consistent with negative in vitro results in HMC3 cells. Most importantly, we found that actin-normalized IQCK protein levels were increased by 2 folds in AD brains relative to normal control (NC) brains. Furthermore, the IQCK protein was found in amyloid plaques, suggesting that IQCK may play a pathogenic role in either Aβ generation or amyloid plaque deposition in AD.

Keywords: Alzheimer’s disease; IQCK; astrocytes; brain regions; colocalization; microglia; neurons; oligodendrocytes.

FIGURE 1

IQCK immunoreactivity is positive in SH-SY5Y and HeLa cells but negative in HMC3 microglial cells and CHO cells. Representative images are shown for different cell types with IQCK-immunoreactive (red) and DAPI-stained nuclei (blue). Merged images indicate IQCK expression in both the cytoplasm and nuclei. Scale bars indicate 20 μm.

FIGURE 2

Immunohistochemical evidence for IQCK protein expression in both human and mouse brains. (A) Shows similar anti-IQCK antibody immunoreactive (green) cytoplasm and nuclei in human and mouse brain cells, while negative controls without the primary antibody lack signals. (B) Shows neuronal processes also expressing the IQCK protein. Scale bars indicate 20 μm in (A) and 50 μm in (B).

FIGURE 3

Different expression levels of the IQCK protein in mouse brain regions as revealed by immunohistochemistry. Representative micrographs are shown for different brain regions, as the images suggest differences in expression levels in the cortex and other brain regions. The lack of signals in the negative controls confirms the specificity of the used antibody for IQCK expression in the brain. Scale bars indicate 50 μm.

FIGURE 4

Demonstration of IQCK protein expression in different brain regions of 12-month-old mice by immunoblots. Mouse brain regions were lysed, and the lysates were subjected to SDS-PAGE electrophoresis. Protein levels of actin utilized as a loading control were used to normalize IQCK protein levels. Relative to the cortex (CX), IQCK expression levels in the hippocampus (HP) was 77%, in the brainstem (BS) was 180% and in the cerebellum (CB) was 123%. Relative to BS, the levels were 55% in the CX, 43% in the HP, and 68% in the CB.

FIGURE 5

IQCK is expressed in different cell types in the cortical region of the mouse brain. Representative micrographs of cortical region serial sections that were immunostained with cell-type-specific antibodies such as Iba1 for microglia, NeuN for neurons, Gfap for astrocytes, and Olig2 for oligodendroglia (shown in red fluorescence) together with the polyclonal IQCK antibody (green fluorescence), and sections were counterstained with DAPI for detecting nuclei (blue fluorescence). IQCK expression was present in neurons, oligodendrocytes, and astrocytes but not in microglia. Control slides without the primary antibodies showed no signals (data not shown). The yellow color in the merged images demonstrates IQCK expression in the specific cell types as indicated by red arrows. White arrows for microglia indicate negativity for IQCK expression. Scale bars in all the images represent 50 μm.

FIGURE 6

IQCK protein levels are significantly increased in the hippocampus of AD brains relative to NC brains by immunoblots. IQCK protein levels were normalized to actin levels used as loading controls, quantified by ImageJ, and compared between NC and AD. Relative to NC, the AD hippocampus showed 109% increase in IQCK levels. Data were statistically analyzed by paired t-test. **, p < 0.01; data are expressed mean + SEM, n = 5 per group.

FIGURE 7

Immunohistochemical staining of NC and AD brain sections with polyclonal anti-IQCK and monoclonal 6E10 antibodies. Representative micrographs of the brain sections show increased IQCK-immunoreactive neurons (green) and 6E10 antibody-reactive Aβ and amyloid plaques (red) in the AD brains relative to the NC brain sections. Visualization of yellow color in the merged images indicates colocalization of the IQCK protein with Aβ and amyloid plaques. Scale bars in all the images represent 100 μm.