Apo J and Apo D: Complementary or Antagonistic Roles in Alzheimer’s Disease?

Abstract

Apolipoprotein D (Apo D) and Apolipoprotein J (Apo J) are among the only nine apolipoproteins synthesized in the nervous system. Apart from development, these apolipoproteins are implicated in the normal aging process as well as in different neuropathologies as Alzheimer’s disease (AD), where a neuroprotective role has been postulated. Different authors have proposed that Apo D and Apo J could be biomarkers for AD but as far as we know, there are no studies about the relationship between them as well as their expression pattern along the progression of the disease. In this paper, using double immunohistochemistry techniques, we have demonstrated that Apo D is mainly located in glial cells while Apo J expression preferentially occurs in neurons; both proteins are also present in AD diffuse and mature senile plaques but without signal overlap. In addition, we have observed that Apo J and Apo D immunostaining shows a positive correlation with the progression of the disease and the Braak’s stages. These results suggest complementary and cell-dependent neuroprotective roles for each apolipoprotein during AD progress.

Keywords

Apolipoproteins amyloid-β peptide Braak’s stage frontal cortex glia

INTRODUCTION

Alzheimer’s disease (AD), the most common form of dementia, is a fatal, irreversible, progressive, and degenerative brain disorder characterized clinically by a loss of memory and cognition. From a pathological point of view, AD is characterized by diffuse (DSP) and mature (MSP) senile plaques (composed of aggregated amyloid-β peptide, Aβ), neurofibrillary tangles (NFT, composed of hyperphosphorylated tau protein), and synapsis loss [1]. Recent evidence suggests that multiple processes are involved in the pathogenic development of AD such as oxidative stress, inflammation, and alterations in lipid metabolism and membrane composition [2, 3].

Alterations in the lipid metabolism are particularly important in the brain and seem to be behind central nervous system (CNS) diseases [4, 5]. The critical role of apolipoproteins in regulating plasma lipid and lipoprotein levels has been further studied during last decades using a variety of techniques. Thus, twenty-two apolipoproteins have been identified in humans and interestingly some of them are expressed in the CNS, related with the normal aging process as well as AD. Apolipoprotein D (Apo D) and Apolipoprotein J (Apo J or clusterin) are secreted glycoproteins, expressed in a wide variety of tissues and found in many human body fluids, acting as lipid transporters, whose exact functions in the brain remain to be fully elucidated.

In non-pathological conditions, Apo J is produced by astrocytes in several brain regions, but also by the pyramidal neurons of the hippocampus and by the Purkinje cells of the cerebellum [6 –8]. Under stressful or pathological situations as ischemia [9] or AD [10], these Apo J levels are increased. During AD, Apo J expression is increased in the frontal cortex [11], hippocampus, and entorhinal cortex [12]. Some authors have described the presence of Apo J in neurons, astrocytes, neuropile, senile plaques, and Aβ deposits in AD brains, but not in NFT [8 , 13]. However, Calero et al. [14] postulated that Apo J is located in some neurons with NFT. Although not clear, it has long been suspected that Apo J plays a protective role in AD, maybe through the clearance of Aβ deposits [15, 16] or by the control of cellular lipid homeostasis [17].

With respect to Apo D, this apolipoprotein is located in the CNS in oligodendrocytes, astrocytes, neurons, and perivascular cells [18, 19]. Its synthesis is increased in different regions of human brain during aging [20], as well as in several neuropathologies: spongiform encephalopathy [21], Niemann-Pick’s disease [22], and AD [23 –25] (see Muffat and Walker for a complete review [26]). In this sense, several studies have demonstrated that Apo D expression is upregulated in cerebrospinal fluid, hippocampus, and cortex in AD patients [25 , 28], and also in the brains of AD transgenic mice that overexpress the human amyloid-β precursor protein [29]. Regarding the neuropathological hallmarks of AD, Apo D has been found in all types of senile plaques [30, 31]. NFT seem to be negative for this protein [32] according to some studies, but positive as shown by others [33]. According with recent in vivo and in vitro studies, Apo D has been proposed as a neuroprotective and antioxidant molecule in the mentioned situations [34 –40].

As we have seen, the implication of Apo J and Apo D in AD is a common finding from evidence in recent years, however, there are no studies about the possible relationship between these two proteins in the disease. Based on the similar expression pattern and role of both apolipoproteins in brain, in normal conditions and during AD, the aim of this study was to investigate whether Apo D and Apo J co-localize in the cerebral cortex of AD brains and their possible functional interaction along the progression of the disease

MATERIAL AND METHODS

Human tissues

Use of human brain tissues were approved by “Comité de Ética de la Investigación Clínica del Principado de Asturias” as follows. These studies were granted waivers of consent on the following bases: 1) samples were gathered retrospectively from pathology archives and resulted from necropsies performed for diagnostic purposes; 2) patient identities were anonymized and completely delinked from unique identifiers; and 3) there was no risk to the participants (only anonymized tissues were used).

All human brain tissues were provided by the Pathologic Anatomy Service of the University Central Hospital of Asturias and the Bank of Neurologic Tissues of the Clinic Hospital of Barcelona, and were obtained from necropsies within 6 h of death. Some of this material was the same that we have used in recent studies of our group [35, 41]. Thirty-six individuals (55–88 years of age) with AD were used in this study. Cases were screened using standardized protocols to confirm the presence of AD and exclude coexisting cerebrovascular and degenerative pathologies. Braak staging of AD-type pathology was performed on sections of the hippocampal formation and temporal cortex according to published criteria [42]. The pieces from human frontal cortex were fixed by immersion in 10% buffered formalin. After fixation, they were washed in distilled water, dehydrated through successive alcohols, cleared in two baths of butyl acetate, embedded in paraffin, and placed in a suitable mold. Transverse sections about 10μm thick were obtained and attached to gelatine-covered slides, deparaffined in xylene, and rehydrated. The present study was conducted according to the Declaration of Helsinki and was approved by the “Comité de Ética de la Investigación Clínica del Principado de Asturias” (Spain).

Neurons, senile plaques, neurofibrillary tangles, and amyloid-β staining

Alternated sections were stained using a Nissl type staining procedure [43]. To visualize the typical cerebral markers of AD neuropathology, the silver technique of Reusche [44] and a modification of the Congo red method developed in our laboratory [41, 43] were used.

Immunohistochemistry

For immunohistochemistry, fixed sections were permeabilized with Triton X (0.01%, 5 min), washed in distilled water, treated with H₂O₂ (3%, 5 min) to quench endogenous peroxidase activity, washed in distilled water, and treated with phosphate-buffered saline (PBS) (2 min). After blocking non-specific binding by incubation in bovine serum (BSA) (30 min), sections were incubated with an rabbit antibody against human Apo D (1 : 2000 dilution, provided by Dr. Carlos López-Otín, Universidad de Oviedo, and used by our group in all studies achieved on Apo D from 1994) [19 , 46], or with a mouse antibody against human Apo J (1 : 500 dilution, NCL-Clusterin, Novocastra). Incubation was carried out overnight at 4°C. After several washes in PBS, sections were incubated with a biotinylated horse universal antibody (Vector, PK-8800), (1 : 50 dilution, 30 min). Afterward, sections were incubated with extravidin (Sigma Extra-3), and peroxidase activity was visualized by incubation with Sigma Fast DAB (Sigma D4 168) at room temperature for 30 min. Finally, sections were counterstained using a modified formaldehyde thionin method [47], dehydrated, cleared in eucalyptol, and mounted with Eukitt. For controls, representative sections were processed in the same way with a non-immune serum or specifically absorbed sera in place of the primary antibody. Under these conditions, no specific immunostaining was observed.

Double immunohistochemistry

In a second group of sections, double immunostaining for Apo D and Apo J was carried out according to the following protocol. The immunodetection of Apo D was achieved as previously described; immunoreactivity was detected using Extravidin–biotin–alkaline phosphatase staining kit (Sigma Extra-1A). Enzyme activity was determined by incubation with Vector-blue substrate (Vector, SK-5300). After that, slides were rinsed in PBS, placed into a plastic coupling jar filled with 0.01 M sodium citrate buffer (pH 6), and heated twice for 5 min at 700W in a household microwave. Microwave procedure involves completely blocking contaminating staining in the double-labeling technique, using primary antibodies from the same species and the same secondary antibody (see Lan et al. [48]). Incubation with a specific antibody against Apo J (1 : 500 dilution) was carried out overnight at 4°C. After several washes in PBS, immunoreactivity was detected using the Extravidin–biotin–peroxidase kit (Sigma Extra-3), and peroxidase activity was visualized in a red AEC reaction (Sigma, A-6926) (0.5 mg AEC, 50μl dimetylformamide, 10μl H₂O₂ (3%) in 940μl acetate buffer).

A third group of sections was used to a fluorescence confocal microscopy study by double immunostaining techniques. Immunostaining for rabbit Apo D or mouse Apo J was carried out as previously described, but in this case and after several washes in PBS, sections were respectively incubated with a donkey anti rabbit secondary antibody labeled with CFL-488 (Santa Cruz Biotechnology, SC-362261, 1:200 dilution) or a donkey anti mouse secondary antibody labeled with CY3 (Jackson Immuno Research, 715-166-150, 1:200 dilution), for 30 min at room temperature. After several washes in PBS, sections immunostained for Apo D were subsequently incubated with a specific mouse antibody against GFAP (Sigma G-3893, 1 : 200 dilution) overnight at 4°C whereas sections immunostained for Apo J were subsequently incubated with a specific rabbit antibody against GFAP (Sigma G-9269, 1 : 500 dilution) overnight at 4°C. Then sections were washed in PBS and respectively incubated with a donkey anti mouse secondary antibody labeled with CY3 (Jackson ImmunoResearch, 715-166-150, 1:200 dilution) or a donkey anti rabbit secondary antibody labeled with CFL-488 (Santa Cruz Biotechnology, SC-362261, 1:200 dilution), for 30 min at room temperature.

Quantification of diffuse and mature senile plaques

For the quantification and characterization of the diffuse and mature senile plaques, six random regions per case of the frontal cortex area were chosen and studied in the field included by a 10x lens. In each sample, the DSP and MSP with positive staining for each protein, Apo J and Apo D, were counted and the mean value obtained in each case was used for the quantification of both types of senile plaques.

Quantification of Apo D and Apo J expression

For quantification of Apo D and Apo J expression, the chromogenic signal was selected using Adobe Photoshop CS 8.0.1 (Adobe Systems Inc., CA, USA) and density measured by ImageJ 1.37c (National Institute of Health, Bethesda, MD, USA) according to a procedure developed by our group [49]. Six random regions per case of the frontal cortex area were photographed using a 20x lens. After acquisition with a digital camera, images are opened in Adobe Photoshop. For subsequent comparison of Apo D expression in different sections, the magnification of images must also be equal. The analysis protocol is as follows: (1) To select the specific chromogenic signal, choose “Color range” in the “Select menu” of Photoshop, and with the “Eyedropper” tool click on any object in the image displaying the desired color/chromogen and all areas within the selected color range will be highlighted in an automatically generated clone image. The tolerance bar of the color range tool makes direct visualization of the selected regions possible with minimal variations in the chromogen selection. Once chromogen selection is finished, the profile used can be archived for use with similar stained sections (2). Close the “Color range” panel and all immunopositive objects in the original image appear highlighted (3). Open the “Edit” menu of Photoshop, select “Copy”, open a “New file”, and “Paste” the selected image. The new image shows only the positive selected areas on a white background (4). Convert the image to greyscale so all selected marked areas are represented in grey tones correlated with chromogen intensity. This greyscale picture must be saved in ‘tif’ format, which can then be opened in Scion Image (or other free image analysis programs such as ImageJ) (5). Open the picture in this program and calculate the “Mean density” under “Uncalibrated” conditions. The measurements obtained show the signal strength and can be copied and exported to a spreadsheet (i.e., Excel). The signal strength varies between 0 and 255 [value 0 corresponds to a nonstained section (white), and 255 corresponds to a fully stained section (black)].

Statistical analyses

The data in the graphs are presented as the mean±SEM. All statistical calculations were conducted using SPSS 21.0 for Windows. Data sets were first tested for normality using the Kolmogorov-Smirnov test with Lilliefors correction. The Mann-Whitney U test, due to non-homogeneity of variance, was used to analyze the differences between the two studied apolipoproteins, and the Kruskal-Wallis test for median analysis. Finally, a Pearson Correlation analysis was used to detect the possible correlation between variables. Significant differences were considered when p < 0.05.

RESULTS

Apo D location

The study of Apo D location shows an apparent increase of the protein level with the disease progression in both neurons and glial cells. As we can observe by the immunohistochemistry analysis, Apo D is present only in scattered neurons but in numerous glial cells in the different layers of the frontal cortex (Fig. 1A, Supplementary Table 1). This apolipoprotein is always located in the cytoplasm surrounding the nucleus, never inside it, showing a blur staining in neurons and a more granular aspect in glia. Notably, Apo D is present in a great amount in astrocytes, oligodendrocytes and microglia (Fig. 1A, Supplementary Table 1) with higher immunostaining in the white matter than in the grey one. This signal shows a great increment in the course of the pathology. Moreover, the intensity of the staining observed is higher in the deeper layers and lower in the outer ones.

Regarding the senile plaques, Apo D is located in all types observed, but with different pattern depending on the maturity of the plaque. In the DSP, Apo D is distributed all over the structure (Fig. 1B, Supplementary Table 1), while in the MSP it is located mainly surrounding the amyloid core. This phenomenon is clearly evidenced when we compare the immunostaining for Apo D shown in Fig. 1C, with the amyloid fluorescence signal that displays the plaque core in Fig. 1D (Supplementary Table 1). In contrast, we found very few neurons with NFT also immunostained for Apo D, and only in those Apo D positive cells with slight signal (Fig. 1E, F, Supplementary Table 1). Moreover, extracellular ghost tangles immunopositive for Apo D were not found. Interestingly and according with our observations, it seems that there is a specific pattern of distribution for the Apo D positive senile plaques in the frontal cortex with a preferential location in the deeper layers of this brain structure, mainly in the multiform layer. Finally, it is important to note that the number of Apo D positive DSP found is lower than the MSP ones.

Apo J location

The study of Apo J location shows, in the same way as Apo D, an apparent protein level increase with the Braak’s stage. Noteworthy, the neuronal signal for Apo J is stronger than the one for Apo D, and appears in a greater number of neurons with a granular distribution all over the cytoplasm but, again, never in the nucleus (Fig. 2A). Nevertheless, Apo J is scarcely present in the glial cells, with a more intense signal in the grey matter (Fig. 2A, Supplementary Table 1). Apo J is located in neurons through the frontal cortex, being the higher immunostaining in the external pyramidal layer.

Related with the AD hallmarks, we have found Apo J expression in both types of senile plaques, DSP and MSP (Fig. 2B). These plaques seem to be preferentially located in the medium layers of the cortex and we have observed a higher number of Apo J positive DSP than MSP ones. Significantly, the chromogenic signal distribution of Apo J in the mature plaques clearly differs from the distribution of Apo D since Apo J is located all over the structure, including the amyloid core (Fig. 2B, Supplementary Table 1). The presence of Apo J was also confirmed, with different staining intensity, in neurons with NFT. The occurrence of Apo J expression and NFT formation in the same neuron was demonstrated by a combination of an immunohistochemistry for Apo J and a Congo red stain for NFT, visualized as a red fluorescence signal, as we can observe in the images of the same sections in Fig. 2C and D and 2E and F (Supplementary Table 1). In this sense, an interesting finding is the fact that all Apo J positive neurons show NFT; only in one of all neurons analyzed was no fluorescence with Congo red stain observed (Fig. 2C, arrow), probably due to a masking effect by the massive signal for this apolipoprotein visualized with DAB. In contrast, not all neurons showing NFT are positives for Apo J; for instance, one neuron that shows NFT detected by fluorescence (Fig. 2F, arrow) is at the same time immuno-negative for Apo J (Fig. 2E). Moreover, the extracellular ghost tangles do not show immunoreactivity for Apo J (Fig. 2F).

Apo D, Apo J, and GFAP co-localization

First, the double immunostaining for Apo D and Apo J showed, as we observed previously, that while most of the neurons are Apo J positives (Fig. 3A, arrows), only a few show immunostaining for Apo D. In contrast, glial cells are mostly positive only for Apo D (Fig. 3A, Supplementary Table 1). In addition, the double immunohistochemistry study demonstrated no co-localization between these two apolipoproteins in neurons and in both types of senile plaques. In Fig. 3B-E, Apo D is located in glial cells and pericytes (as was previously published by several studies), whereas Apo J is present in the DSP and in the thickened wall of an amyloid-laden vessel. The co-localization between Apo J and amyloid peptide is also observed in the case of MSP, where the amyloid core shows an intense and homogeneous staining for Apo J (Supplementary Table 1). As expected, Apo D is located in the periphery of these plaques in association with the glial cells surrounding the structure and in the long processes of astroglial cells included in all types of senile plaques (Fig. 3D, E, Supplementary Table 1). Notably, the immunostaining for Apo J in all types of senile plaques seems to be higher than that observed for Apo D in these structures (Fig. 3B-D). Accordingly, the absence of co-localization of Apo J and GFAP in the senile plaques is clearly observed in Fig. 3F.

Apo D and Apo J protein levels in frontal cortex of AD brain in relation with Braak’s stage

In order to analyze the Apo D and Apo J expression in the frontal cortex of AD patients, the samples were grouped according to Braak’s stage. The densitometric analysis of the immunohistochemical signal showed an increase of Apo D and Apo J signal with the progression of the disease. A positive correlation was found between the levels of the two proteins and Braak’s stage (Apo D, r = 0.7015, p < 0.05; Apo J, r = 0.9893, p < 0.05) (Fig. 4).

Apo D and Apo J presence in diffuse and mature senile plaques according to Braak’s stage

To investigate and compare the Apo D and Apo J expression in both types of senile plaques we count the number of DSP and MSP with positive staining for Apo J and for Apo D, also grouped according to Braak’s stage.

Regarding DSP, we observed that the behavior of both proteins is almost equal, showing a positive correlation (Apo D, r = 0.941, p < 0.05; Apo J, r = 0.8585, p < 0.05) with the progression of the disease without statistically significant differences between them (Fig. 5A).

In the quantification of the MSP, we have found a similar pattern in the immunolabeling for Apo D and Apo J, but with some differences. The number of plaques stained with any of the proteins shows a positive correlation with Braak’s staging, that is more dramatic in the case of Apo D, with an average number of positive plaques per area studied of 0.2 in Braak’s I and an average of 4.5 in Braak’s VI (Apo D, r = 0.9575, p < 0.05; Apo J, r = 0.9159, p < 0.05) (Fig. 5B). Moreover, as we can observe in the graph, the accumulation of Apo J in the MSP occurs mainly in the first stages of AD but continues until the last ones. Interestingly, the number of Apo J positive MSP is significantly higher than Apo D ones in Braak’s stage I (p < 0.05), as we show in Fig. 5B.

DISCUSSION

The compensation roles of apolipoproteins in neurodegenerative diseases like AD have been under investigation for years. To elucidate the possible related functions of some of these proteins in the AD brains, the first step is to study their possible complementary/similar expression patterns. In this work, we have observed that Apo D is present in most of the cellular types of the different cortices under study. As we previously described for the non-pathological brains [19, 20], we have detected Apo D presence in the cytoplasm of few cortical neurons but preferentially in the glial cells, as in astrocytes and with a stronger immunostaining in oligodendrocytes. Hence, it seems that the pattern of cellular location for Apo D is not altered in AD, although an increment in the immunostaining intensity has been observed with Braak’s stage, mainly in the multiform layer of the cortex. This fact could be related to the increase of the reactivity of glial cells of the white matter in the immediate vicinity of the deepest layer of the cortex that we observed in these situations. Apo D is also present in some AD hallmarks, such as the DSP and MSP, where its localization seems to be independent of the grade of maturity being detected in both diffuse and mature plaques, confirming previous observations of our group [30, 31]. In contrast, we found very few neurons with NFT and positives for Apo D.

Since the discovery of Apo D in the 1970s, it has been attributed to many functions. The current assumption is that Apo D is an “age-related” protein whose expression is highly regulated at the promoter level and that could have an important function for the proper CNS response to physiological and pathological brain oxidative stress, as it occurs in neurodegenerative diseases, such as AD, and in aging [24 , 40]. In this sense, it is shown that the absence of Apo D expression appears to enhance neuronal vulnerability to death [46, 50]. In contrast, its overexpression in cell lines and in several transgenic mouse and fly models or its direct exogenous addition protects against oxidants by the control of lipid peroxidation and seems to be critical for neural function and homeostasis [29 , 51–53]. However, the causal link that triggers Apo D synthesis and the possible function that it can exert are unclear. As a lipid transporter, this protein could prevent Aβ aggregation or promote its clearance in AD brains. In this sense, our group has recently documented that Aβ_25 - 35 induces Apo D expression in hippocampal cells in response to stress-induced growth arrest [40].

Nowadays, the role of Apo J in the brain is less known. Although there are not many studies about the immunolocation of Apo J in non-pathological brains, it has been demonstrated by different authors that Apo J expression in the CNS displays a regional and cell-type specific pattern. In fact, high levels of Apo J have been found in astrocytes as well as in the pyramidal neurons of hippocampus and in the Purkinje cells of cerebellum. However, its expression is low in microglia [7, 8]. In 1990, May et al. [10] demonstrated for the first time, the possible link between Apo J and AD. Their in situ hybridization observations confirmed that Apo J expression is increased in AD brains. Later studies confirmed the previous ones and also reported the presence of Apo J in the pathological hallmarks of AD, such as DSP and MSP, neuritic plaques, or cerebrovascular amyloid deposits [12 , 54–56] and, in contrast, to a lesser extent in NFT-containing neurons [12]. However, Lidstörm et al. [11] did not find a correlation between Apo J levels and the number of senile plaques, reporting the presence of Apo J only in a small portion of these structures.

In the present work, we have observed by immunohistochemical techniques, that Apo J is expressed in both glial and neuronal cells in AD brains, with the immunosignal appearing stronger in the latter. Moreover, our analysis showed a significant increase in Apo J levels correlated with Braak’s stages and the presence of this protein in all types of senile plaques, congophilic vessels, and some NFT. Growing evidence indicates that Apo J seems to be related to Aβ but its exact function is unknown (see Nuutinen et al. [8], for review). It could prevent the cytotoxic effect of Aβ [57] or could promote it, as a facilitator of the conversion of diffuse Aβ deposits into amyloid fibrils and enhancer of tau phosphorylation [58 –61]. In this study, we have observed, in contrast with previous reports [11], an increase in the number of Apo J positive senile plaques in relation with Braak’s stage. Interestingly, we have also found Apo J immunosignal in some NFT. Notwithstanding, not all the NFT are positive for Apo J and not all the positive neurons contain NFT. This observation was previously made by Giannakopoulos et al. [12] and proposed that Apo J-immunoreactive NFT-free neurons appear to be relatively resistant to neuronal death in AD, and in contrast, Apo J immunoreactivity is rarely observed in NFT-containing neurons. These findings together with the fact that in MSP Apo J colocalizes with Aβ in the plaque core seem to corroborate the protective role proposed for Apo J by several authors.

The ability of Apo J to bind and sequester misfolded and toxic oligomers formed during aggregation and disaggregation processes of Aβ_1 - 40 fibrils is a common finding from evidence over the last few years [16 , 63]. This protective function is related to its potential role as a potent ATP-independent extracellular chaperone protein, similar to that of the small heat shock proteins [62, 64]. Moreover, Apo J can also prevent Aβ aggregation by enhancing its transport across the blood-brain barrier via megalin receptor [65, 66] or its endocytosis within glial cells [67, 68]. Noteworthy, it has been shown that the APO E 4/4 allele, another apolipoprotein that seems to promote fibrillogenesis, significantly decreases the amount of Apo J in the frontal lobe in AD patients [56, 66].

According to our data, co-localization between Apo D and Apo J was negative, reinforcing our observations with individual immunostaining techniques. As mentioned previously, Apo D is expressed preferentially in glial cells both in astrocytes and oligodendrocytes, whereas Apo J shows a distribution almost opposite, with its immunosignal stronger in neurons. With respect to the AD hallmarks, Apo J is located preferentially in the DSP, in association with dystrophic neurites, while Apo D locates mainly in astrocytes and microglia of the mature ones. Therefore, being even in the same senile plaque, Apo D and Apo J occupy different places, i.e., Apo J in the plaque core and Apo D in the periphery. This could mean that Apo J is a neuroprotective protein in AD and, consequently, its increase an adaptive response to neuronal damage. In contrast, the Apo D overexpression would depend on the glial response after an initial damage, maybe involved in reinnervation processes.

CONCLUSIONS

Bearing in mind all our evidence, the increased expression of Apo D and Apo J with Braak’s stage could make them good markers of the progression of the disease. Thus, Apo J could act as a neuronal marker and Apo D as a glial marker of AD progression. In this sense, the two apolipoproteins seem to have complementary and cell-dependent neuroprotective roles in both normal and pathological conditions of the CNS. Further experimental efforts will be needed to elucidate the exact functions of these apolipoproteins in the neuronal and glial responses to a progressive brain injury and, more important, whether they can modulate each other at a transcriptional level in order to exert a coordinated response.

Footnotes

ACKNOWLEDGMENTS

This work was supported by FISS Instituto de Salud Carlos III and FEDER (Fondo Europeo de Desarrollo Regional) (PI15/00601) grant.

Authors’ disclosures available online ().

Appendix

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