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Journal of Neuropathology & Experimental Neurology:
doi: 10.1097/NEN.0b013e3181d53d98
Original Articles

Paxillin and Hydrogen Peroxide-Inducible Clone 5 Expression and Distribution in Control and Alzheimer Disease Hippocampi

Caltagarone, John PhD; Hamilton, Ronald L. MD; Murdoch, Geoffrey MD; Jing, Zheng PhD; DeFranco, Donald B. PhD; Bowser, Robert PhD

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Author Information

From the Department of Pathology (JC, RLH, GM, RB), Center for Neuroscience (ZJ), Department of Pharmacology (JC, DBD), and Pittsburgh Institute for Neurodegeneration (DBD, RB), University of Pittsburgh, Pennsylvania.

Send correspondence and reprint requests to: Robert Bowser, PhD, Department of Pathology, University of Pittsburgh School of Medicine, BST S-420, 200 Lothrop St, Pittsburgh, PA 15261; E-mail: Bowserrp@upmc.edu

This work was supported by Grant No. NS042724 (to Robert Bowser) from the National Institutes of Health.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.jneuropath.com).

Online-only figures are available at http://www.jneuropath.com.

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Abstract

Hydrogen peroxide-inducible clone 5 (Hic-5) and paxillin are members of the Group III LIM domain protein family that localize toboth cell nuclei and focal adhesions and link integrin-mediated signaling to the actin cytoskeleton. Prior in vitro studies have implicated paxillin in β-amyloid-induced cell death, but little is known about the expression and function of Hic-5 and paxillin in the brain. We performed a blinded retrospective cross-sectional study of Hic-5 and paxillin expression in the hippocampi of Alzheimer disease (AD) and control subjects using immunohistochemistry and laser scanning confocal microscopy. The analysis included assessment of the expression of phosphorylated isoforms of paxillin that reflect activation of distinct signaling pathways. We found changes in the subcellular distribution of Hic-5, paxillin, and specific phosphorylated isoforms of paxillin in the AD brains. The Hic-5 and phosphorylated isoforms of paxillin colocalized with neurofibrillary tangles. Paxillin was predominantly found in reactive astrocytes in the AD hippocampi, and activated paxillin was also detected in granulovacuolar degeneration bodies in AD. These data indicate that these important scaffolding proteins that link various intracellular signaling pathways to the extracellular matrix are modified and have altered subcellular distribution in AD.

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INTRODUCTION

The participation of focal adhesion proteins in the pathogenesis of Alzheimer disease (AD) has been postulated based upon integrin-mediated interactions with β-amyloid (Aβ) in vitro (1-5). The Aβ-induced activation of integrins in AD might trigger downstream signaling events that are linked to focal adhesion complexes, thereby differentially affecting cell functions and survival. Therefore, an analysis of focal adhesion proteins that link integrin activation to downstream signaling cascades may provide insight into cellular responses in AD.

Focal adhesions are macromolecular protein complexes that link integrin-mediated signaling to the actin cytoskeleton (6-9). Upon cell adhesion, integrins use both structural and signaling cell adhesion molecules to impact cellular motility and viability (7-10). During the initial stage of cell adhesion to the extracellular matrix (ECM), numerous proteins are required for focal adhesion assembly; the activity of each of these may be regulated by specific post-translational modifications such as phosphorylation (7-9, 11-14). For example, integrin activation leads to the phosphorylation and sequestration of LIM domain-containing proteins, such as paxillin, that may create an intracellular scaffold for many interacting cytoskeleton signaling proteins (6, 15, 16). Paxillin and the closely related protein, hydrogen peroxide-inducible clone 5 (Hic-5), are members of the Group III LIM domain family that contain 7 zinc fingers, at least 4 N-terminus leucine-rich motifs termed LD motifs, and numerous sites modified by phosphorylation (6, 15-20). Hydrogen peroxide-inducible clone 5 and paxillin share many interacting partners. Because of the close relatedness of their LIM domains, they are believed to act competitively or antagonistically in many signal transduction pathways (15, 17, 21-24). Paxillin is phosphorylated on tyrosine amino acid residues 31 and 118 (pY31 and pY118) upon the initial activation of ECM-integrin signaling and the formation of intracellular focal adhesion sites (5, 15, 25-28). Specific phosphorylation of paxillin is required for activation of distinct downstream signaling pathways (26, 27, 29-31). The integration of a variety of signaling cascades is further influenced by the ability of some focal adhesion proteins to regulate gene expression (10, 16). Indeed, both Hic-5 and paxillin localize within both cytoplasmic focal adhesions and the nucleus (10). The ability to act both in the nucleus and focal adhesions suggests that intracellular trafficking impacts their biologic effects. Hydrogen peroxide-inducible clone 5 contains 2 nuclear export signal sequences, but oxidative injury induces Hic-5 nuclear import in cultured fibroblasts (18). Hydrogen peroxide-inducible clone 5 has also been shown to be both a positive and a negative coregulator of gene expression (32-38).

Hydrogen peroxide-inducible clone 5 and paxillin are expressed in numerous regions of the rat brain including cerebellum, striatum, prefrontal cortex, hippocampus, hypothalamus, thalamus, and spinal cord (39). Although little is known about the specific roles of paxillin and Hic-5 in regulating focal adhesion signaling and gene expression within the brain, nongenomic roles for both paxillin and Hic-5 in the brain have been described. For example, paxillin is rapidly phosphorylated in the presence of fibrillar Aβ and colocalizes with hyperphosphorylated tau in cultured neurons (1). This altered subcellular distribution increases focal adhesion turnover and altered cell mobility and plasticity associated with a loss of synaptic integrity (1, 40). Hydrogen peroxide-inducible clone 5 decreases cell surface levels of dopamine transporter (DAT) in rat midbrain neuronal cultures and negatively affects dopamine uptake (39). A direct interaction between Hic-5 and DAT may be responsible for this effect. These reports suggest biologic functions of Hic-5 and paxillin in the brain, but a detailed analysis of paxillin and Hic-5 expression and distribution in control and AD brains has not been performed.

In view of the in vitro association between paxillin and Aβ-induced toxicity and Hic-5 response to oxidative stress and because the generation of amyloid plaques and neurofibrillary tangles (NFTs), the main hallmarks of AD, is influenced by reactive oxygen species and altered cell signaling pathways (41-43), we hypothesized that these associations may be involved in AD pathogenesis. Therefore, we performed a blinded retrospective analysis of the expression and subcellular distribution of Hic-5 and paxillin in hippocampi of 10 AD and 5 age-matched non-neurological disease control subjects using immunohistochemistry (IHC). Laser scanning confocal microscopy was used to colocalize Hic-5, paxillin, and phosphorylated isoforms of paxillin with the pathological hallmarks of AD. Our results provide novel insights regarding the expression and distribution of Hic-5 and paxillin in control and AD brains and of the phosphorylated isoforms of paxillin that are altered in AD.

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MATERIALS AND METHODS

Human Brain Tissue

The Committee for Oversight of Research Involving the Dead at the University of Pittsburgh School of Medicine approved the use of human tissue from the University of Pittsburgh Tissue Bank. A blinded retrospective cross-sectional study involving age-matched postmortem brains was performed using 5 cases with no clinical history of a neurodegenerative disease or AD pathology, 5 cases of pathologically defined early-stage AD, and 5 cases of pathologically defined late-stage AD (Table 1). All cases were categorized according to Braak staging and grouped for analysis into 3 categories: control (Braak Stages 0-I); Braak Stages II to III; and Braak Stages V to VI. The study consisted of 10 patients who were clinically diagnosed as having probable AD using the National Institute of Neurological and Communication Disorder and Stroke/Alzheimer's Disease and Related Disorders Association criteria with subsequent postmortem neuropathologic confirmation of AD using the Consortium to Establish a Registry for AD, Braak criteria, and the National Institute on Aging-Reagan Institute criteria (41, 44-47). Bielschowsky impregnations were performed on all cases.

Table 1
Table 1
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Western Blot

Tissue extracts were prepared essentially as previously described (48). Briefly, fresh-frozen human hippocampi from control and AD subjects were homogenized in 20% wt/vol of ice-cold Tris-Triton X-100 (TX-100) buffer with protease (Sigma, St Louis, MO) and phosphatase (Calbiochem, La Jolla, CA) inhibitors. The homogenate was centrifuged at 500 × g to remove debris and then centrifuged at 100,000 × g at 4°C. The supernatant was saved containing the TX-100-soluble fraction. The pellet containing the TX-100-insoluble fraction was resuspended in TX-100 buffer. The protein concentration for TX-100-soluble and -insoluble fractions was quantitated using the BCA Protein Assay Kit (Pierce). Fifty micrograms protein per lane for each subject was separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. Nonspecific antigens were blocked with 5% dry milk for 1 hour at room temperature and incubated overnight at 4°C with primary antibodies. Immunoblots were then incubated with horseradish peroxidase-conjugated secondary antibodies (Chemicon International, Temecula, CA) and developed using enhanced chemiluminescence (PerkinElmer Life Sciences, Boston, MA). Protein levels were quantified after scanning the appropriate molecular weight on the film using NIH Image 1.63. Each lane was normalized to actin, and the most intense band was arbitrarily set to 100%. Antibodies used for Western blot and IHC are listed in Table 2.

Table 2
Table 2
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IHC and Laser Scanning Confocal Microscopy

Tissue was immersion fixed in 10% buffered formalin and embedded in paraffin. Hippocampal tissue sections, 8-μm thick, were heated to 60°C for 1 hour before deparaffinization via 100% xylene and rehydration. Endogenous peroxidase activity in the tissue was blocked with 0.3% H2O2 for 30 minutes. To expose antigenic epitopes, sections were steamed for 20 minutes in target retrieval solution (DAKO, Carpinteria, CA). To reduce nonspecific primary antibody labeling for endogenous biotin, biotin receptors, and avidin binding sites in tissue, the avidin/biotin blocking kit was used according to company specifications (Vector Laboratories, Burlingame, CA). To block general nonspecific tissue antibody binding sites in tissue, SuperBlock (ScyTek Laboratories, Logan, UT) was added to tissue for 10 minutes. A no-primary antibody control slide was used to determine nonspecific immunoreactivity associated with the secondary antibodies. Tissue slides across all cases for each antibody were performed simultaneously to avoid day-to-day variability and were also not counterstained to permit accurate interpretation of the staining patterns. Commercial blocking peptides for phosphorylated Y118 (pY118) and pS126 paxillin were used to demonstrate specificity of the immunostaining. For the blocking peptides, brain tissue was incubated with antibody and 100-fold molar excess of either specific blocking peptides or nonspecific blocking peptides. Immunoreactivity on the tissue slides was visualized with Nova-Red chromogen (Vector Laboratories), dehydrated, and mounted with Permount (Fisher, Pittsburgh, PA).

Confocal microscopy was performed as previously described, except that secondary antibodies were conjugated to Alexa Fluor-488 and -568. Triple labeling was performed by incubation with the far-red fluorescent nucleic stain TO-PRO-3 (Invitrogen, Carlsbad, CA). Pyramidal neurons in human brain are known to contain elevated levels of lipofuscin granules typically localized within the soma, which exhibit significant autofluorescence at longer wavelengths. Therefore, to minimize the contribution of autofluorescence to laser scanning confocal microscopy signals, secondary antibodies conjugated to Alexa Fluor-488 were used for Hic-5 or paxillin, whereas secondary antibodies conjugated to Alexa Fluor-568 were used for colocalization with well-characterized markers for AD pathology.

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Immunoprecipitation

Fresh-frozen hippocampi from control and AD subjects were homogenized in 5 volumes of cold lysis buffer (50 mmol/L Tris HCl pH 7.4, 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate-stage, 0.1% sodium dodecyl sulfate, protease [Sigma], and phosphatase [Calbiochem] inhibitors) in a glass-Teflon homogenizer (500 rpm, 10 strokes). One hundred microliters of total homogenate from control and AD subjects were preincubated with 4 μL primary antibodies for 2 hours. The tissue lysate was then mixed with Protein G Magnetic Microbeads for 30 minutes. To activate the μMACS columns (Miltenyi Biotec, Auburn, CA), 200 μL of lysis buffer was added before the immunoprecipitates were captured using the magnetic field. After the tissue lysate flowed through the columns, they were washed 4× 200-μL lysate buffer, 1× 200-μL high-salt buffer (500 mmol/L NaCl, 1% NP-40, 50 mmol/L Tris HCl pH 8.0), and 1× 200-μL low-salt buffer (20 mmol/L Tris HCl pH 7.5). To elute the bound complexes, 20 μL of preheated (95°C) 1× sample buffer (200 mmol/L Tris HCl pH 6.8, 0.2% sodium dodecyl sulfate, 10% glycerol, 0.01% bromphenol blue) was added to the column for 5 minutes. Eighty microliters of 1× sample buffer was then added, and the flow-through was saved. To control for nonspecific binding of protein to μMACS columns, control or AD tissue lysates were combined with Microbeads without prior incubation with primary antibodies and monitored proteins that eluted from the column.

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Rank Order and Statistical Analysis

The rank intensity order test was used as a nonparametric statistical method (49). The intensity of protein expression was ranked from lowest to highest based on blinded qualitative observations and assigning values of 1 to 15. The rank intensity order was then divided into groups based on the unblinded clinical information. The Mann-Whitney U test was used when comparing intensity differences in control versus AD subjects; the Kruskal-Wallis (analysis of variance) test was used when comparing control (Braak Stages 0-I), Braak Stages II to III, and Braak Stages V to VI subjects (GraphPad Prism 4.0a).

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RESULTS

Immunoblot Analysis for Hic-5 and Paxillin

Immunoblot analysis of cell extracts from cultured prostate stromal cells (WPMY-1) and mouse embryonic fibroblast cells (MEF) known to express either Hic-5 (WPMY-1) or paxillin (MEF) was used to demonstrate specificity of each antibody (50, 51). Small-interfering RNA to Hic-5 or genetic ablation of the paxillin gene in cultured cells demonstrated specific immunoreactive bands of the appropriate molecular mass for each antibody (Figure, Supplemental Digital Content 1, http://links.lww.com/NEN/A89). Also included in each gel are the TX-100-soluble (100,000 × g) and -insoluble tissue extracts prepared from control and AD hippocampi. A nonspecific lower-molecular weight band was detected by many of the anti-paxillin antibodies, but specific bands of the appropriate mass were observed in control and AD brain extracts.

Both TX-100-soluble and -insoluble fractions of control and AD hippocampi revealed a specific Hic-5-positive band of the appropriate molecular weight (Fig. 1). Hydrogen peroxide-inducible clone 5 levels in the TX-100-soluble fraction trended to decrease (p < 0.087) in Braak Stages II to III and Braak Stages V to VI compared with control subjects (Figs. 1A, C). Hydrogen peroxide-inducible clone 5 protein levels in the TX-100-insoluble fraction were significantly decreased (p < 0.012) in Braak Stages II to III compared with control and Braak Stages V to VI AD subjects (Figs. 1B, D). Paxillin was present in the TX-100-soluble fractions in all cases, but we did not detect any overall changes in AD compared with controls (data not shown).

Figure 1
Figure 1
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Hic-5 and Paxillin Distribution in the Hippocampus

Classical plaques are composed of a heterogeneous population of extracellular Aβ deposits, dystrophic neurites, NFTs, activated astrocytes, and microglia (42, 43). Therefore, double label confocal microscopy with markers specific to plaques (Aβ), NFTs (AT8), activated astrocytes (glial fibrillary acidic protein [GFAP]) and microglia (CD68) was used to determine the localization of Hic-5 and paxillin in AD brains. In control and AD hippocampi, Hic-5 immunoreactivity was observed predominantly in cell bodies of pyramidal neurons throughout the stratum pyramidale layer (Fig. 2A), in dentate gyrus neurons, and round cells of the stratum oriens layer (data not shown). Hydrogen peroxide-inducible clone 5 was occasionally detected within pyramidal neuron nuclei in both AD and control hippocampi. Hydrogen peroxide-inducible clone 5 immunoreactivity in astrocytes was not observed in controls. By rank order analysis, Hic-5 immunoreactivity was determined to be significantly greater in CA1 pyramidal neurons in AD versus control subjects (Figs. 2B, C). This observation contradicts in vitro studies in which Hic-5 was found to be predominantly in the nucleus. We also detected Hic-5 in endothelial and smooth muscle cell nuclei of surrounding arteries and large capillaries (data not shown).

Figure 2
Figure 2
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By double label confocal microscopy with markers specific to Aβ plaques, NFTs, activated astrocytes, and microglia, Hic-5 immunofluorescence was observed within pyramidal neuron neurites, cell bodies, and nuclei throughout AD hippocampi (Fig. 3). Hydrogen peroxide-inducible clone 5-positive pyramidal neurons were also adjacent to Aβ-containing plaques (Figs. 3a-c); it was also detected in beaded neuritic processes throughout the hippocampi and in a subset of plaques (Figs. 3a, j). A majority of AT8-positive NFTs including those adjacent to plaques were Hic-5 positive (Figs. 3d-i). Colocalization of Hic-5 in AT8-positive NFTs was evident in perinuclear regions or limited to large processes proximal to the cell body (Figs. 3d-i). AT8-positive neuropil threads were negative for Hic-5, and it was not observed in extracellular ghost tangles (Figs. 3d-i). Hydrogen peroxide-inducible clone 5 was often seen within plaque cores (Figs. 3a-c); it was not localized to GFAP-positive astrocytes (not shown), but there was some colocalization with CD68 in microglia (Figs. 3j-l).

Figure 3
Figure 3
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By light microscopy, paxillin was predominantly located to the outer white matter closest to the lateral ventricle and the stratum lacunosum in control hippocampi (Figs. 4Aa, b). Paxillin was detected in the nucleus and cytoplasm of reactive astrocytes and neuropil within the hilus/CA4 region of all cases and was also present in astrocytes within hippocampal subfields in AD cases (Figs. 4Ac, d). At higher-power magnification, paxillin was observed in focal nuclear granules of CA1 pyramidal neurons in controls; these were present to a lesser degree in AD (Figs. 4Ae, f). By rank order analysis, there was a statistically significant increase in paxillin immunoreactivity in AD (Fig. 4B), but this difference did not reach statistical significance when the AD subjects were separately categorized to Braak stages (data not shown). We also detected paxillin in the nuclei of choroid plexus cells in control subjects, with loss of nuclear immunostaining in AD (Figure, Supplemental Digital Content 2, http://links.lww.com/NEN/A90). Therefore, paxillin immunoreactivity is elevated in reactive astrocytes and decreased in the nuclei of CA1 pyramidal neurons and choroid plexus cells in AD.

Figure 4
Figure 4
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To define cell types expressing paxillin and any association with AD pathology, we performed double label confocal microscopy. Paxillin was elevated in cells containing numerous processes surrounding and penetrating Aβ-containing plaques in AD (Figs. 5a-c). These cells colocalized with GFAP (Figs. 5d-f), consistent with observations by light microscopy (Fig. 4A). Colocalization of paxillin and GFAP was not evenly distributed and appeared in discrete locations within the glial processes (Fig. 5f). Paxillin was not detected in AT8-positive NFTs (Figs. 5g-i).

Figure 5
Figure 5
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Distribution of Activated Paxillin in AD

To identify cell types that exhibit activated paxillin (i.e. pY31 and pY118), we performed light microscopy and confocal microscopy. In control subjects, pY31 paxillin immunoreactivity was distributed throughout the soma, processes, and in nuclei of hippocampal pyramidal neurons (Fig. 6Aa). In AD, pY31 paxillin was observed in the cytoplasm and as punctate structures within the soma, with comparatively lower levels in neuronal nuclei (Fig. 6Ab). The pY31 paxillin immunoreactivity was observed in both Hirano bodies and plaques (Figs. 6Ac, d). By confocal microscopy, pY31 paxillin was observed in pyramidal neurons and colocalized with AT8 in soma and neuritic processes (Figs. 6Ba-f). Within plaques, pY31 paxillin was present in both AT8-positive and -negative neuritic processes (Figs. 6Bd-f).

Figure 6
Figure 6
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Paxillin pY118 immunoreactivity was predominantly located in the nuclei of hippocampal pyramidal neurons in control subjects (Fig. 7Aa). In AD, pY118 paxillin immunoreactivity was decreased within nuclei and present in granulovacuolar degeneration (GVD) bodies (Fig. 7Ab). The specificity of the pY118 paxillin immunoreactivity was confirmed using phosphorylated and nonphosphorylated blocking peptides (Figs. 7Ac, d). By confocal microscopy, pY118 paxillin was observed in AT8-positive NFTs and plaques but not in neuritic threads (Fig. 7B). Abundant pY118 paxillin was observed within nuclei of neuronal and non-neuronal cells (Fig. 7B).

Figure 7
Figure 7
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We next examined phosphorylation of serine residues in paxillin within control and AD brains. Phosphorylation of serine residues is regulated by the c-Jun N-terminal kinase pathway and modulates cell migration. Immunoreactivity for paxillin phosphorylated on serine residue 126 (pS126) was detected in the somata and nuclei of neurons throughout the hippocampi of control subjects (Fig. 8A). Within AD hippocampi, pS126 paxillin was detected in structures resembling GVD bodies in the cytoplasm of CA1 pyramidal neurons (Fig. 8Ab). The specificity of the pS126 paxillin antibody was confirmed using phosphorylated and nonphosphorylated blocking peptides (Figs. 8Ac, d). By confocal microscopy, pS126 paxillin was observed in pyramidal neurons, and it was colocalized with AT8-positive soma (Figs. 8Ba-c); it was largely absent from AT8-positive neuritic processes within plaques (Figs. 8Bd-f). Phosphorylated serine 178 (pS178) paxillin immunoreactivity was similarly detected in neuronal somata and nuclei throughout the hippocampi of control subjects; it was absent from the nuclei and was located in GVD bodies in AD (Fig. 9A). The pS178 paxillin was observed in neuronal processes, GVD bodies, and NFTs, but not AT8-positive neuritic threads (Fig. 9B).

Figure 8
Figure 8
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Figure 9
Figure 9
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Finally, we examined whether Hic-5 and paxillin are associated with phosphorylated tau (pTau) using a coimmunoprecipitation assay. Phosphorylated tau was contained within immunoprecipitates of Hic-5, paxillin, or activated isoforms of paxillin (i.e. pY31, pY118) in AD but not control subjects (Fig. 10). Thus, Hic-5, paxillin, and at least 2 phosphorylated isoforms of paxillin are stably associated with pTau in AD brain.

Figure 10
Figure 10
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DISCUSSION

We postulated that the focal adhesion proteins paxillin and Hic-5 (which regulate integrin signaling in AD) may exhibit altered activation and subcellular distribution and that this might modulate neuronal function and contribute to its pathogenesis. Immunoblot analysis demonstrated Hic-5 and paxillin expression in human hippocampus but failed to show significant alterations in detergent extracts from whole tissue; however, there were reduced Hic-5 levels in membrane fractions in Braak Stages II to III AD subjects. By IHC, Hic-5 was predominantly expressed in neurons within hippocampi and localized to NFTs in AD. Paxillin was detected in reactive astrocytes and CA1 pyramidal neurons, whereas phosphorylated isoforms of paxillin were observed in pyramidal neurons and NFTs throughout the AD hippocampi. Activated paxillin was also detected in NFTs and GVD bodies in AD hippocampi. These data suggest that the scaffolding proteins that link various intracellular signaling pathways to the ECM are altered in AD, possibly early in the disease.

Our data provide the first characterization of Hic-5 and paxillin expression and distribution in the human brain. Hydrogen peroxide-inducible clone 5 is expressed predominantly in pyramidal neurons of human hippocampus. To date, the only known function of Hic-5 in neurons is its regulation of surface levels of DAT in rat midbrain neuronal cultures and inhibition of dopamine uptake (39). Within the midbrain, a direct interaction between Hic-5 and DAT might be responsible for this effect. Our observation that Hic-5 protein levels are significantly elevated within the cytoplasm of CA1 pyramidal neurons in AD suggests that it may function similarly in sequestering cell surface receptors or transporters away from the plasma membrane to alter function and intracellular signaling. A recent study analyzed the expression and localization of dopamine receptor (DR1-5) subtypes in control brain versus AD brain, indicating that the dopaminergic system is altered in AD (52). Whether Hic-5 plays any role in regulating these dopamine receptor subtypes or other neuronal cell surface receptors during AD is not known at present.

Hydrogen peroxide-inducible clone 5 was predominantly located within the cell body of pyramidal neurons and excluded from the nucleus in most cells in both control and AD hippocampi. Hydrogen peroxide-inducible clone 5 was observed in the nuclei of a subset of CA1 pyramidal neurons and non-neuronal cells in AD subjects, and it colocalized with pTau in NFTs. Hydrogen peroxide-inducible clone 5 was also observed in the core of many amyloid plaques. Both oxidative stress and transforming growth factor-β are known regulators of Hic-5 expression and subcellular distribution, but further studies are required to determine the potential role of oxidative stress in modulating Hic-5 distribution in the brain.

Paxillin was observed to be expressed predominantly in activated astrocytes throughout the hippocampi and elevated in AD subjects. Previous studies have not detected paxillin in astrocytes, but rodent models of brain trauma exhibit increased paxillin only in reactive glia of the white matter, suggesting that initial glial reactions to trauma generate new "focal contacts" requiring GFAP and paxillin (53). Thus, it is possible that colocalization of paxillin in GFAP-positive astrocytes reflects the generation of new focal adhesions required for astrocytic motility and activation in AD. Interestingly, paxillin was detected in focal granules in the nucleus of both CA1 pyramidal neurons and cells of the choroid plexus in controls, whereas paxillin immunoreactivity in the nucleus was decreased in AD subjects. Although alterations in CA1 pyramidal neurons have been extensively observed in various neurodegenerative disorders and trauma, only more recent studies have observed alterations in the structure and function of the choroid plexus in AD (54-58). The choroid plexus is known to undergo morphological alterations in late-stage AD (55, 56), and it has also been postulated that it may be responsible for removing Aβ from the cerebrospinal fluid (59). Changes in the localization of paxillin out of focal nuclear granules and the nucleus in both CA1 neurons and choroid plexus cells might reflect activation of key regulatory signaling pathways in AD.

Paxillin is phosphorylated on tyrosine residues Y31 and Y118 sites upon the initial activation of ECM-integrin signaling and the formation of intracellular focal adhesion sites. In cultured neurons, paxillin is also rapidly phosphorylated on tyrosine residues in the presence of fibrillar Aβ (1). Phosphorylation of paxillin on serine residues S126 and S178 is regulated by c-Jun N-terminal kinase signaling pathways (29-31, 60). We observed all phosphorylated isoforms of paxillin in the nucleus, soma, and processes of pyramidal neurons in control subjects, whereas the nuclear localization of all phosphorylated paxillin examined was reduced in AD. The pY31 was rarely detected in NFTs, but pY118 was detected not only in NFTs, but also in GVD bodies within soma of pyramidal neurons. The differences in staining of these 2 phosphorylated paxillin isoforms may reflect activation of distinct signaling pathways in AD. Both pS126 and pS178 were present in GVDs and NFTs in AD hippocampus, with highest levels in CA1. These results suggest that paxillin phosphorylation occurs during AD, and that paxillin protein is redistributed to intracellular protein aggregates and NFTs upon phosphorylation. We also noted the presence of phosphorylated paxillin in nuclei of non-neuronal cells.

We demonstrated an association between Hic-5, paxillin, pY31, and pY118 paxillin with pTau in AD brains by coimmunoprecipitation assays using tissue lysates. These results suggest that paxillin and Hic-5 may modulate tau phosphorylation or associate with hyperphosphorylated tau in vivo. Further studies are required to determine whether the interactions between Hic-5, paxillin, and tau occur before the formation of cytoplasmic aggregates or result only after the accumulation of proteins within aggregates and tangles. Our data suggest that altered Hic-5 and paxillin distribution in AD hippocampi occurred concurrent with tau and amyloid pathology but not before the accumulation of typical AD neuropathology. Biochemical data supporting direct interactions of pS126 and pS178 paxillin with tau await future studies.

The localization of phosphorylated paxillin within nuclei is a novel finding that warrants further investigation. Interestingly, there was decreased paxillin in CA1 pyramidal neuron nuclei and in choroid plexus cells in AD. The role of paxillin in the nucleus is poorly understood, although it can function as a coactivator of gene expression and can bind poly(A) binding proteins to facilitate mRNA transport from the nucleus (35, 37). It is possible that phosphorylated paxillin may also regulate gene expression or mRNA trafficking in the hippocampus.

We note that antibodies to nonphosphorylated paxillin predominantly detected glial cells, whereas antibodies to activated isoforms of paxillin predominantly labeled neurons and nuclei of non-neuronal cells. The basis for this difference may reflect the differential state of paxillin phosphorylation in neurons and glia. Prior in vitro models demonstrated that activation of astrocytes generated a loss of paxillin phosphorylation on tyrosine residues (61). This may result in the loss of immunoreactivity to activated astrocytes with antibodies to phosphorylated forms of paxillin.

In summary, our results provide novel insights into the distribution and activation state of focal adhesion proteins in AD, suggesting that altered signals from the ECM may modulate neuronal function, motility, and survival in neurodegenerative diseases. Future studies will examine the functional role of these focal adhesion proteins in Aβ-induced cell death as well as potential therapeutic targets for AD, and determine the function of Hic-5 and paxillin within neuronal nuclei.

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ACKNOWLEDGMENTS

The authors thank Jonette Werley and Wanda Wang for expert technical assistance.

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Keywords:

Alzheimer disease; Amyloid plaque; Focal adhesions; Granulovacuolar degeneration; Hippocampus; Neurofibrillary tangles

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