Differential regulation of Nedd4 ubiquitin ligases and their adaptor protein Ndfip1 in a rat model of ischemic stroke

Experimental Neurology 235 (2012) 326–335

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Differential regulation of Nedd4 ubiquitin ligases and their adaptor protein Ndfip1 in a rat model of ischemic stroke Jenny Lackovic a, Jason Howitt a, Jennifer K. Callaway b, John Silke c, Perry Bartlett d, Seong-Seng Tan a,⁎ a

Brain Development and Regeneration Laboratory, Florey Neuroscience Institutes, The University of Melbourne, Parkville, Australia Department of Pharmacology, The University of Melbourne, Parkville, Australia Department of Biochemistry, La Trobe University, Bundoora, Australia d Queensland Brain Institute, University of Queensland, St. Lucia, Australia b c

a r t i c l e

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Article history: Received 30 December 2011 Revised 20 February 2012 Accepted 25 February 2012 Available online 5 March 2012 Keywords: Nedd4 Itch Ndfip1 E3 ubiquitin ligase Ubiquitination Cerebral ischemia Stroke Neuroprotection

a b s t r a c t Ubiquitin-modification of proteins by E3 ubiquitin ligases is an important post-translational mechanism implicated in neuronal survival and injury following cerebral ischemia. However, of the 500 or so E3s thought to be present in mammalian cells, very few specific E3s have been identified and associated with brain ischemia. Here, we demonstrate endogenous induction of HECT-type E3 ligases of the Nedd4 family and their adaptor Nedd4-family interacting protein 1 (Ndfip1) following transient focal cerebral ischemia in rats. Ndfip1 is upregulated in surviving cortical neurons and its neuroprotective activity is correlated with Nedd4-2 upregulation, but not two other Nedd4 family members examined (Nedd4-1 and Itch). Immunoprecipitation assays confirmed biochemical binding of Ndfip1 with Nedd4-2 in the brain, with or without ischemic stroke, indicating their endogenous interaction. While Ndfip1 and Itch have been previously shown to interact outside of the nervous system, ischemic induction of Itch in the present study was associated with cellular survival independent of Ndfip1. Together, these findings demonstrate specific and differential regulation of Nedd4 family E3 ligases under ischemic conditions, and identify two E3 ligases and their adaptor that potentially regulate ubiquitination in ischemic stroke to provide neuroprotection. © 2012 Elsevier Inc. All rights reserved.

Introduction Ubiquitin is a highly conserved 76-amino-acid polypeptide that can attach to one or more lysine residues of proteins through an enzymatic cascade known as ubiquitination (Hershko and Ciechanover, 1998). This post-translational modification of proteins can lead to either protein degradation in the 26S proteasome, or protein sorting and trafficking through various cell machinery. Ubiquitinmodification of proteins regulates diverse physiological processes including receptor internalization, transcriptional regulation, protein quality control, and apoptosis. In the nervous system, ubiquitination is a hallmark feature of neurodegenerative disease, and more recently, has also been implicated in the pathobiology of ischemic stroke. Following brain ischemia, protein ubiquitination is increased within the first few hours of reperfusion and is accompanied by ubiquitin upregulation, depletion of free ubiquitin and changes in ubiquitin distribution (Hu et al., 2001; Ide et al., 1999; Liu et al., 2004, 2006; Noga et al., 1997). Within ischemic neurons destined for death, ubiquitinated proteins (ubi-proteins) are found in protein aggregates ⁎ Corresponding author at: Brain Development and Regeneration Laboratory, Florey Neuroscience Institutes, The University of Melbourne, Parkville Victoria 3010, Australia. Fax: + 61 3 9347 0446. E-mail address: stan@florey.edu.au (S.-S. Tan). 0014-4886/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2012.02.014

that accumulate and persist (Hu et al., 2000, 2001; Liu et al., 2004), and ubi-protein deposition is thought to be caused by underlying post-ischemic proteasome dysfunction and failure to remove proteins marked for proteasome degradation (Asai et al., 2002; Ge et al., 2007; Keller et al., 2000). While the build up of ubi-proteins due to proteasomal failure is correlated with neuronal death (Hu et al., 2000, 2001; Liu et al., 2004), neuron populations resistant to ischemic death paradoxically show early, but transient, increases in protein ubiquitination (Ide et al., 1999; Liu et al., 2004). Furthermore, ubiquitin-conjugation and degradation of proteins (e.g. proapoptotic factor Bim) also underlie ischemic tolerance in neurons; an endogenous protective response to lethal ischemia that is induced by prior exposure to a brief and non-harmful ischemic event known as preconditioning (Meller et al., 2006, 2008; Pradillo et al., 2005). Ubiquitin-proteasome regulation also renders tolerant neurons resistant to excitotoxicity through synaptic reorganization (Meller et al., 2008) and reduces brain infarction by mediating downstream TNF-α signaling events (Pradillo et al., 2005). Thus, ubiquitin modification and signaling of diverse protein targets is a multifaceted, but poorly understood, mechanism mediating both neuron death and survival during ischemia. A major class of enzymes involved in the conjugation of ubiquitin to their targets are the E3 ligases with HECT (homology to the E6associated protein carboxyl terminus domain) domains (Huibregtse

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et al., 1995). Nedd4-1 and Nedd4-2 are archetypal members of this family (nine members in total); with the ability to recognize and ubiquitinate protein targets containing PPxY motifs (Pirozzi et al., 1997; Staub et al., 1996). Besides proteasomal degradation of targeted proteins, Nedd4 proteins also participate in a number of cellular trafficking activities including viral budding, protein sorting, and cell signaling (for reviews, see Ingham et al., 2004; Shearwin-Whyatt et al., 2006). The protein targets of Nedd4-mediated ubiquitination are diverse in function and location (Shearwin-Whyatt et al., 2006). In the nervous system, Nedd4-mediated ubiquitination is required for downregulating voltage-gated K + and Na + channels (Ekberg et al., 2007; Fotia et al., 2004), axon-guidance proteins (Myat et al., 2002), and TrkA neurotrophin receptor (Arevalo et al., 2006). E3 ligases such as Nedd4 can further extend their sphere of influence to ubiquitinate proteins that they do not normally recognize. For example, Nedd4-family proteins (e.g. Nedd4-1, Nedd4-2, and Itch) can ubiquitinate proteins lacking PPxY motifs by employing Nedd4 adaptors which contain PPxY motifs. One such adaptor is Ndfip1, a transmembrane protein that is richly expressed in the Golgi and post-Golgi vesicles of cells, including neurons (Harvey et al., 2002; Putz et al., 2008; Sang et al., 2006). Ndfip1 upregulation associated with Nedd4 co-expression has been previously demonstrated to benefit surviving neurons after traumatic brain injury (Sang et al., 2006). In human neurons, protein ubiquitination by Ndfip1 via Nedd4-2 is instrumental for improving the survival of neurons under stress from metal toxicity (Co++, Fe ++) (Howitt et al., 2009). More recently, it has been demonstrated that injection of a synthetic cobalt compound can induce upregulation of Ndfip1 in cortical neurons (Schieber et al., 2010). Together, the above studies point to the importance of Ndfip1 and Nedd4 proteins for improving neuron survival under stress conditions, including cerebral ischemia (Howitt et al., 2012). In the present work, we investigated the expression profile of Ndfip1 in surviving neurons and its relationship with three members of the Nedd4-family (Nedd4-1, Nedd4-2, and Itch) in a rat model of transient focal cerebral ischemia. We found that Ndfip1 upregulation is strongly correlated with neuron survival following ischemic stroke, and Nedd4-2, but not Nedd4-1 or Itch, was co-upregulated with Ndfip1. We provide evidence for biochemical interaction between Nedd4-2 and Ndfip1 in brain tissues under normal conditions and also in ischemic stroke. Further, we show Itch is associated with survival of a cellular subpopulation, but this is independent of Ndfip1 upregulation. We conclude that upregulation of certain E3 ligases with HECT domains, together with its Ndfip1 adaptor, may be important for survival of post-ischemic neurons by modifying the ubiquitin status of target proteins. Materials and methods Animals All procedures were performed in accordance with the Prevention of Cruelty to Animals Act 1986 and National Health and Medical Research Council of Australia (NH & MRC) Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Hooded Wistar rats were obtained from Adelaide University, Australia. C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were obtained from the Animal Resources Centre, Australia. All animals were housed under a 12 h light/dark cycle in temperature controlled rooms (22 °C) and allowed free access to standard chow and water. Endothelin-1-induced middle cerebral artery occlusion (ET-1-induced MCAo) Adult male hooded Wistar rats (weighing 421 ± 11.4 g) were randomly assigned to receive ET-1-induced MCAo or sham treatment. Focal transient MCAo was induced in conscious rats using

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intracerebral injection of the vasoconstrictive peptide endothelin-1 (ET-1; American Peptide Company, Inc., Sunnyvale, CA). Cannula implantation and induction of ET-1-induced MCAo was performed as previously described (Callaway et al., 1999). Animal behavior was monitored during ET-1 administration and an ischemic stroke severity score (1–5; 1 = mild, 5 = severe) was assigned to each animal based on a defined scale of neurological deficits as previously described (Roulston et al., 2008). Animals that received ET-1 but did not show stroke behavior were excluded from further analysis. Sham-operated animals were subjected to cannula implantation but received an equal volume of saline instead of ET-1 (Callaway et al., 1999). Saline administration did not induce stroke-behaviors and resulted in only a small amount of brain damage associated with the cannula tract (analysis was not performed on sham or ischemic tissue in the vicinity of cannula insertion). Core (rectal) temperature was monitored with a thermistor probe before ischemia and at regular intervals for 2 h following ischemia to ensure maintenance of normothermia. Immunohistochemistry Rats were sacrificed by pentobarbital overdose and transcardially perfused with phosphate-buffered saline (PBS, pH 7.4) after MCAo before embedding brains in Tissue-Tek O.C.T. compound (Sakura Finetek, Tokyo, Japan). Fresh frozen coronal sections (14 μm) were fixed with 4% PFA in 0.1 M PB, blocked and permeabilized in 10% normal goat serum with 0.3% Triton X-100 in 0.1 M PB. Sections were incubated with primary antibodies overnight followed by appropriate secondary antibodies for 1 h at room temperature. DNA fragmentation as an indicator of cell death was detected using terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) as per the manufacturer's instructions (Roche, Basel, Switzerland). Sections were counterstained with DAPI (1:10,000, Dako, Carpinteria, CA) before mounting under glass cover slips with anti-fade mounting reagent. Immunostaining was visualized by a blinded observer using a Zeiss Axiovert 200-LSM 5-Pascal laser scanning confocal microscope, an Olympus FluoView FV1000 laser scanning confocal microscope, an Olympus BX51 fluorescence microscope or Leica MZ16F stereo microscope. Primary antibodies were: purified rabbit polyclonal anti-Ndfip1 (1:200, Howitt et al., 2009); rat monoclonal anti-Ndfip1, clone 1G5 (1:200, Howitt et al., 2012); mouse monoclonal anti-neuronal-specific nuclear protein (NeuN; 1:500, Chemicon, Temecula, CA); purified rabbit polyclonal anti-Nedd4-2 (1:1,000, a gift from Prof. S. Kumar, Center for Cancer Biology, SA Pathology); mouse monoclonal anti-Nedd4-1, clone 15 (1:100, BD Transduction Laboratories, San Jose, CA) and mouse monoclonal anti-Itch, clone 32 (1:200, BD Transduction Laboratories). Secondary antibodies were: Alexa Fluor 594-conjugated goat antirabbit IgG (1:500, Molecular Probes, Eugene, OR); Alexa Fluor 594conjugated goat anti-rat IgG (1:500, Molecular Probes); Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:500, Molecular Probes); Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:500, Molecular Probes); and Alexa Fluor 594-conjugated donkey anti-mouse IgG (1:500, Molecular Probes). Quantification of Ndfip1 upregulation and TUNEL Double Ndfip1 and TUNEL immunohistochemistry was performed on coronal sections from sham-operated and ET-1-induced MCAo rats as described above and visualized by a blinded investigator using an Olympus BX51 fluorescence microscope. Contiguous microscopic fields covering the entire area of the ipsilateral cortex were analyzed to quantify the number of Ndfip1-upregulated neurons and TUNELpositive cells. Briefly, cells were manually counted on screen for each field (1.069 mm 2) at 200× magnification with the aid of a counting grid overlay. Cell counts in each field were then tallied to

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obtain the total number of Ndfip1-upregulated or TUNEL-positive cells per ipsilateral cortex. Two random sections were chosen in the range of +1.20 to + 1.00 mm relative to Bregma for each animal. Protein lysate preparation and immunoprecipitation assays Frozen rat (peri-infarct cortical region of ipsilateral and corresponding contralateral hemisphere region) or mouse brain hemispheres were homogenized by passing through fine gauge needles in ice-cold RIPA lysis buffer (50 mM Tris, pH 7.2, 0.15 M NaCl, 2 mM EDTA, 1% NP40, 0.1% SDS) with Complete Mini protease inhibitor cocktail (Roche) for 20 min at 4 °C. Brain homogenates were cleared of insoluble debris by centrifugation at 13,000 rpm for 15 min at 4 °C. Rat/mouse brain lysates were co-immunoprecipitated with polyclonal or monoclonal anti-Ndfip1 (1:100), anti-Nedd4-2 (1:100) or control IgG (anti-β-actin) for 1 h at room temperature followed by pull down with Protein G beads (Pierce, Rockford, IL) for precipitation of Ndfip1, or Protein A beads (Zymed, Carlsbad, CA) for precipitation of Nedd4-2 for 2 h at room temperature. For each experiment, beads were washed 3 times with RIPA buffer before elution using the manufacturer's instructions and eluted proteins were suspended in Laemmli buffer for SDS-PAGE and analyzed by Western blotting. Antibodies were: purified rabbit polyclonal anti-Ndfip1 (Howitt et al., 2009); rat monoclonal anti-Ndfip1, clone 1G5 (Howitt et al., 2012); purified rabbit polyclonal anti-Nedd4-2 (a gift from Prof. S. Kumar); and mouse monoclonal anti-β-actin, clone AC-40 (Sigma, St. Louis, MO). For comparison of cortical lysates from ET-1-induced MCAo rats, total protein concentration was measured using the detergent-compatible (DC) protein assay according to manufacturer's instructions (Bio-Rad, Hercules, CA) and 50 μg of protein loaded per well. Western blotting Immunoprecipitates or lysates were resolved on SDS-PAGE gels (10 or 12%) followed by transfer onto Hybond C nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK). Membranes were blocked for 1 h at room temperature in 5% non-fat milk in Trisbuffered saline, 0.05% tween-20 (TBST). Blots were incubated overnight with primary antibodies at 4 °C followed by appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Proteins were detected using Amersham enhanced chemiluminescence reagent (GE Healthcare) as per the manufacturer's instructions and visualized by exposure to X-ray film. Primary antibodies were: purified rabbit polyclonal anti-Ndfip1 (1:1,000, Howitt et al., 2009); rat monoclonal anti-Ndfip1, clone 1G5 (1:1,000, Howitt et al., 2012); purified rabbit polyclonal anti-Nedd4-1 (1:2,000, a gift from Prof. S. Kumar); purified rabbit polyclonal anti-Nedd4-2 (1:1,000, a gift from Prof. S. Kumar); mouse monoclonal anti-Itch, clone 32 (1:2,000, BD Transduction Laboratories); and mouse monoclonal anti-β-actin, clone AC-15 (1:5,000, Sigma). HRP-conjugated secondary antibodies were: goat polyclonal anti-rabbit (1:10,000, Millipore, Billerica, MA); goat polyclonal anti-rat (1:5,000, Millipore); and goat polyclonal anti-mouse (1:10,000, Millipore). Western blots were scanned at 400 dpi for densitometry of Ndfip1, Nedd4-1, Nedd4-2, Itch and β-actin protein bands using ImageJ 1.44 p (National Institutes of Health, Bethesda, MD) (Abramoff et al., 2004). Statistical analysis All results are presented as mean + SEM. Data were analyzed using GraphPad Prism software version 5 (GraphPad Software, San Diego, CA) and p b 0.05 considered statistically significant. Where appropriate, normality of data sets was assessed prior to statistical analysis using the D'Agostino and Pearson test.

Results Ndfip1 is upregulated in response to transient focal cerebral ischemia in vivo To determine if Ndfip1 was regulated in response to ischemia, immunostaining for Ndfip1 was carried out (in concert with TUNEL to detect cell death) on coronal rat brain sections following ET-1induced MCAo of the right hemisphere (stroke, n = 14; sham, n = 6). Stroke injury following MCAo occurs within the MCAsupplied cortex and striatum ipsilateral to the occlusion and can be detected with TUNEL (Harhausen et al., 2010). Similar studies using the ET-1 model have indicated TUNEL as a marker for apoptotic cells (Al-Jamal et al., 2011), although TUNEL is known to also mark DNA fragmentation in necrotic cells (Charriaut-Marlangue and BenAri, 1995). Following ET-1 induction, Ndfip1 was consistently upregulated in cortical neurons throughout the ipsilateral cortex (+3.20 to −6.80 mm relative to Bregma) of all ischemic animals that sustained TUNEL (n = 14) (Figs. 1C and D) while sham-treated animals (n = 6) displayed only basal levels of Ndfip1 in cortical neurons as previously reported in brain trauma (Sang et al., 2006) (Figs. 1A and B). This confirms our previous observation that Ndfip1 is only expressed at low levels in neurons but not in glia (Sang et al., 2006). Basal expression of Ndfip1 under physiological (non-injury) conditions may be detected by immunocytochemistry in the contralateral hemisphere of ischemic animals, and also in both hemispheres of sham-operated animals (Figs. 1A and C). This strongly suggests that Ndfip1 upregulation on the ipsilateral cortex is an ischemia-specific response. These results were verified by Western analysis of ischemic cortices (periinfarct region), demonstrating significant Ndfip1 upregulation in ipsilateral versus contralateral lysates (Figs. 6A and B). Elevated levels of Ndfip1 were most prominently observed in layer 5 neurons of the cortex (Fig. 1C, arrows), presumably more detectable due to their larger soma sizes, although neurons in other cortical layers also upregulated Ndfip1. Higher power views revealed that upregulation of Ndfip1 in neurons is associated with intense cytosolic staining after ischemic stroke (Figs. 1I and J), compared to a diffused punctate appearance (representing basal staining) in the non-ischemic hemisphere (Figs. 1F and G). Throughout the study, Ndfip1 basal expression and upregulation (in ischemia) was only ever detected in NeuN-positive cells, confirming that Ndfip1 expression is neuronspecific (Figs. 1E–J). Upregulated Ndfip1 does not colocalize with TUNEL-positive neurons Neurons with increased Ndfip1 were consistently found in juxtaposed positions with neighboring regions rich in TUNEL-positive cells (Fig. 1D), suggesting that Ndfip1 upregulation is an injuryrelated response. Although some neurons in overlapping regions were occasionally doubled-labeled for Ndfip1/TUNEL; in the majority of instances, the two markers were mutually exclusive. This suggests that Ndfip1 upregulation is rarely associated with cell death that ensues following ischemic stroke (Fig. 1D). Higher power views confirmed this observation; while the contralateral cortex showed only basal levels of Ndfip1 and complete absence of TUNEL (Figs. 2A–C); the ipsilateral cortex exhibited numerous TUNEL-positive cells that possessed low Ndfip1 expression (Figs. 2D–F). Conversely, neurons that showed upregulated Ndfip1 invariably did not stain for TUNEL suggesting that these neurons have survived (Figs. 2D–F). To ascertain whether there is a numerical relationship between the degree of cell death and upregulation of Ndfip1, immunohistochemical quantification of Ndfip1-upregulated neurons and TUNEL-positive cells was performed on ipsilateral cortices following MCAo. Using this approach, we found a strong positive correlation between the number of TUNEL-positive cells and the number of neurons with upregulated Ndfip1 (r = 0.936, p b 0.0001, n = 20 cortices), indicating that

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Fig. 1. Ndfip1 protein is upregulated in cortical neurons following ET-1-induced MCAo. Stereo (A) and confocal (B) microscope images reveal that Ndfip1 is normally expressed at low level in the brain (A) and cortex (B) in sham-operated animals. (B) The cortex in sham animals shows only basal Ndfip1 expression but no TUNEL staining. (C) Following ET-1 induction of ischemia, Ndfip1 is upregulated in neurons of the ipsilateral cortex. In this specimen, Ndfip1 is strongly expressed in the deep cortical layers bounded by the arrows at 24 h following mild ischemic damage. (D) Higher magnification of cortical area in the vicinity of the infarct shows strong Ndfip1 upregulation in neurons lying adjacent to TUNEL-positive cells. (E–G) Confocal images of Ndfip1 and NeuN following double-immunolabelling demonstrate basal Ndfip1 expression in cortical neurons situated in the non-ischemic (contralateral) hemisphere (24 h shown). (H–J) Double immunocytochemistry for Ndfip1 and NeuN in the ipsilateral (ischemic) cortex shows strong Ndfip1 upregulation in a number of neurons (arrows) of the peri-infarct region. Scale bars: A and C: 1 mm; B and D: 50 μm; E–J: 30 μm.

increased Ndfip1 levels in neurons are coincident with increased cellular damage. These results indicate that Ndfip1 upregulation following cerebral ischemia is a survival response to ischemic conditions causing neuronal death. Immunohistochemical quantification of Ndfip1 upregulation and TUNEL was also employed to quantify the extent of the Ndfip1 response in relation to ischemia severity. This analysis confirmed that Ndfip1 was significantly upregulated in a greater number of neurons in the ipsilateral cortex of ischemic stroke animals

(n = 14) compared to sham-operated animals (n = 6) (p b 0.01, unpaired two-tailed Student's t test; Fig. 2G). Furthermore, the number of Ndfip1-overexpressing neurons per cortical hemisphere significantly increased with ischemic stroke severity (p b 0.001, one-way ANOVA followed by post test for linear trend; Fig. 2H). This trend was mirrored by an increase in the number of TUNEL-positive cells with increasing ischemia severity (p b 0.001, one-way ANOVA followed by post test for linear trend; Fig. 2H). These results confirm that Ndfip1 upregulation is modulated by the severity of the insult. A

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Fig. 2. Ndfip1 upregulation following MCAo is associated with neuron survival. (A–C) Confocal microscopy demonstrates Ndfip1 expression and no TUNEL-positive cells in the contralateral cortex following ischemia. (D–F) In the peri-infarct region of the ipsilateral cortex, Ndfip1 is upregulated in neurons (arrows) that have survived (lack of TUNEL staining) (48 h time point shown). Scale bar: A–F: 20 μm. (G) In the ischemic cortex, Ndfip1 is significantly upregulated in cortical neurons of ET-1-induced animals (n = 14) compared to sham-operated controls (n = 6). The number of TUNEL-positive cells in ischemic stroke animals (n = 14) is also significant compared to sham (n = 6). **p b 0.01 compared to sham (unpaired two-tailed Student's t test), ***p b 0.001 compared to sham (unpaired two-tailed Mann–Whitney t test). (H) The number of Ndfip1-upregulated neurons and TUNEL-positive cells is also increased with ischemic stroke severity (p b 0.01, one-way ANOVA followed by linear trend analysis). *p b 0.05 compared to sham, †p b 0.05 compared to ischemia rating 3.0 (one-way ANOVA followed by Tukey's test). n = 4 for sham, n = 2 for ischemia rating 2.0, n = 4 for ischemia rating 3.0, n = 6 for ischemia rating 4.0, and n = 2 for ischemia rating 4.5. (I) The Ndfip1 upregulation response is detected from 12 h (n = 3) onwards, with maximum expression observed between 24 h and 72 h. There was no significant difference in the number of Ndfip1-upregulated neurons between 24 h (n = 4), 48 h (n = 3), and 72 h (n = 4) following MCAo (one-way ANOVA followed by Tukey's test). *p b 0.05 compared to sham (n = 6), **p b 0.01 compared to sham.

Fig. 3. Nedd4-1 expression is not co-upregulated with Ndfip1 following MCAo. (A–C) Nedd4-1 and Ndfip1 are co-expressed at low levels in the same neurons (arrows) in the contralateral cortex. (D–F) Following MCAo at 48 h, neurons in the peri-infarct ipsilateral cortex showed upregulation of Ndfip1, but these neurons showed only low levels of Nedd4-1 (arrows). Scale bar: A–F: 20 μm.

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temporal assessment of Ndfip1 upregulation also demonstrated a positive correlation with ischemic stroke progression. Ndfip1 upregulation in neurons was clearly detectable 12 h post ischemia, but maximal upregulation was reached at 24 h and maintained at 48 h and 72 h (p b 0.01, one-way ANOVA followed by Tukey's test; Fig. 2I) even though the ET-1 model is known to cause cortical lesion expansion during this period (Weston et al., 2007). This would suggest that the upper limit to the number of Ndfip1-overexpressing neurons per cortical hemisphere was achieved at 24 h and maintained thereafter. In addition, the strong correlation between the Ndfip1 response and level of TUNEL for up to 72 h would suggest a durable neuroprotective response during the infarct maturation stage (Fig. 2I). Nedd4-2, a biochemical partner of Ndfip1, is also upregulated following ischemic stroke Ndfip1 is an adaptor for target proteins ubiquitinated by the Nedd4-family of E3 ligases, including Nedd4-1, Nedd4-2, Itch, and WWP2 (Foot et al., 2008; Harvey et al., 2002; Howitt et al., 2009; Oliver et al., 2006). Ndfip1 has also been shown to associate with Nedd4 in the brain following trauma (Sang et al., 2006). To ascertain whether or not Ndfip1 upregulation in ischemia was accompanied by

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increased expression of certain Nedd4 family proteins, Ndfip1 double-immunolabelling with Nedd4-1 and Nedd4-2 was performed on coronal sections following ET-1 MCAo. Immunohistochemical analysis of the control (contralateral cortex) showed only basal levels of Nedd4-1 and Ndfip1, often in the same neurons (Figs. 3A–C). In the ipsilateral (ischemic) cortex, Nedd4-1 remained low in all neurons examined, despite Ndfip1 upregulation in some of these neurons in response to the injury (Figs. 3D–F). These observations would suggest that Ndfip1 upregulation following ischemia is not accompanied by concomitant Nedd4-1 upregulation. This issue was further tested by biochemical analysis of both proteins by Western blot quantification of protein lysates from ipsi- and contralateral cortices (Figs. 6A and B). The results showed that Nedd4-1 levels were not altered following MCAo (Figs. 6A and B). A similar comparison with Nedd4-2 showed contrasting results (Figs. 4 and 6). While the contralateral cortex showed ubiquitous low level expression of both Ndfip1 and Nedd42 in neurons (Figs. 4A–C), the ipsilateral (ischemic) cortex showed co-extensive upregulation of both Ndfip1 and Nedd4-2 in the same neurons (Figs. 4D–F), implying parallel molecular responses of both proteins to ischemia. Because Ndfip1 and Nedd4-1 have been shown to interact (Shearwin-Whyatt et al., 2006), we hypothesized that this coordinate upregulation involved a biochemical interaction

Fig. 4. Nedd4-2 is co-upregulated and interacts with Ndfip1 in cortical neurons following MCAo. (A–C) Confocal images of the contralateral cortex show Nedd4-2 and Ndfip1 are normally expressed at low levels in all cortical neurons examined. (D–F) In the ipsilateral cortex, Nedd4-2 and Ndfip1 are co-upregulated in the same neurons (arrows) following ET-1 ischemia at 48 h. Inset shows cytoplasmic upregulation of both proteins and extensive colocalization in the same neuron. Scale bar: A–F: 30 μm. (G) Under physiological conditions, Nedd4-2 is found to bind to Ndfip1. Co-immunoprecipitation assays of uninjured mouse brain lysates demonstrate Ndfip1 and Nedd4-2 are reciprocally immunoprecipitated by Ndfip1 or Nedd4-2 antibody. (H) Following MCAo, binding of Ndfip1 with Nedd4-2 can be detected by immunoprecipitation of cortical lysates from either the ipsilateral (ischemic) or contralateral (non-ischemic) hemisphere.

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between Ndfip1 and Nedd4-2. To test this, we performed immunoprecipitation assays from brain lysates sourced from control and ischemic brain tissues. In uninjured brains, immunoprecipitation of Ndfip1 and subsequent probing for Nedd4-2 or the reverse (i.e. immunoprecipitation of Nedd4-2 and probing for Ndfip1) indicated an interaction between the two proteins (Fig. 4G). This biochemical interaction was maintained after ischemia, revealed by analysis of brain lysates from both the contralateral and ipsilateral cortices of ET-1-induced MCAo rats (Fig. 4H). Furthermore, Western analysis of these proteins revealed increased Ndfip1 and Nedd4-2 (but not Nedd4-1) levels in ipsilateral (ischemic) cortices compared to contralateral controls (Figs. 6A and B). Together, these multiple strands of evidence point to Ndfip1 and Nedd4-2 upregulation and biochemical interaction as key molecular responses to promote neuron survival during ischemia.

Itch is upregulated and localized following ischemic stroke but not co-upregulated together with Ndfip1 While the above results demonstrate coordinate interaction and upregulation of Ndfip1 with Nedd4-2 in ischemic stroke, Ndfip1 is also known to interact with other Nedd4 family members including Itch (Harvey et al., 2002; Oliver et al., 2006). Therefore, it would be of interest to determine whether or not upregulation of Ndfip1 in neurons was also associated with Itch upregulation following ischemia. Immunocytochemical analysis revealed Itch expression in the non-ischemic (contralateral) cortex was low and dispersed throughout the cytosol (Figs. 5A and G; inset in Fig. 5A); however, in the ischemic (peri-infarct) cortex, the Itch staining was intense and localized to a specific intracellular compartment within the soma of cortical cells (Figs. 5D and J; inset in Fig. 5D). This would suggest

Fig. 5. Itch protein upregulation in surviving cells during ischemia is independent of Ndfip1 upregulation. (A and G) In non-ischemic contralateral cortex, Itch expression is low and barely detectable. Inset (A) shows high-powered confocal view of low, but diffuse, cytoplasmic staining for Itch protein. (D and J) Following ischemia, Itch protein staining in the peri-infarct cortex becomes intense and is localized to a discrete region of cytoplasm (48 h time point shown). Inset (D) shows an ischemic cell with localized and upregulated Itch protein. (D–F) Itch upregulation is not coincident with other cells that upregulate Ndfip1 (arrows) following ischemia. (G–I) In non-ischemic cortex, Itch levels are barely detectable and no TUNEL cells are present. (J–L) Following ischemia, Itch protein is upregulated and localized to a condensed cytoplasmic spot. Cells that upregulate Itch have not undergone cell death as indicated by the absence of TUNEL (arrows) (L). (48 h time point shown). Scale bars: A–L: 20 μm; A and D insets: 5 μm.

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Fig. 6. Western analysis of Ndfip1, Nedd4-1, Nedd4-2, and Itch protein levels following MCAo. (A) Western blotting of ipsilateral (peri-infarct) and corresponding contralateral cortical lysates demonstrates increased expression of Ndfip1, Nedd4-2, and Itch, but not Nedd4-1, following cerebral ischemia (24 h). Representative blot from three independent animals shown. (B) Quantification of contralateral versus ipsilateral protein expression from Western blots represented in (A). All data were analyzed using the paired two-tailed Student's t test (n = 3). *p b 0.05 compared to contralateral. β-actin was used as a standard for normalization.

that Itch is upregulated in a localized cytosolic region following ischemic damage. Biochemical comparison of ipsilateral and contralateral cortices from ET-1 MCAo rats confirmed there was a significant increase in Itch expression following ischemia (Figs. 6A and B). Together, these data demonstrate that induction of Itch in ischemic stroke is characterized by both upregulation and subcellular localization. Surprisingly, while induction of Itch and upregulation of Ndfip1 was evident within the same peri-infarct area of the ipsilateral cortex, there was no overlap of both proteins within the same cells (Figs. 5D–F). This would suggest that ischemia is able to elicit different molecular responses in different cell types. We also observed that Itch upregulation/localization rarely colocalized with TUNEL-positive cells after ischemia (Figs. 5J–L, arrows), suggesting that Itch, like Ndfip1, is upregulated in surviving, not dying (TUNEL-positive), cells. Together, these results would suggest that Itch upregulation is closely associated with cell survival following ischemia. However, it would appear that Itch upregulation as a survival response is controlled by mechanisms that are separate to those that upregulate Ndfip1. Discussion In brain ischemia, ubiquitin-modification of proteins has been demonstrated to be an important post-translational mechanism that

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is correlated with neuron survival or death (Hu et al., 2001; Ide et al., 1999; Liu et al., 2004, 2006; Vannucci et al., 1998). However, of the five hundred or so E3 ubiquitin ligases present in the mammalian cell, very few specific E3 enzymes have been identified and associated with cerebral ischemia. In the present study, we demonstrate for the first time that members of the Nedd4 family i.e. Nedd4-2, Itch, and their adaptor protein, Ndfip1, are strongly upregulated in the cortex following transient MCAo. In surviving cortical neurons, only Nedd4-2, but not Nedd4-1 or Itch, was co-upregulated with Ndfip1 suggesting specific and differential regulation of Nedd4family proteins under ischemic conditions. Further confirmation was obtained biochemically using co-immunoprecipitation assays, demonstrating that Nedd4-2 and Ndfip1 can interact in these tissues. Interestingly, Itch by itself was also strongly upregulated in surviving neurons (devoid of TUNEL) in peri-infarct areas. The observation that these neurons did not co-upregulate Ndfip1 suggests a parallel neuron survival mechanism that is independent of Ndfip1. Thus, our results identify two distinct endogenous survival pathways that utilize Nedd4 family-mediated ubiquitination following cerebral ischemia. The number of surviving neurons that upregulate Ndfip1 is correlated with the severity of disease, but only up to a point in animals with increasing ischemia severity. Thus, the proportion of Ndfip1upregulated neurons appeared to plateau out 24 h after ischemic stroke, suggesting that after this period, there is equilibrium; with the addition of new neurons that upregulate Ndfip1 being canceled out by other neurons that succumbed to death. Nonetheless, once the number of Ndfip1-upregulated cells was maximally attained, there was no decline in the number of upregulated neurons for a further 48 h despite infarct expansion that is known to take place during this period (Weston et al., 2007). So while Ndfip1 elevation in neurons is strongly correlated with neuroprotection, this effect appears to be restricted to a subpopulation of neurons of uncertain identity and properties. Future exploration of these properties will be beneficial for expanding the proportion of neurons capable of upregulating Ndfip1 for survival. How may Ndfip1 upregulation be neuroprotective? The colocalization and interaction of Ndfip1 and Nedd4-2 in these surviving neurons suggest that Ndfip1 may improve survival of cortical neurons by promoting Nedd4-2-mediated ubiquitination, followed by degradation, of harmful proteins. This notion is drawn from the observation that in yeast, the homologues of Ndfip1 and Nedd4 (known as Bsd2 and Rsp5, respectively) are key players for removing damaged and misfolded proteins during cellular stress resulting in cell survival (Hettema et al., 2004). This is consistent with our previous study demonstrating that Ndfip1 promotes survival of human neurons, in vitro, against metal toxicity by binding Nedd4-2 and increasing protein ubiquitination of the harmful divalent metal transporter 1 (DMT1) (Howitt et al., 2009). In vivo, the current study confirmed that Nedd4-2, but not Nedd4-1, was upregulated and colocalized with Ndfip1 following cerebral ischemia, suggesting that only Nedd4-2 is physiologically relevant for Ndfip1-mediated neuroprotection in ischemia. The current study does not go far enough to identify the downstream targets of Ndfip1/Nedd4-2 for neuroprotection, but they are likely to be substrates lacking the classical PPxY motifs necessary for binding to WW domains of Nedd4-family E3 ligases. It has been shown that both Ndfip1 and Ndfip2 function as adaptors for recruiting targets normally incapable of binding to WW domains of Nedd4 proteins such as WWP2 (Foot et al., 2008). Despite the fact that Ndfip1 can bind to Itch in tissue outside of the nervous system (Harvey et al., 2002; Oliver et al., 2006), our study showed that induction of Itch and Ndfip1 following ischemia were mutually exclusive in peri-infarct brain areas. We previously observed a similar phenomenon in the regulation of the divalent metal transporter DMT1 in human cortical neurons, whereby Ndfip1 recruits Nedd4-2, but not Itch, for DMT1 ubiquitination (Howitt et al., 2009). Nonetheless, induction of Itch following MCAo in the present

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study was correlated with cell survival. Although the neuroprotective mechanism is not clear, other published work shows that Itch is a key regulatory protein in the immune response, known to ubiquitinate and downregulate transcription factors JunB (Fang et al., 2002; Oliver et al., 2006) and c-Jun (Fang and Kerppola, 2004; Fang et al., 2002) in a c-Jun N-terminal kinase (JNK)-dependent manner (Fang et al., 2002; Gao et al., 2004). In experimental ischemia, JunB and c-Jun are upregulated immediate early genes (An et al., 1993; Gass et al., 1992; Kinouchi et al., 1994a, 1994b) and c-Jun upregulation and phosphorylation by JNK has been correlated with cell death in MCAo and other ischemia models (Borsello et al., 2003; Herdegen et al., 1998; Kuan et al., 2003). Thus, it will be of interest to determine whether Itch promotes cellular survival by downregulating c-Jun following cerebral ischemia. In conclusion, our findings provide specific identification of proteins that are upregulated in surviving neurons, and known to promote ubiquitination of downstream targets involving HECT-type ubiquitin ligases. These finding have important implications for understanding how protein ubiquitination pathways may be manipulated as a therapeutic strategy in ischemic stroke. Several studies demonstrate that global proteasome inhibition within the first few hours of experimental ischemia is neuroprotective (Buchan et al., 2000; Henninger et al., 2006; Phillips et al., 2000; Williams et al., 2003; Zhang et al., 2001). However, the precise mechanisms for this short-term effect remain obscure as other studies also report that the ubiquitin-proteasome system can mediate endogenous mechanisms that protect against cerebral ischemia (Meller et al., 2006, 2008; Pradillo et al., 2005). In the current study, we demonstrate that in the intermediate term (beyond 24 h), the Nedd4-family of E3 ligases has the opposite effect in promoting survival. This would suggest that in the post-ischemic environment, different cellular processes are activated to defend neurons against death. By specifically identifying the enzymes involved, we have opened up the way for future studies aimed at targeting the specific pathways to promote neuron survival.

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Glossary DMT1: divalent metal transporter 1 ET-1: endothelin-1 HECT: homology to the E6-associated protein carboxyl terminus domain JNK: c-Jun N-terminal kinase MCAo: middle cerebral artery occlusion Ndfip1: Nedd4-family interacting protein 1