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Oxidative Stress, Nitric Oxide, and the Mechanisms of Cell Death in Lurcher Purkinje Cells Rebecca McFarland,1,2 Andrei Blokhin,2 James Sydnor,2 Jean Mariani,3,4 Michael W. Vogel2 1

Department of Biology, University of Maryland Baltimore County, Baltimore, Maryland 21250

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Department of Psychiatry, Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore, Maryland 21228

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Equipe De´veloppement et Vieillissement du Syste`me Nerveux, UMR NPA 7102, CNRS et Universite Pierre and Marie Curie, 9, Quai St. Bernard, 75005 Paris, France

4

AP-HP, Hopital Charles Foix, UEF, Ivry sur Seine, 94200 France

Received 6 October 2006; revised 22 December 2006; accepted 25 December 2006

ABSTRACT: Oxidative stress is postulated to play a role in cell death in many neurodegenerative diseases. As a model of neonatal neuronal cell death, we have examined the role of oxidative stress in Purkinje cell death in the heterozygous Lurcher mutant (þ/Lc). Lurcher is a gain of function mutation in the d2 glutamate receptor (GluRd2) that turns the receptor into a leaky membrane channel, resulting in chronic depolarization of þ/Lc Purkinje cells starting around the first week of postnatal development. Virtually, all þ/Lc Purkinje cells die by the end of the first postnatal month. To investigate the role of oxidative stress in þ/Lc Purkinje cell death, we have examined nitric oxide synthase (NOS) activity and the expression of two markers for oxidative stress, nitrotyrosine and manganese super oxide dismutase (MnSOD), in wild type and þ/Lc Purkinje cells at P10, P15, and P25.

This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/1932-8451/ suppmat. Correspondence to: M.W. Vogel ([email protected]. edu). Contract grant sponsor: NINDS; contract grant number: NS34309. ' 2007 Wiley Periodicals, Inc. Published online 6 March 2007 in Wiley InterScience (www. interscience.wiley.com). DOI 10.1002/dneu.20391

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The results show that NOS activity and immunolabeling for nitrotyrosine and MnSOD are increased in þ/Lc Purkinje cells. To determine whether peroxynitrite formation is a prerequisite for þ/Lc Purkinje cell death, þ/Lc mutants were crossed with an a-nNOS knockout mutant (nNOSa/) to reduce the production of NO. Analysis of the double mutants showed that blocking a-nNOS expression does not rescue þ/Lc Purkinje cells. However, we present evidence for sustained NOS activity and nitrotyrosine formation in the GluRd2þ/Lc:nNOS/ double mutant Purkinje cells, which suggests that the failure to rescue GluRd2þ/Lc: nNOS/ Purkinje cells may be explained by the induction of alternative nNOS isoforms. ' 2007 Wiley Periodicals, Inc. Develop Neurobiol 67: 1032–1046, 2007

Keywords: excitotoxicity; nitric oxide; cell death; caspase-3; d2 glutamate receptors; nitrotyrosine

INTRODUCTION The Lurcher mouse mutant has an autosomal dominant mutation in the d2 glutamate receptor (GluRd2) that causes the cell autonomous death of virtually all cerebellar Purkinje cells (Phillips, 1960; Caddy and Biscoe, 1979; Yue et al., 2002). GluRd2 is preferentially expressed in high levels at parallel fiber synap-

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ses in cerebellar Purkinje cells and at lower levels in hindbrain neurons (Araki et al., 1993; Lomeli et al., 1993; Takayama et al., 1996; Landsend et al., 1997). The Lurcher mutation in GluRd2 is a base-pair substitution that changes an alanine to threonine in the highly conserved third hydrophobic segment of GluRd2 (Zuo et al., 1997). The mutation changes the GluRd2 receptor into a leaky membrane channel that carries a constitutively-active inward cation current into the cells that express the subunit, causing a chronic depolarization. Homozygous Lc/Lc pups die around birth (Cheng and Heintz, 1997), while in the þ/Lc mutant the chronic depolarization of Purkinje cells starts by at least P5-6 (Selimi et al., 2003). þ/Lc Purkinje cell death begins around P7 to P10 and nearly all have died by the end of the first postnatal month (Caddy and Biscoe, 1979). þ/Lc Purkinje cell death has been alternately described as necrotic, apoptotic, and autophagic based on a variety of criteria. Ultrastructural descriptions have suggested necrotic cell death (DumesnilBousez and Sotelo, 1992), but GluRd2Lc receptors may also trigger an autophagic cell death pathway through their failure to sequester Beclin (Yue et al., 2002). There is also evidence for apoptotic pathways: TUNEL labeled þ/Lc Purkinje cells have been detected in three studies (Norman et al., 1995; Wullner et al., 1995; Selimi et al., 2000) and BAX and Bcl-x expression are increased in dying þ/Lc Purkinje cells (Wullner et al., 1995). Pro-caspase 3 levels are increased in *25% of þ/Lc Purkinje cells at P12 to P20 and activated caspase-3 is expressed in a much lower percentage of þ/Lc Purkinje cells at P12 to P25 (Selimi et al., 2000). Presumably, the percentage is low because the þ/Lc Purkinje cells die soon after pro-caspase-3 is activated. In addition, there is increased c-Jun phosphorylation in þ/Lc Purkinje cells along with increases in caspase-8 and -9 expression (Lu and Tsirka, 2002). The goal of this study is to test the hypothesis that oxidative stress and nitric oxide (NO) production induce þ/Lc Purkinje cell death. The chronic cation leak mediated by the GluRd2Lc channel suggests that excitotoxicity is a likely mechanism for the induction of þ/Lc Purkinje cell pathology (Zuo et al., 1997). As a test of this hypothesis, we have examined nitric oxide synthase (NOS) activity and the expression of two markers for oxidative stress, nitrotyrosine and manganese super oxide dismutase (MnSOD), in wild type and þ/Lc Purkinje cells. Nitrotyrosine in the product of peroxynitrite nitration of tyrosine residues on target proteins. MnSOD is the principal scavenger of superoxide in mitochondria and is induced by a variety of cytotoxic and proapoptotic agents (Fridovich,

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1975). To determine whether peroxynitrite formation is a prerequisite for þ/Lc Purkinje cell death, þ/Lc mutants were crossed with a-nNOS knockout mutants to reduce the production of NO. Analysis of the double mutants showed that blocking a-nNOS expression might not rescue þ/Lc Purkinje cells from death because there is an induction of alternate NOS activities.

METHODS Animals GluRd2þ/Lc mutant and wild type pups were generated by mating B6CBACa Aw-J/A-Grid2Lc/J males with wild type females (C57BL/6J), both from Jackson Laboratories (GRID2 is an alternative abbreviation for GluRd2). GluRd2þ/Lc; NOS/ double mutants and heterozygous controls were generated by mating male GluRd2þ/Lc mutants with female B6;129S4-Nos1tm1Plh/J NOS homozygotes (from Jackson Laboratories; nNOS/). The F1 generation of mice were genotyped and males and females identified as double heterozygotes (GluRd2þ/Lc:NOSþ/) were harem mated with GluRd2þ/þ:NOSþ/ heterozygotes to generate F2 generations that contained GluRd2þ/Lc: NOS/ double mutants as well as heterozygous and homozygous controls (GluRd2þ/Lc:nNOSþ/ or GluRd2þ/Lc: nNOSþ/þ and GluRd2þ/þ:nNOSþ/ or GluRd2þ/þ:nNOSþ/þ). All animals were housed in standard conditions (14 h light, 10 h dark) in the animal facilities at the Maryland Psychiatric Research Center and provided with food and water ad libitum. Males were harem mated with 1 male to 2 or 3 females. The day of birth was counted as postnatal day 0 (P0). The animal facilities are fully accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC) and the studies were conducted in accordance with the Guide for Care and Use of Laboratory Animals provided by the NIH. Depending on the requirements of the experiment, mice were either euthanized by cardiac perfusion with 0.9% saline followed by 4% paraformaldehyde (while deeply anesthetized with Avertin) or by decapitation when fresh brains were required for measuring protein levels in Western blots. Following the perfusions with 4% paraformaldehyde, the brains were removed from the skull and postfixed for 2– 24 h. For freshly dissected brains, the cerebellum and frontal cortex was removed and quickly frozen in microcentrifuge tubes on crushed dry ice.

Immunocytochemistry Fixed brains were either cut at 30 lm and collected as floating sections in 10 mM PBS or were cut at 12 lm on a Leica cryostat and collected directly on slides. The slide-mounted sections were stored at 708C until stained. For immunolabeling experiments, the floating sections or slides were Developmental Neurobiology. DOI 10.1002/dneu

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rinsed in 10 mM PBS, followed by incubation in two changes of 0.1 M glycine for 5 min each. Endogenous fluorescence was reduced by incubating the sections in 50 mM ammonium chloride for 1 h. The sections were then rinsed three times in 10 mM PBS and then incubated for an hour in blocking solution containing 3% normal goat serum and 0.3% Triton X-100. Sections were then incubated in the primary antibodies overnight at 48C. All sections were double labeled with mouse anticalbindin (Sigma: 1/10,000) or rabbit anticalbindin (Calbiochem: 1/1000) and either mouse antinitrotyrosine (Upstate: 1/1000), rabbit anti-N-terminal nNOS (Zymed: 1/250), rabbit anti-C-terminal nNOS (Upstate: 1/100), or rabbit anti-MnSOD (Stressgen: 1/ 1000). The sections were rinsed three times in PBS and then incubated for 2 h with fluorescent labeled secondary antibodies (anti-mouse or anti-rabbit Alexa 594 and Alexa 488: Molecular Probes: 1/200). After incubation, they were rinsed three times in 10 mM PBS, once in distilled water, and then coverslipped with gelmount. The finished slides were then photographed using either a Leica TCS scanning confocal microscope or Olympus BH-2 or Zeiss Axioplan fluorescence microscopes. All immunolabeling experiments included slides (wild type and þ/Lc) with no 18 antibody incubation as a control for nonspecific immunolabeling. Digital images of immunolabeling for each antigen in wild type and þ/Lc cerebella were taken with the same exposure times, adjusted for hue, brightness and contrast the same amount and cropped in Photoshop and then assembled in photo plates with Adobe Illustrator. For the semi-quantitative comparison of MnSOD immunolabeling in wild type and þ/Lc cerebella digital images of MnSOD and calbindin immunolabeling were taken at 403 on a Zeiss Axioplan with an Olympus DP70 CCD camera within the first 2 days after staining all sections (to avoid uneven fading artifacts). Images were taken from more than five sections per cerebellum systematically randomly selected from throughout the medial to lateral extent of the vermis in randomly selected lobules that still contained Purkinje cells. The raw images were then analyzed using Metamorph Version 7.0r1. Threshold and colocalization functions were used to selectively measure the intensity of MnSOD immunolabeling in areas that colocalized with calbindin-stained Purkinje cells in the same section. In the deep cerebellar nuclei (DCN), the threshold and colocalization functions were used to measure the intensity of MnSOD immunolabeling in the DCN excluding calbindinpositive axons. The intensity of MnSOD labeling at P15 and P25 is expressed as a percent change from the baseline labeling in control regions at P15.

NADPH Histochemistry Fixed brains (2 h postfixation) were cut at 12 lm on a cryostat and collected on slides. The sections were rinsed in 10 mM PBS and then incubated with 0.5 mg/mL nitroblue tetrazolium, 1 mg/mL NADPH, 0.3% Triton X-100 in phosphate buffer for 3 h at 378C. The sections were rinsed again with PBS and coverslipped with gelmount. Developmental Neurobiology. DOI 10.1002/dneu

Purkinje Cell Counts The number of Purkinje cells per cerebellum were estimated in þ/Lc:nNOS/ double mutants and controls using the optical fractionator technique (West, 1999; Fan et al., 2001). Fixed cerebella, embedded in paraffin, were sectioned sagittally at 25 lm on a Leitz microtome. Every 20th to 25th section (every 4th or 5th slide) from a random start was selected for counts and stained for cresyl violet. Purkinje cells were counted on a BH-2 Olympus microscope using Nomarski optics and an oil immersion 1003 objective and an oil immersion condenser. The cerebellar cortex was used as reference volume for counting Purkinje cells. The optical dissector used to count Purkinje cells was 15 lm deep and 500 lm2, spaced 200 lm apart. These parameters were chosen to obtain a coefficient of error for the cell counts of less than 0.1. The nuclei of Purkinje cells at their maximum diameter within the counting box or touching the top and/or right sides were included in the counts.

Western Blot Analysis Wild type and þ/Lc cerebella were collected from freshly dissected brains, rapidly frozen in dry ice, and stored at 708C until processed. Each cerebellum was homogenized in a buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% SDS, 10 lL/mL Protease inhibitor cocktail (Sigma), 1 mM PMSF, and 1 mM NaVO4. The homogenate was centrifuged at 15,000 rpm for 15 min. Protein concentration in the supernatant was measured using a BioRad protein assay kit. Protein extracts were diluted in Laemmli sample buffer with b-mercaptoethanol and 20 lg of protein per well was resolved on a Tris-glycine gel. Protein was transferred overnight at 48C onto a PVDF membrane. The membrane was rinsed with 5% nonfat dry milk dissolved in 13 TBS. It was then incubated in the primary antibody, diluted in TBS/0.1% Tween (TBS-T) with 1% milk overnight at 48C and then rinsed 3 3 10 min each with PBS-T. The sample membrane was incubated in alkaline phosphatase-conjugated secondary antibody, diluted, and the protein detected using Bio-Rad immunStar chemiluminescence kit. Film exposed to the chemiluminescent signal was digitized using a light box and Pixera digital camera connected to a PowerMacintosh. The optical density of the images was calibrated using a photographic calibration step tablet (Kodak) so that data is collected in the linear range of the O.D. The relative density of the labeled protein bands was determined using densitometric measurements with the AIS image analysis system. In all studies, once data from the antigen of interest had been collected, the membrane was stripped and labeled for total protein with India ink as a loading control. The density of protein bands was expressed as a percent of the density of protein bands from P10 controls and corrected for total amounts of protein.

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Statistical Analyses Statistical comparisons between experimental and control groups were made using two-way or one-way analysis of variance (ANOVA) and post hoc comparisons were made using Fisher’s PLSD (Statview 5.01).

RESULTS nNOS Histochemistry in þ/Lc Purkinje Cells As an assay for NO production in the þ/Lc cerebellum, the histochemical activity of formaldehyde-stable NADPH diaphorase (NADPH-d) was examined in fixed, frozen sections of wild type and þ/Lc cerebella from P5 through P25. Formaldehyde-stable NADPH-d histochemistry is considered a marker for the presence of NOS (Matsumoto et al., 1993). No differences were observed between wild type and þ/Lc cerebella at P5 (data not shown), so we have concentrated on analyzing older age groups. Images of NADPH-d labeling at P10, P15, and P25 are shown in Figure 1. Multiple sections from at least three separate wild type and þ/Lc cerebella were examined at each age. As has been reported previously (Bruning, 1993), NADPH-d staining is high throughout the wild type cerebellar cortex at P10. Both wild type and þ/Lc Purkinje cells at P10 show high levels of staining throughout their cell bodies and dendritic trees [Fig. 1(A,B)]. By P15, however, NADPH-d staining has disappeared in wild type Purkinje cells although labeling remains high in the granule cell and molecular layers [Fig. 1(C,E)]. White arrows indicate NADPH-d labeled interneurons (basket or stellate neurons) in the molecular layer of wild type cerebella. In contrast, virtually all þ/Lc Purkinje cells still stain for NADPH-d histochemical activity at P15 [Fig. 1(D)] and many surviving Purkinje cells are still labeled at P25 [Fig. 1(F)]. A single labeled þ/Lc Purkinje cell is shown in Figure 1(D,F) for P15 and P25, respectively. NADPH-d staining remains especially high in the þ/Lc Purkinje cells dendrites through P25. The difference in the pattern of NADPH-d between wild type and þ/Lc Purkinje cells is apparent in comparing Figure 1(C,E) with (D,F) wherein the cell bodies and primary dendrites of wild type Purkinje cells are clear with their outlines defined by NADPH-d staining in the surrounding molecular layer. In contrast, the cell bodies and dendrites of þ/Lc Purkinje cells stand out against the background because the NADPH-d staining is within the cell.

Figure 1 Developmental progression of NADPH-d histochemistry activity in the cerebellar cortex of a wild type mouse (A, C, E) and þ/Lc mutant (B, D, F) at P10 (A, B), P15 (C, D), and P25 (E, F). NADPH-d activity disappears in wild type Purkinje cells before P15, but þ/Lc Purkinje cell bodies and dendrites remain heavily stained through P25. Purkinje cell bodies are indicated by black arrowheads and dendrites by black arrows. The white arrows indicate molecular layer interneurons heavily stained for NADPH-d activity. Scale bar is 20 lm.

Oxidative Stress in þ/Lc Purkinje Cells To assay for evidence of oxidative stress in þ/Lc Purkinje cells, we have examined the distribution of nitrotyrosine immunolabeling in wild type and þ/Lc cerebella at P10, P15, and P25. (Fig. 2; n > 3 for each genotype at each age). The red immunolabeling in Figure 2 shows the expression of Purkinje cell calbindin, while the green staining represents immunolabeling for nitrotyrosine. In control experiments to confirm the specificity of the antibody, the mouse monoclonal antibody to nitrotyrosine was preabsorbed with either free nitrotyrosine or phosphotyrosine. No immunolabeling was observed in control þ/ Developmental Neurobiology. DOI 10.1002/dneu

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Figure 2 Developmental progression of immunolabeling for nitrotyrosine in the cerebellar cortex of a wild type mouse and þ/Lc mutant at P10, P15, and P25. The microphotographs on the far left and right (A, D, E, H, I, L) show calbindin (red) labeling of Purkinje cells, while the central photographs show nitrotyrosine immunolabeling (green) in the same microscope field. The cell bodies of Purkinje cells are indicated by white arrowheads. Scale bar is 20 lm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

þ or þ/Lc sections with the antibody preabsorbed with nitrotyrosine (data not shown), indicating that the antibody specifically recognizes nitrotyrosine antigens. In contrast, no change was seen in the pattern of immunolabeling in þ/Lc cerebellar sections with nitrotyrosine antibodies preabsorbed with phosphotyrosine, indicating that the nitrotyrosine antibody does not cross react with phosphotyrosine residues. At P10, nitrotyrosine is primarily localized in Purkinje cells in both wild type and þ/Lc mutant mice (Fig. 2). However, the amount of nitrotyrosine labeling in þ/þ Purkinje cells appears to decline with age [Fig. 2(B,F,J)], especially in the Purkinje cell dendrites. The þ/þ Purkinje cell bodies (white arrowheads) are still lightly labeled for nitrotyrosine at Developmental Neurobiology. DOI 10.1002/dneu

P15, but by P25, only a low level of residual labeling is present in the þ/þ Purkinje cell somas. In contrast, virtually all þ/Lc Purkinje cells are labeled with antinitrotyrosine antibodies at P15 with intense labeling in the dendrites and somas [Fig. 2(G)]. By P25, significant numbers of þ/Lc Purkinje cells have degenerated and most of the surviving Purkinje cells remain labeled for nitrotyrosine [Fig. 2(K)]. The dendrites of þ/Lc Purkinje cells that survive to P25 assume a characteristic short, stubby appearance [Fig. 2(K,L)]. The distal dendrites often appear swollen and the immunolabeling for nitrotyrosine is especially intense in the distal dendrites. There was no obvious, consistent regional heterogeneity in the distribution of nitrotyrosine-labeled þ/Lc Purkinje cells at P25 in sagittal sec-

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Figure 3 Confocal images of overlays of calbindin (red) and nitrotyrosine (green) immunolabeling of wild type (A) and þ/Lc Purkinje cells (B) at P15. The arrowheads indicate double labeled (yellow) þ/Lc Purkinje cell bodies while the arrow indicates a Purkinje cell axonal swelling that is colabeled for nitrotyrosine.

tions. However, the medial to lateral distribution of immunolabeled þ/Lc Purkinje cells was not examined in coronal sections so parasagittal patterns of þ/ Lc Purkinje cell degeneration may have been missed.

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A second hallmark of the þ/Lc cerebellum is the appearance of swellings on Purkinje cell axons in the granule cell layer, white matter, and in the DCN. A few of these axonal swellings are apparent by P7 to P10 (see Dumesnil-Bousez and Sotelo, 1992; Wang et al., 2006) and they are present in large numbers by 15. By 25, most of the swellings have disappeared, presumably as many of the cell bodies and axons of þ/Lc Purkinje cells have degenerated. Overlays of confocal images of calbindin (red) and nitrotyrosine (green) labeling in the Purkinje cell layer from a P15 wild type (A) and þ/Lc cerebella (B) are shown in Figure 3. This figure illustrates that there is virtually no nitrotyrosine immunolabeling in the dendritic trees of wild type Purkinje cells, although there is a low level of labeling in the cell bodies (white arrowheads). In contrast, the yellow dendritic and cell body labeling (white arrowheads) in þ/Lc Purkinje cells shows that there is spatial overlap between calbindin in Purkinje cells and proteins that have been nitrated by peroxynitrite. Many of the calbindin-positive

Figure 4 Immunolabeling for calbindin and MnSOD in the cerebellar cortex of wild type controls and þ/Lc mutants at P10, P15, and P25. The panels on the far left and right are merged images of calbindin (red) and MnSOD (green) immunolabeling, while the central panes show the same field with only MnSOD immunolabeling. The white arrowheads indicate Purkinje cell bodies. The white arrows in D and L indicated calbindin positive axonal swellings. ML, molecular layer; GCL, granule cell layer. Scale bar 20 lm. Developmental Neurobiology. DOI 10.1002/dneu

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swellings on þ/Lc Purkinje cell axons are also labeled for nitrotyrosine [white arrow in Fig. 3(B)], indicating that there is a source of reactive oxygen species (ROS) and NO in the þ/Lc Purkinje cell axonal swellings. As an independent measure of oxidative stress in þ/Lc Purkinje cells, we have examined the cellular distribution and expression levels of MnSOD (Figs. 4–6). MnSOD is the principal scavenger of superoxide in mitochondria (Fridovich, 1975) and is induced by a variety of cytotoxic and proapoptotic agents. The expression pattern of MnSOD is dramatically altered in þ/Lc cerebella compared with the wild type as shown by immunolabeling for MnSOD in Figure 4. At P10, MnSOD immunolabeling in the cerebellum is primarily found in Purkinje cells (Fig. 4) and DCN neurons (data not shown). MnSOD immunolabeling is distributed throughout the Purkinje cell body (arrowheads) and dendrites in both wild type and þ/Lc Purkinje cells [Fig. 4(A–D)]. The arrow in Figure 4(D) indicates one of the calbindin positive axonal swellings that start to appear in the þ/Lc cerebellum by P10. By P15, punctate immunolabeling for MnSOD is most apparent in the cell bodies of wild type Purkinje cells while the labeling in the molecular layer has become more diffuse so it is no longer possible to resolve individual Purkinje cell dendrites based on MnSOD immunolabeling [Fig. 4(E,F)]. In contrast, P15 þ/Lc Purkinje cells retain the intense immunolabeling for MnSOD throughout their cell bodies and dendrites as shown by double labeling for calbindin (red) and MnSOD [green; Fig. 4(G,H)]. There are many calbindin positive axonal swellings in the white matter and DCN and some are also double labeled for MnSOD, indicating that the swellings contain mitochondria (data not shown). By P25, MnSOD immunolabeling has become even more diffuse in the molecular layer of wild type cerebella, although punctate labeling is still present in wild type Purkinje cell bodies. Relatively few þ/Lc Purkinje cells survive to P25 and there is a great deal of variability in the appearance of their dendritic trees [Fig. 4(K,L)]. However, the dendrites and cell bodies of many of the degenerating þ/Lc Purkinje cells are still delineated by intense MnSOD immunolabeling. There is relatively little labeling in the þ/Lc granule cell layer through P25 compared to wild type cerebella. Immunolabeling for MnSOD is present in the cell bodies of all DCN neurons through P25 in both wild type and þ/Lc cerebella (Fig. 5). The intensity of MnSOD immunolabeling within the DCN cell bodies appears higher at P15 when compared with P25, especially in the þ/Lc mutant cerebella. Developmental Neurobiology. DOI 10.1002/dneu

Semi-quantitative comparisons of the intensity of MnSOD immunolabeling between þ/þ and þ/Lc Purkinje cells at P15 and P25 throughout lobules in the cerebellar vermis showed that there is a significant increase in the overall intensity of MnSOD labeling in the þ/Lc Purkinje cells [Fig. 6(A)]. Calbindin labeling was used to identify þ/þ and þ/Lc Purkinje cells in cerebellar sections double labeled for calbindin and MnSOD immunohistochemistry and the colocalize function in Metamorph was used to measure the intensity of MnSOD immunolabeling in calbindin labeled Purkinje cells. A two-way analysis of variance showed that there was a significant genotype effect (F1,13 ¼ 17.4, p < 0.002), but no significant effect of age or age by genotype interaction (p > 0.5). If the results are split by age, a one-way analysis of variance showed that MnSOD immunolabeling in þ/ Lc Purkinje cells is significantly different from wild type at both P15 and P25 (P15; F1,7 ¼ 16.3, p < 0.005; P25; F1,6 ¼ 6.7, p < 0.05). Although the mean intensities of MnSOD immunolabeling are similar in þ/Lc Purkinje cells at P15 and P25, the variance is considerably higher in the P25 Purkinje cells. There are considerably fewer þ/Lc Purkinje cells by P25 and some of these did not show any MnSOD immunolabeling. A similar analysis of MnSOD immunolabeling was performed among the DCN neurons, except that MnSOD immunolabeling in calbindin labeled axons

Figure 5 Immunolabeling for MnSOD in the DCN of wild type controls (A, C) and þ/Lc mutants (B, D) at P15 (A, B) and P25 (C, D). The white arrowheads indicate the cell bodies of DCN neurons. Scale bar 20 lm. A color photomontage of double labeling for MnSOD and calbindin in the DCN is shown in a supplementary figure.

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Figure 6 Semi-quantitative analysis of MnSOD immunohistochemistry and protein expression in wild type controls and þ/Lc mutants. (A, B) Graphs showing the percent change in the intensity of MnSOD immunolabeling in þ/Lc Purkinje cells (A) and DCN neurons (B) when compared with wild type levels. *P15; F1,7 ¼ 16.3, p < 0.005; #P25; F1,6 ¼ 6.7, p < 0.05; **P15; F1,7 ¼ 12.4, p < 0.02 C) Western blots for MnSOD from wild type and þ/Lc mutants at P10, P15, and P25. A single band at 24 kD was obtained as shown in the top panel in (C). The relative density of the bands was compared to wild type values at P10 within each Western blot and the results are plotted in the lower panel, after correcting for total protein levels (stained with India ink) in the transfer blot.

in the DCN were excluded from the measurements [Fig. 6(B)]. A two-way analysis of variance showed that there were significant effects for genotype (F1,9 ¼ 13.8, p < 0.005), age (F1,9 ¼ 13.2, p < 0.01), and age 3 genotype interactions (F1,9 ¼ 6.1, p < 0.05). The intensity of MnSOD immunolabeling in the

DCN of þ/Lc mutants was significantly increased when compared with controls at P15 (one-way ANOVA, F1,7 ¼ 12.4, p < 0.02), but by P25 MnSOD immunolabeling in the þ/Lc DCN had decreased to control levels (one-way ANOVA, F1,4 ¼ 4.3, p > 0.1). Developmental Neurobiology. DOI 10.1002/dneu

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Figure 7 Estimates of Purkinje cell number per hemicerebellum in GluRd2þ/þ:nNOSþ/þ,þ/ (WT:WT), GluRd2þ/þ: nNOS/ (WT:NOS/), and GluRd2þ/Lc:nNOSþ/þ, þ/ controls (þ/Lc:NOS-WT) and GluRd2þ/Lc:nNOS/ double mutants (þ/Lc:NOS/) at P23 to P25.

The increased intensity of immunolabeling for MnSOD in þ/Lc Purkinje cells from P10 through P25 is consistent with previous studies, suggesting that mitochondrial activity or density is increased in þ/Lc Purkinje cells, especially in the distal dendrites (DumesnilBousez and Sotelo, 1992; Vogel et al., 2001). To determine whether relative MnSOD protein levels are affected by the þ/Lc mutation, MnSOD protein levels from whole wild type and þ/Lc cerebella were analyzed by Western blots [Fig. 6(C); n ¼ 5 per each genotype and age group]. The overall relative expression levels of MnSOD protein increase with age with significant effects for both genotype and age (Genotype: two-way ANOVA, F1,24 ¼ 5.0, p < 0.05; Age: F2,24 ¼ 5.4, p < 0.02), although the genotype by age interaction is not significant (Genotype 3 Age ANOVA, F2,24 ¼ 1.4, p > 0.1). If the analysis is split by age, the relative MnSOD protein levels are not significantly different between wild type and þ/Lc cerebella at P10 and P15, although they approach statistical significance by P25 (one-way ANOVA, F1,8 ¼ 5.3, p ¼ 0.051).

Role of nNOS in þ/Lc Purkinje Cell Death The increased expression of markers for oxidative stress and NO production in þ/Lc Purkinje cells sugDevelopmental Neurobiology. DOI 10.1002/dneu

gest that the production of peroxynitrites may be a key step in the pathway for þ/Lc Purkinje cell degeneration. To test this hypothesis, we crossed þ/Lc mutants with a nNOS knockout mutant (nNOS/) to reduce the production of NO (Huang et al., 1993). The nNOS knockout mice have a targeted deletion of the first translated exon, which includes the coding region for the PDZ domain of the a nNOS isoform. The deletion of this exon dramatically reduces NOS activity in the cerebellum as shown in a previous study (Huang et al., 1993) and by the lack of NADPH-d staining of cerebellar sections from GluRd2þ/þ:nNOS/ mutants in this study (data not shown). Counts of the number of surviving Purkinje cells at P24 to P26 in control and GluRd2þ/Lc: nNOS/ double mutants showed that the loss of the a-subunit of nNOS does not prevent the death of þ/ Lc Purkinje cells (Fig. 7). Estimates of Purkinje cell numbers in wild type cerebella (GluRd2þ/þ: nNOSþ/þ and GluRd2þ/þ:nNOS þ/) and nNOS knock-out mice (GluRd2þ/þ:nNOS/) were significantly higher when compared with all þ/Lc mutants, regardless of the nNOS genotype (GluRd2þ/Lc: nNOSþ/þ, n ¼ 3, GluRd2þ/Lc:nNOSþ/, n ¼ 4; GluRd2þ/Lc:nNOS/, n ¼ 6; ANOVA, F3,14 ¼ 124.2, p < 0.0001). There was no significant difference between the number of þ/Lc Purkinje cells in nNOS þ/þ or þ/ cerebella (GluRd2þ/Lc:nNOSþ/þ and GluRd2þ/Lc:nNOSþ/) and GluRd2þ/Lc:nNOS/ double mutants (Fisher’s PLSD, p > 0.1) and there was also no significant difference between the numbers of Purkinje cells in wild type cerebella þ/þ (GluRd2 :nNOSþ/þ and GluRd2þ/þ:nNOSþ/) and nNOS knock-out mice (GluRd2þ/þ:nNOS/, Fisher’s PLSD, p > 0.1). The failure to rescue þ/Lc Purkinje cells by deleting a nNOS expression does not necessarily rule out a role for NO production in þ/Lc Purkinje cell death. A subsequent examination of NADPH-d activity in the cerebella of GluRd2þ/Lc:nNOS/ double mutants showed that the GluRd2þ/Lc:nNOS/ Purkinje cells are still stained for NADPH-d activity, suggesting that there is persistent NOS activity in the degenerating neurons [Fig. 8(A,B)]. The rest of the brain in the GluRd2þ/Lc:nNOS/ double mutants showed very little NADPH-d activity, confirming that most a nNOS activity was deleted (data not shown). The NADPH-d labeling appears more distinctly in the GluRd2þ/Lc:nNOS/ Purkinje cells at least in part because there is virtually no background labeling of granule cell parallel fibers and molecular layer interneurons [see Fig. 8(D)]. The persistence of NADPHd staining in GluRd2þ/Lc:nNOS/ Purkinje cells with no labeling of surrounding cells supports our

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Figure 8 NADPH-d histochemistry (P25; A, D), nitrotyrosine (P25; B, E), and activated caspase-3 (P15; C, F) immunolabeling in GluRd2þ/Lc:nNOSþ/þ (A, B, C) and GluRd2þ/Lc:nNOS/ mutants (D, E, F). Arrowheads indicate Purkinje cell bodies, while arrows indicate labeled Purkinje cell dendrites. Scale bar is 20 lm.

conclusion that the NADPH-d staining is within þ/Lc Purkinje cells and does not represent staining on the exterior surface of Purkinje cell membranes. The continued expression of NO in GluRd2þ/Lc: nNOS/ Purkinje cells is also shown by the persistence of nitrotyrosine immunolabeling in the double mutant [Fig. 8(E)]. The pattern of nitrotyrosine labeling is noticeably altered in the double mutant with less nitrotyrosine immunolabeling in the dendrites and more in the cell body of the Purkinje cells [compare Fig. 8(B), P25 GluRd2þ/Lc:nNOSþ/þ with Fig. 8(E), P25 GluRd2þ/Lc:nNOS/]. The mechanism of cell death does not appear to be altered in GluRd2þ/Lc: nNOS/ mutants because occasional GluRd2þ/Lc: nNOS/ Purkinje cells (1–3 per section) are still immunolabeled for activated caspase-3 as in þ/Lc mutants [Fig. 8(C,F)]. Formaldehyde stable-NADPH-d activity in GluRd2þ/Lc:nNOS/ Purkinje cells could be due to inducible NOS (iNOS), endothelial NOS (eNOS), or nNOS enzyme activity. Immunohistochemical staining for iNOS and eNOS showed no labeling of either WT or þ/Lc Purkinje cells at any age or background

genotype so we focused on the possible expression of other nNOS isoforms (data not shown). There are three main isoforms of nNOS in the rodent, a, b, and c. The most prevalent is the a isoform, which accounts for *95% of the nNOS in the cerebellum (Eliasson et al., 1997). The nNOS/ knockout mutant used in this study only deletes the a isoform, leaving the b- and c-subunits (Eliasson et al., 1997). In wild type mice, b isoforms make up *5% of the nNOS expressed in the cerebellum and deletion of the a isoform does not change the expression level of the b form (Eliasson et al., 1997; Putzke et al., 2000). However, we hypothesized that the deletion of the asubunit of nNOS fails to block þ/Lc Purkinje cell death because other NOS enzymes are upregulated in the absence of a nNOS. To test this hypothesis we labeled sections of GluRd2þ/þ:NOSþ/þ, GluRd2þ/þ: NOS/, GluRd2þ/Lc:nNOSþ/þ, and GluRd2þ/Lc: nNOS/ cerebella with antibodies to the N- and Cterminus of nNOS. The N-terminal antibody labels only the a isoform while the C-terminus antibody labels all three major isoforms, a, b, and c. Photomicrographs of nNOS immunolabeling of the þ/Lc cerDevelopmental Neurobiology. DOI 10.1002/dneu

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of the b and c isoforms which lack the PDZ binding domain that links a nNOS to the membrane. The qualitative intensity of C-terminus labeling appears similar in both GluRd2þ/Lc:nNOSþ/þ and GluRd2þ/Lc: nNOS/ Purkinje cells, suggesting that there is an upregulation of the b and/or c isoforms in both GluRd2þ/Lc:nNOSþ/þ and GluRd2þ/Lc:nNOS/ Purkinje cells.

DISCUSSION

Figure 9 Immunolabeling for N-terminal (A, C) and Cterminal (B, D) nNOS in GluRd2þ/Lc:nNOSþ/þ (A, B) and GluRd2þ/Lc:nNOS/ Purkinje cells (C, D). Arrowheads indicate Purkinje cell bodies, while arrows indicate labeled Purkinje cell dendrites. The immunolabeling for the N-terminal of nNOS is relatively indistinct when compared with the NADPH-d histochemistry, possibly due to the relatively poor binding qualities of many nNOS antibodies (see coers et al., 1998). We have tried at least three different commercially available antibodies to the N-terminal of nNOS and obtained the best results with Zymed (1/250). No N-terminal nNOS immunolabeling was observed in the GluRd2þ/Lc: nNOS/ cerebellar cortex (C), but C-terminal nNOS immunlabeling is observed in the Purkinje cells of the GluRd2þ/Lc:nNOSþ/þ (B) and GluRd2þ/Lc:nNOS/ (D) mutants. Scale bar is 20 lm.

ebella are shown in Figure 9. As expected, the Nterminus antibody labels the molecular and granule cell layer in the GluRd2þ/Lc:nNOSþ/þ cerebella [Fig. 9(A)], but there is no labeling in the GluRd2þ/Lc: nNOS/ cerebella, indicating that the a isoform has been deleted in the a nNOS knock-out mutant [Fig. 9(C)]. A similar pattern of labeling was observed in the GluRd2þ/þ:NOSþ/þ and GluRd2þ/þ:nNOS/ cerebella (N-terminal labeling in nNOSþ/þ cerebella and no N-terminal labeling in nNOS/ cerebella; data not shown). In contrast, C-terminus immunolabeling shows distinct labeling of the þ/Lc Purkinje cell body and, to a lesser extent, labeling of the dendrites in both GluRd2þ/Lc:nNOSþ/þ and GluRd2þ/Lc: nNOS/ cerebella [Fig. 9(B,D)]. The C-terminus labeling appears to be more broadly distributed throughout the cytoplasm of the Purkinje cell soma in GluRd2þ/Lc:nNOS/ Purkinje cells [Fig. 9(D)], which is consistent with the cytoplasmic distribution Developmental Neurobiology. DOI 10.1002/dneu

There is evidence for oxidative stress in a number of neurodegenerative diseases, but it is not always clear whether the oxidative stress is a cause of neuronal death or the result of the degenerative processes (Klein and Ackerman, 2003; Andersen, 2004). The altered expression of NADPH-d activity, and MnSOD and nitrotyrosine immunolabeling in degenerating þ/Lc Purkinje cells suggests that these neurons are experiencing an increase in oxidative stress as a result of their chronic depolarization. However, deletion of a major neuronal source of NO in the CNS, the a-subunit of nNOS, does not affect þ/Lc Purkinje cell survival. Although unexpected, this result does not rule out a role for oxidative stress in þ/Lc Purkinje cell death since there is evidence for an increase in alternative NOS activity that compensates for the missing a nNOS subunit activity. These results highlight the plasticity of cellular metabolic and cell death pathways in response to stress. Previous studies of the cerebellum have shown that NOS activity is transiently expressed during early postnatal development in cerebellar Purkinje cells, but it can also be induced in mature Purkinje cells in response to trauma or injury (Bruning, 1993; Chen and Aston-Jones, 1994; O’Hearn et al., 1995; Saxon and Beitz, 1996; Ikeda et al., 1999). In this study, we observed increased NADPH-d activity and immunolabeling for isoforms of nNOS in þ/Lc Purkinje cells after P10. The leak current mediated by the GluRd2Lc receptor begins around P5-P6 (Selimi et al., 2003), so we hypothesize that the sustained NADPH-d activity and nNOS expression is a response to the chronic depolarization of the þ/Lc Purkinje cells. The NADPH-d activity is abolished in cerebellar neurons in the a-nNOS knockout mutant, indicating that virtually all of the NADPH-d activity in cerebellar neurons is normally supplied by the asubunit of nNOS. In contrast, in the GluRd2þ/Lc: nNOS/ double mutant, NADPH-d labeling persists in Purkinje cells, suggesting that cellular injury from the chronic cation leak has induced alternate sources of NOS activity. The induction of NOS in þ/Lc

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Purkinje cells by chronic depolarization may be similar to the induction of NOS in adult Purkinje cells by overstimulation of olivary fiber inputs with ibogaine (O’Hearn and Molliver, 1993, 1997; O’Hearn et al., 1995). There are two main classes of NOS enzymes, iNOS and constitutively expressed nNOS and eNOS (Forstermann et al., 1998; Rodrigo et al., 2001). The expression of iNOS is normally triggered in response to cellular stress while n- and eNOS expression is regulated by intracellular calcium levels (Rodrigo et al., 2001). We have not detected the expression of iNOS in þ/Lc Purkinje cells but we cannot rule out the possibility that the antibodies we tried could not detect iNOS expression in our tissue. Immunolabeling did detect evidence of nNOS expression. Each of the genes for nNOS has many isoforms based on differential splicing of the N- and C-terminal regions and they are often preferentially expressed in specific tissues (Eliasson et al., 1997; Putzke et al., 2000). The three principal isoforms, a, b, and c, are differentiated by alternate splicing in the N-terminal domain. The a isoform contains the PDZ domain that allows for a nNOS binding to PSD-95 at synaptic membranes (Brenmand et al., 1996). The b and c isoforms lack the PDZ domain and are distributed in the cytoplasm, but they are only 80 and 1% as active as the a form, respectively (Eliasson et al., 1997; Putzke et al., 2000). The immunolabeling of þ/Lc Purkinje cells in the a nNOSþ/þ and / double mutants with N-terminal and C-terminal nNOS antibodies (see Fig. 9) suggests that while the a isoform is deleted in the knock-out mutants, it has been replaced by the upregulation of the b and/or c isoforms in þ/ Lc Purkinje cells. This hypothesis is supported by the apparent change in the distribution of C-terminal immunolabeling in the GluRd2þ/Lc:nNOSþ/þ Purkinje cells to a more cytoplasmic localization in GluRd2þ/Lc:nNOS/ Purkinje cells. The apparent changes in nNOS expression in þ/Lc Purkinje cells are accompanied by increases in two markers for oxidative stress, nitrotyrosine and MnSOD. Nitrotyrosine immunolabeling indicates the presence of both reactive oxygen (O 2 ) and NO, while MnSOD is a major mitochondrial superoxide scavenger and its expression is induced by a variety of cytotoxic and proapoptotic agents (Fridovich, 1975). The increased labeling for both is especially pronounced in the distal dendrites of þ/Lc Purkinje cells, where previous studies have noted an increase in the density of mitochondria (Caddy and Biscoe, 1979; Dumesnil-Bousez and Sotelo, 1992). Cytochrome oxidase histochemical labeling is also increased in þ/Lc Purkinje cell dendrites, which suggests that mito-

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chondrial activity is increased in the depolarized cells (Vogel et al., 2001). The qualitative and semi-quantitative analysis of MnSOD immunolabeling intensity shows that the distribution of MnSOD is significantly altered in þ/Lc Purkinje cells with more intense labeling in the dendritic tree. The quantitative Western blot data does not show a significant change in overall MnSOD concentrations at P10 and P15 in þ/ Lc cerebella, although by P25 the difference between mean MnSOD levels in the þ/Lc and wild type cerebella approaches statistical significance. However, specific increases in MnSOD protein levels in þ/Lc Purkinje cells may be obscured by the overall variation in MnSOD levels in the whole cerebellum. Taken together, the immunolabeling and Western blot data suggests that, at the very least, there is a significant redistribution of MnSOD in þ/Lc Purkinje cells. In particular, there is an increase in dendritic localization of MnSOD and it is likely that overall MnSOD protein levels are increasing in þ/Lc Purkinje cells through the period of þ/Lc Purkinje cell death. The continued rise in MnSOD levels through P25 þ/Lc cerebella is striking considering that many Purkinje cells and granule cells have died by P25. The analysis of MnSOD immunolabeling in the DCN suggests that the continued rise in MnSOD protein levels in þ/Lc cerebella cannot be attributed to increases in MnSOD protein expression in þ/Lc DCN neurons since the levels of MnSOD immunolabeling decrease to wild type levels by P25 in the þ/Lc cerebella. The increase in the density of mitochondria and the expression of markers for oxidative stress in þ/Lc Purkinje cell dendrites suggests a cell death pathway linked to energy metabolism. It seems likely that mitochondrial oxidative respiration is increased in response to the greater demand for ATP in depolarized þ/Lc Purkinje cells. The chronic cation leak current mediated by the GluRd2Lc channel will increase not only intracellular Naþ levels, but may also increase intracellular Ca2þ levels by activating voltage-gated Ca2þ channels (VGCCs) (Mouginot et al., 1997). The increased Naþ and Ca2þ load will increase metabolic demands for ATP as the þ/Lc Purkinje cells try to maintain their membrane potential and intracellular Naþ and Ca2þ levels. ATP is required for the ion pumps that restore intracellular Naþ and Ca2þ levels; about 50% of CNS ATP is used for the outward transport of Naþ by Naþ/Kþ-ATPase (Ames, 1997). There are metabolic costs associated with increased mitochondrial respiratory activity. Approximately 1–2% of the oxygen not consumed by mitochondrial cytochrome c oxidase is reduced to O 2 and H2O2 at mitochondrial and extra-mitochondrial sites (Radi et al., Developmental Neurobiology. DOI 10.1002/dneu

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1997). Therefore, any increases in cellular respiration rates may increase ROS production as a by-product. Increased Ca2þ levels and ROS production may stimulate the production of NO by nNOS, leading to the formation of peroxynitrite (ONOO; Beckman and Crow, 1993; Kamii et al., 1996; Keller et al., 1998; Bredt, 1999). Peroxynitrite will oxidize thiol groups, nitrate tyrosines, or nitrosylate cysteines in both structural proteins and enzymes. It was originally thought that peroxynitrite would indiscriminately react with any available protein or lipid substrates, but there is evidence that it may specifically nitrate or nitrosylate specific signaling proteins, with the effect of either aberrantly activating or deactivating the proteins (Klotz et al., 2002). In particular, the reaction of peroxynitrite with select target proteins may trigger cell death pathways by activating key pathways that promote cell death (e.g. stress activated MAP kinase pathways) or by inactivating pathways that inhibit cell death (e.g. PI/AKT; Minetti et al., 2002; Monteiro, 2002). A number of cell death pathways have been implicated in the death of þ/Lc Purkinje cells. The discovery that Beclin 1 is constitutively activated by the GluRd2Lc channel has led to the suggestion that excess autophagy plays a role in þ/Lc Purkinje cell death (Yue et al., 2002). While there is clear evidence for the induction of autophagosomes in the swellings of þ/Lc Purkinje cell axons, the incidence of autophagosomes was much less within the cell bodies and dendrites of þ/Lc Purkinje cells (Wang et al., 2006). The results of this study show that þ/Lc Purkinje cells express signs of oxidative stress that may trigger apoptotic cell death pathways. It is likely that there are a variety of cellular mechanisms that contribute to þ/Lc Purkinje cell death with oxidative stress as one contributing factor. Autophagy may be induced in þ/Lc Purkinje cells, not only by the failure of GluRd2Lc channels to sequester Beclin 1, but also as a cellular response to metabolic stress, including decreasing ATP levels as þ/Lc Purkinje cells mature with a chronic overload of Naþ and Ca2þ ions. This raises the possibility that if the induction of limited autophagy cannot rescue the þ/Lc Purkinje cells from death, autophagy regulatory mechanisms may also activate or enhance apoptotic pathways (Furuya et al., 2005). The excessive stimulation of autophagy alone may induce Purkinje cell death as suggested by the dissociation between the leak current and Purkinje cell death in Lurcher/hotfoot (Lc/ho) double mutants (Selimi et al., 2003). Nevertheless, the conditions for cell death in the Lc/ho mutant do not rule out a role for oxidative stress and apoptosis in the þ/Lc mutant. Developmental Neurobiology. DOI 10.1002/dneu

We thank Drs. Ann Lohof and Hadi Zanjani for their helpful comments on the manuscript.

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