Oxidative Stress, Nitric Oxide, and the Mechanismsof 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

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

3 Equipe Developpement et Vieillissement du Systeme Nerveux, UMR NPA 7102, CNRS etUniversite 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 Pur-

kinje 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 recep-

tor into a leaky membrane channel, resulting in

chronic depolarization of þ/Lc Purkinje cells starting

around the first week of postnatal development. Virtu-

ally, 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.

The results show that NOS activity and immunolabel-

ing 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 produc-

tion of NO. Analysis of the double mutants showed

that blocking a-nNOS expression does not rescue þ/LcPurkinje cells. However, we present evidence for sus-

tained 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 domi-

nant 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 preferen-

tially expressed in high levels at parallel fiber synap-

Correspondence to: M.W. Vogel (mvogel@mprc.umaryland.edu).

Contract grant sponsor: NINDS; contract grant number:NS34309.

This article contains supplementary material available via theInternet at http://www.interscience.wiley.com/jpages/1932-8451/suppmat.

' 2007 Wiley Periodicals, Inc.Published online 6 March 2007 in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/dneu.20391

1032

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 substi-

tution 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/LcPurkinje 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 descrip-

tions have suggested necrotic cell death (Dumesnil-

Bousez 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; Wull-

ner et al., 1995; Selimi et al., 2000) and BAX and

Bcl-x expression are increased in dying þ/Lc Pur-

kinje 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 expres-

sion (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 ox-

ide synthase (NOS) activity and the expression of two

markers for oxidative stress, nitrotyrosine and manga-

nese super oxide dismutase (MnSOD), in wild type

and þ/Lc Purkinje cells. Nitrotyrosine in the product

of peroxynitrite nitration of tyrosine residues on tar-

get proteins. MnSOD is the principal scavenger of

superoxide in mitochondria and is induced by a vari-

ety of cytotoxic and proapoptotic agents (Fridovich,

1975). To determine whether peroxynitrite formation

is a prerequisite for þ/Lc Purkinje cell death, þ/Lcmutants were crossed with a-nNOS knockout mutants

to reduce the production of NO. Analysis of the dou-

ble mutants showed that blocking a-nNOS expression

might not rescue þ/Lc Purkinje cells from death

because there is an induction of alternate NOS activ-

ities.

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 homozy-

gotes (from Jackson Laboratories; nNOS�/�). The F1 gen-

eration 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 homo-

zygous 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 Psychiat-

ric Research Center and provided with food and water adlibitum. 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 Amer-

ican Association for the Accreditation of Laboratory Ani-

mal Care (AAALAC) and the studies were conducted in ac-

cordance 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% sa-

line followed by 4% paraformaldehyde (while deeply anes-

thetized 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 fron-

tal cortex was removed and quickly frozen in microcentri-

fuge tubes on crushed dry ice.

Immunocytochemistry

Fixed brains were either cut at 30 lm and collected as float-

ing 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 immuno-

labeling experiments, the floating sections or slides were

Lurcher Purkinje Cells 1033

Developmental Neurobiology. DOI 10.1002/dneu

rinsed in 10 mM PBS, followed by incubation in two

changes of 0.1 M glycine for 5 min each. Endogenous fluo-

rescence was reduced by incubating the sections in 50 mMammonium 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 pri-

mary antibodies overnight at 48C. All sections were double

labeled with mouse anticalbindin (Sigma: 1/10,000) or rab-

bit 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 immu-

nolabeling 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 ran-

domly selected from throughout the medial to lateral extent

of the vermis in randomly selected lobules that still con-

tained Purkinje cells. The raw images were then analyzed

using Metamorph Version 7.0r1. Threshold and colocaliza-

tion functions were used to selectively measure the inten-

sity 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 coloc-

alization functions were used to measure the intensity of

MnSOD immunolabeling in the DCN excluding calbindin-

positive 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 cryo-

stat 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 phos-

phate buffer for 3 h at 378C. The sections were rinsed again

with PBS and coverslipped with gelmount.

Purkinje Cell Counts

The number of Purkinje cells per cerebellum were esti-

mated 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 ran-

dom 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 cer-

ebellar cortex was used as reference volume for counting

Purkinje cells. The optical dissector used to count Pur-

kinje cells was 15 lm deep and 500 lm2, spaced 200 lm

apart. These parameters were chosen to obtain a coeffi-

cient 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 su-

pernatant 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 dis-

solved 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 immun-

Star chemiluminescence kit. Film exposed to the chemi-

luminescent 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 anti-

gen 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.

1034 McFarland et al.

Developmental Neurobiology. DOI 10.1002/dneu

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/LcPurkinje Cells

As an assay for NO production in the þ/Lc cerebel-

lum, the histochemical activity of formaldehyde-sta-

ble NADPH diaphorase (NADPH-d) was examined

in fixed, frozen sections of wild type and þ/Lc cere-

bella 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 previ-

ously (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 Pur-

kinje cells although labeling remains high in the gran-

ule 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/LcPurkinje cells still stain for NADPH-d histochemical

activity at P15 [Fig. 1(D)] and many surviving Pur-

kinje 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 out-

lines defined by NADPH-d staining in the surround-

ing 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.

Oxidative Stress in þ/Lc Purkinje Cells

To assay for evidence of oxidative stress in þ/Lc Pur-

kinje cells, we have examined the distribution of

nitrotyrosine immunolabeling in wild type and þ/Lccerebella 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 cal-

bindin, while the green staining represents immuno-

labeling for nitrotyrosine. In control experiments to

confirm the specificity of the antibody, the mouse

monoclonal antibody to nitrotyrosine was preab-

sorbed with either free nitrotyrosine or phosphotyro-

sine. No immunolabeling was observed in control þ/

Figure 1 Developmental progression of NADPH-d histo-

chemistry 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.

Lurcher Purkinje Cells 1035

Developmental Neurobiology. DOI 10.1002/dneu

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 pat-

tern of immunolabeling in þ/Lc cerebellar sections

with nitrotyrosine antibodies preabsorbed with phos-

photyrosine, indicating that the nitrotyrosine antibody

does not cross react with phosphotyrosine residues.

At P10, nitrotyrosine is primarily localized in Pur-

kinje cells in both wild type and þ/Lc mutant mice

(Fig. 2). However, the amount of nitrotyrosine label-

ing in þ/þ Purkinje cells appears to decline with age

[Fig. 2(B,F,J)], especially in the Purkinje cell den-

drites. The þ/þ Purkinje cell bodies (white arrow-

heads) are still lightly labeled for nitrotyrosine at

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 anti-

nitrotyrosine antibodies at P15 with intense labeling

in the dendrites and somas [Fig. 2(G)]. By P25, sig-

nificant numbers of þ/Lc Purkinje cells have degen-

erated 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 char-

acteristic short, stubby appearance [Fig. 2(K,L)]. The

distal dendrites often appear swollen and the immuno-

labeling for nitrotyrosine is especially intense in the

distal dendrites. There was no obvious, consistent re-

gional heterogeneity in the distribution of nitrotyro-

sine-labeled þ/Lc Purkinje cells at P25 in sagittal sec-

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 photo-

graphs 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.]

1036 McFarland et al.

Developmental Neurobiology. DOI 10.1002/dneu

tions. However, the medial to lateral distribution of

immunolabeled þ/Lc Purkinje cells was not exam-

ined in coronal sections so parasagittal patterns of þ/

Lc Purkinje cell degeneration may have been missed.

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 arrow-

heads). 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 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 dou-

ble labeled (yellow) þ/Lc Purkinje cell bodies while the arrow

indicates a Purkinje cell axonal swelling that is colabeled for

nitrotyrosine.

Figure 4 Immunolabeling for calbindin and MnSOD in the cerebellar cortex of wild type con-

trols 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.

Lurcher Purkinje Cells 1037

Developmental Neurobiology. DOI 10.1002/dneu

swellings on þ/Lc Purkinje cell axons are also la-

beled 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 axo-

nal swellings.

As an independent measure of oxidative stress

in þ/Lc Purkinje cells, we have examined the cellu-

lar 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 dramat-

ically 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 Pur-

kinje 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 calbin-

din 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 dif-

fuse so it is no longer possible to resolve individual

Purkinje cell dendrites based on MnSOD immuno-

labeling [Fig. 4(E,F)]. In contrast, P15 þ/Lc Pur-

kinje 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 cal-

bindin positive axonal swellings in the white matter

and DCN and some are also double labeled for

MnSOD, indicating that the swellings contain mito-

chondria (data not shown). By P25, MnSOD immu-

nolabeling has become even more diffuse in the mo-

lecular layer of wild type cerebella, although punc-

tate 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 cere-

bella. 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.

Semi-quantitative comparisons of the intensity of

MnSOD immunolabeling between þ/þ and þ/LcPurkinje cells at P15 and P25 throughout lobules in

the cerebellar vermis showed that there is a signifi-

cant increase in the overall intensity of MnSOD label-

ing 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 calbin-

din and MnSOD immunohistochemistry and the

colocalize function in Metamorph was used to mea-

sure the intensity of MnSOD immunolabeling in cal-

bindin 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 inten-

sities of MnSOD immunolabeling are similar in þ/LcPurkinje cells at P15 and P25, the variance is consi-

derably higher in the P25 Purkinje cells. There are con-

siderably 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 pho-

tomontage of double labeling for MnSOD and calbindin in

the DCN is shown in a supplementary figure.

1038 McFarland et al.

Developmental Neurobiology. DOI 10.1002/dneu

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).

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.

Lurcher Purkinje Cells 1039

Developmental Neurobiology. DOI 10.1002/dneu

The increased intensity of immunolabeling for

MnSOD in þ/Lc Purkinje cells from P10 through P25

is consistent with previous studies, suggesting that mi-

tochondrial activity or density is increased in þ/Lc Pur-

kinje cells, especially in the distal dendrites (Dumesnil-

Bousez and Sotelo, 1992; Vogel et al., 2001). To deter-

mine whether relative MnSOD protein levels are

affected by the þ/Lc mutation, MnSOD protein levels

from whole wild type and þ/Lc cerebella were ana-

lyzed by Western blots [Fig. 6(C); n ¼ 5 per each ge-

notype and age group]. The overall relative expression

levels of MnSOD protein increase with age with signif-

icant effects for both genotype and age (Genotype:

two-way ANOVA, F1,24 ¼ 5.0, p < 0.05; Age: F2,24 ¼5.4, p < 0.02), although the genotype by age interac-

tion 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 dif-

ferent 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 sug-

gest that the production of peroxynitrites may be a

key step in the pathway for þ/Lc Purkinje cell degen-

eration. To test this hypothesis, we crossed þ/Lcmutants 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 signifi-

cantly 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 ¼124.2, p < 0.0001). There was no significant differ-

ence 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 num-

bers of Purkinje cells in wild type cerebella

(GluRd2þ/þ

:nNOSþ/þ and GluRd2þ/þ:nNOSþ/�) and

nNOS knock-out mice (GluRd2þ/þ:nNOS�/�, Fish-

er’s PLSD, p > 0.1).

The failure to rescue þ/Lc Purkinje cells by delet-

ing 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 degenerat-

ing 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 anNOS 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 inter-

neurons [see Fig. 8(D)]. The persistence of NADPH-

d staining in GluRd2þ/Lc:nNOS�/� Purkinje cells

with no labeling of surrounding cells supports our

Figure 7 Estimates of Purkinje cell number per hemicer-

ebellum 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�/� dou-

ble mutants (þ/Lc:NOS�/�) at P23 to P25.

1040 McFarland et al.

Developmental Neurobiology. DOI 10.1002/dneu

conclusion that the NADPH-d staining is within þ/LcPurkinje 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 persist-

ence of nitrotyrosine immunolabeling in the double

mutant [Fig. 8(E)]. The pattern of nitrotyrosine label-

ing is noticeably altered in the double mutant with

less nitrotyrosine immunolabeling in the dendrites

and more in the cell body of the Purkinje cells [com-

pare 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/Lcmutants [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 stain-

ing 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 mu-

tant 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 a-

subunit 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 la-

beled sections of GluRd2þ/þ:NOSþ/þ, GluRd2þ/þ:

NOS�/�, GluRd2þ/Lc:nNOSþ/þ, and GluRd2þ/Lc:

nNOS�/� cerebella with antibodies to the N- and C-

terminus 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. Photomi-

crographs of nNOS immunolabeling of the þ/Lc cer-

Figure 8 NADPH-d histochemistry (P25; A, D), nitrotyrosine (P25; B, E), and activated cas-

pase-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.

Lurcher Purkinje Cells 1041

Developmental Neurobiology. DOI 10.1002/dneu

ebella are shown in Figure 9. As expected, the N-

terminus 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 immunolab-

eling 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

of the b and c isoforms which lack the PDZ binding

domain that links a nNOS to the membrane. The qual-

itative intensity of C-terminus labeling appears simi-

lar 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�/� Pur-

kinje cells.

DISCUSSION

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-sub-

unit of nNOS, does not affect þ/Lc Purkinje cell sur-

vival. 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 alter-

native NOS activity that compensates for the missing

a nNOS subunit activity. These results highlight the

plasticity of cellular metabolic and cell death path-

ways 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 Pur-

kinje 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/LcPurkinje 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 a-

subunit 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

Figure 9 Immunolabeling for N-terminal (A, C) and C-

terminal (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-ter-

minal 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 commer-

cially 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 im-

munlabeling is observed in the Purkinje cells of the

GluRd2þ/Lc:nNOSþ/þ (B) and GluRd2þ/Lc:nNOS�/� (D)

mutants. Scale bar is 20 lm.

1042 McFarland et al.

Developmental Neurobiology. DOI 10.1002/dneu

Purkinje cells by chronic depolarization may be simi-

lar 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. Immunolabel-

ing did detect evidence of nNOS expression. Each of

the genes for nNOS has many isoforms based on dif-

ferential 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 differen-

tiated 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 mem-

branes (Brenmand et al., 1996). The b and c isoforms

lack the PDZ domain and are distributed in the cyto-

plasm, but they are only 80 and 1% as active as the aform, 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 Pur-

kinje cells to a more cytoplasmic localization in

GluRd2þ/Lc:nNOS�/� Purkinje cells.

The apparent changes in nNOS expression in þ/LcPurkinje cells are accompanied by increases in two

markers for oxidative stress, nitrotyrosine and

MnSOD. Nitrotyrosine immunolabeling indicates the

presence of both reactive oxygen (O2�) and NO, while

MnSOD is a major mitochondrial superoxide scav-

enger and its expression is induced by a variety of cy-

totoxic and proapoptotic agents (Fridovich, 1975).

The increased labeling for both is especially pro-

nounced 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 ox-

idase histochemical labeling is also increased in þ/LcPurkinje cell dendrites, which suggests that mito-

chondrial activity is increased in the depolarized cells

(Vogel et al., 2001). The qualitative and semi-quanti-

tative 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 West-

ern 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 cere-

bella approaches statistical significance. However,

specific increases in MnSOD protein levels in þ/LcPurkinje cells may be obscured by the overall varia-

tion in MnSOD levels in the whole cerebellum.

Taken together, the immunolabeling and Western

blot data suggests that, at the very least, there is a sig-

nificant 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 Pur-

kinje 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/Lccerebella.

The increase in the density of mitochondria and

the expression of markers for oxidative stress in þ/LcPurkinje cell dendrites suggests a cell death pathway

linked to energy metabolism. It seems likely that mi-

tochondrial oxidative respiration is increased in

response to the greater demand for ATP in depolar-

ized þ/Lc Purkinje cells. The chronic cation leak cur-

rent mediated by the GluRd2Lc channel will increase

not only intracellular Naþ levels, but may also

increase intracellular Ca2þ levels by activating volt-

age-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 intracel-

lular 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 trans-

port of Naþ by Naþ/Kþ-ATPase (Ames, 1997). There

are metabolic costs associated with increased mito-

chondrial respiratory activity. Approximately 1–2%

of the oxygen not consumed by mitochondrial cyto-

chrome c oxidase is reduced to O2� and H2O2 at mito-

chondrial and extra-mitochondrial sites (Radi et al.,

Lurcher Purkinje Cells 1043

Developmental Neurobiology. DOI 10.1002/dneu

1997). Therefore, any increases in cellular respiration

rates may increase ROS production as a by-product.

Increased Ca2þ levels and ROS production may stim-

ulate 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 struc-

tural 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 per-

oxynitrite with select target proteins may trigger cell

death pathways by activating key pathways that pro-

mote 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 impli-

cated in the death of þ/Lc Purkinje cells. The discov-

ery 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 auto-

phagosomes 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/Lcmutant.

We thank Drs. Ann Lohof and Hadi Zanjani for their

helpful comments on the manuscript.

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