American Journal ofPathology, Vol. 151, No. 6, December 1997Copyright © American Society for Investigative Pathology

Diverse Cell Death Pathways Result from a SingleMissense Mutation in weaver Mouse

Antonio Migheli,* Roberto Piva,* Jianjun Wei,$Angelo Attanasio,* Stefania Casolino,*Marion E. Hodes,t Stephen R. Dlouhy,t*Shirley A. Bayer,§ and Bernardino Ghettit:From the Department ofNeuroscience,* Laboratory ofNeuropathology, University of Turin, Turin, Italy, and theDepartments ofMedical and Molecular Geneticst and Pathologyand Laboratory Medicine,' Laboratory of Cellular and MolecularNeuropathology, Indiana University School ofMedicine, and theDepartment of Biology,§ Indiana-Purdue University,Indianapolis, Indiana

Neuronal death affects selectively granule celi precur-sors ofthe cerebelium and the dopaminergic neuronsof midbrain in the weaver mutant mouse. The weaverphenotype is associated with a missense mutation inthe gene coding for the GIRK2 potassium channel,which results in chronic depolarization. Using DNAgel electrophoresis, electron microscopy (EM), the insitu end-labeling (ISEL) technique at the light and EMlevel, and inmmunohistochemistry for apoptosis-re-lated proteins c-Jun and proliferating cell nuclear an-tigen (PCNA), we have investigated the mechanismsof celi death in cerebellum and substantia nigra. Be-tween postnatal day Pl and P21, in the external ger-minal layer of the cerebellum, most degeneratinggranule celi precursors were found to aggregate toform clusters. Degenerating cells exhibited strong nu-clear staining for ISEL, c-Jun, and PCNA and had atypical apoptotic morphology by EM. Increased c-Junand ISEL staining were also occasionally seen in Pur-kinje cells. Between P14 and P21, when dopaminergicneurons start to degenerate, staining for ISEL, c-Jun,and PCNA in weaver substantia nigra was the same asin controls. By EM, however, we found only inweaver mice numerous dopaminergic cells thatshowed extensive vacuolar and autophagic changesof cytoplasm, preservation of membrane and or-ganelle integrity, and absence of chromatin conden-sation or DNA fragmentation by EM-ISELs The combi-nation of vacuolar and autophagic changes identifiesa novel type of non-necrotic, nonapoptotic cell death.After biochemical analysis of DNA, a clear-cut ladder-ing, suggestive of oligonucleosomal fragmentation,was present in samples from weaver cerebellum. Celldeath diversity appears to be influenced by specificfeatures oftarget cells. These findings may be relevantfor understanding the mechanisms of cell death in

neurodegenerative diseases. (Am J Pathol 1997,151:1629-1638)

The weaver mutation in the laboratory mouse has beenidentified as a missense mutation of the Girk2 gene,which encodes the G-protein-activated inwardly rectify-ing potassium (GIRK) channel GIRK2.1 Of the five GIRKchannels that have been identified so far, GIRK1, -2, and-3 are expressed in the cerebellum, whereas only GIRK2is expressed in the substantia nigra.25 GIRK subunitsassemble as hetero- or homotetrameric channels in vitroand presumably also in vivo, producing functionally dis-tinct channels.6 The wv mutation results in a glycine toserine substitution at residue 156 in the putative pore-forming region.1 Functional consequences of the muta-tion include loss of normal selectivity for K+ over Na+ andCa2+, constitutive activation of channels, and cell deathafter chronic depolarization.7 1The weaver mutation has an incomplete dominant pat-

tern of expression.12 Homozygous (wv/wv) mice displayinstability of gait and both resting and intention tremor.wv/wv males are infertile, whereas females reproducenormally. This complex clinical picture includes extensivecell loss in the cerebellum,13 the dopamine (DA) projec-tion system of the midbrain,14 and the germinal epithe-lium of the testis.15'16 Heterozygous (wvl+) mice show aless severe involvement of the same regions.The fact that GIRK2 dysfunction is a key factor in the

process of cell death is supported by the observation thatthe areas most affected by cell loss show a high expres-sion of Girk2 mRNA and by in vitro studies showing thatexpression of the mutated gene leads to markedly re-duced cell survival.8 Recent data have shown that mostgranule cell precursors forming the external germinallayer (EGL) in weaver cerebellum die by apoptosis withinthe first two postnatal weeks,17-19 whereas a type of celldeath morphologically distinct from apoptosis was ob-served in the substantia nigra.2021 On the other hand, nostudy has investigated the possible involvement in thedegenerative process of Purkinje cells (PCs), which areabnormally located and, according to several investiga-tions, also reduced in number.2223

Supported by USPHS grants P01 NS27613 and R01 NS14426 and by ECCopernicus Programme grant CIPA-CT93-0210.Accepted for publication August 27, 1997.Address reprint requests to Dr. Antonio Migheli, Department of Neuro-

science, University of Turin, Laboratory of Neuropathology, Via Cherasco15, 10126 Turin, Italy.

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1630 Migheli et alAJP December 1997, Vol. 151, No. 6

The aim of this study was to investigate some of thecellular mechanisms leading to cell death in weaver cer-ebellum and substantia nigra. Degenerative changeshave been evaluated using 1) an in situ end-labeling(ISEL) technique for the demonstration of DNA fragmen-tation, a hallmark of apoptotic cell death,24"2 2) electronmicroscopy (EM), and 3) an application of the ISEL tech-nique at the EM level (EM-ISEL).26 The expression ofmolecules that appear to be critical for neuronal celldeath via apoptosis, ie, transcription factor c-Jun27'28and the proliferating cell nuclear antigen (PCNA),2930has been investigated by immunohistochemistry. Finally,DNA gel electrophoresis has been performed to detectDNA fragmentation biochemically.

Materials and Methods

Animals and Experimental DesignThe weaver and wild-type animals were obtained from acolony established at Indiana University Medical Centerfrom mice heterozygous for the weaver gene that origi-nated from Jackson Laboratory (Bar Harbor, ME). Mutantand wild-type mice are maintained on a B6CBA-Aw-J/Ahybrid stock. Genotype analysis was performed as de-scribed.22 Sixteen +/+ and sixteen wv/wv mice com-bined in four age groups (P1, P7, P14, and P21) werestudied by light (n = 16) and electron (n = 16) micros-copy.

Tissue PreparationLight Microscopy

Animals were anesthetized with sodium pentobarbital(50 mg/kg intraperitoneally) and perfused transcardiallywith 10 ml of normal saline and subsequently with 60 to120 ml of 4% paraformaldehyde in 0.1 mol/L phosphatebuffer (PB), pH 7.2. Brains were removed and post-fixedin the same fixative for 30 to 60 minutes. Tissues weredehydrated in graded ethanols, cleared in xylene, andembedded in paraffin. Midsagittal sections of the cere-bellum and transverse sections of midbrain were cut at 6,um and mounted on poly-L-lysine-coated slides.

Electron Microscopy

Animals were perfused with 2.5% glutaraldehyde in PBas previously reported.18 Brains were post-fixed in glu-taraldehyde for 1 to 3 hours, and slices of representativeareas were post-fixed in 2% osmium in PB for 2 hours,dehydrated in graded ethanols, cleared in propyleneoxide, and embedded in Epon.

ImmunohistochemistryTyrosine Hydroxylase (TH)

Recognition of DA neurons was accomplished usingan antiserum raised in rabbit against bovine TH (Eugene

Tech, Allendale, NJ). The reaction was revealed with thephosphatase anti-alkaline phosphatase method, usingdiaminobenzidine (DAB) as substrate.

c-Jun

An affinity-purified rabbit antibody raised against apeptide corresponding to residues 73 to 87 in the amino-terminal region of c-Jun (Oncogene Sciences, Uniondale,NJ) was used. The antibody recognizes mouse, rat, andhuman c-Jun and was used at 0.1 ,ug/ml concentration.To achieve significant immunostaining, the sections wereplaced in 0.01 mol/L citrate buffer, pH 6, and heated in amicrowave oven (Whirlpool) at 750 W until boiling andthen at 350 W for 15 minutes. The reaction was revealedwith the avidin-biotin complex technique, using cobalt-chloride-intensified DAB as substrate. Specificity of thereaction was tested by preabsorbing the antibody with a10-fold excess of the immunizing peptide (OncogeneSciences). Sections were counterstained with hematoxy-lin.

PCNA

A monoclonal anti-rat PCNA antibody (Dako, Carpin-teria, CA), which reacts with PCNA from all vertebratespecies, insects, and Schizosaccharomyces pombe, wasused at 1/30,000 dilution after microwave treatment, asdescribed above.

ISELThe ISEL technique is used to demonstrate in single cellsthe occurrence of DNA fragmentation, a hallmark of ap-optosis.24'25 Terminal deoxynucleotidyl transferase (TdT)is applied to tissue sections to incorporate labeled dUTPin the 3'-hydroxyl recessed termini of DNA breaks. Sec-tions were deparaffinized and the endogenous peroxi-dase activity blocked with 3% H202 for 10 minutes. Sec-tions were then rinsed in TdT buffer (25 mmol/L Tris/HCI,200 mmol/L sodium cacodylate, 5 mmol/L cobalt chlo-ride) and incubated with the labeling mix (20 U of TdT(Boehringer Mannheim, Indianapolis, IN) and 1 nmol offluorescein-1 1-dUTP (Boehringer Mannheim) in 100 li1 ofTdT buffer) for 120 minutes at 370C. The reaction wasterminated by rinsing in 300 mmol/L sodium chloride/30mmol/L sodium citrate (2X SSC) for 10 minutes at roomtemperature. The sections were placed in Tris-bufferedsaline (TBS) containing 0.3% Triton X-100 and 2% bovineserum albumin for 30 minutes at room temperature andthen incubated with anti-fluorescein sheep antibody con-jugated with peroxidase (Boehringer Mannheim), diluted1/500 in TBS/Triton, for 30 minutes at 370C. After washingin TBS, the reaction was revealed with cobalt-chloride-intensified DAB. Sections were counterstained with he-matoxylin and eosin (H&E) or methyl green, dehydrated,and mounted in Permount. Control of the reaction wasperformed by omitting TdT from the labeling mix. In somecases the sections were visualized under the fluores-cence microscope immediately after the TdT reaction.

Cell Death in weaver Mice 1631AJP December 1997, Vol. 151, No. 6

ISEL, c-Jun, and PCNA labeling were quantitativelyanalyzed in the EGL. For each staining, five midsagittalsections were used for each animal. Cell counts weremade with an oil-immersion objective at X1000 magnifi-cation. On each section, six areas (0.02 mm2 each) con-taining the whole thickness of the EGL were chosen, sothat the anterior to posterior axis of the cerebellum wasequally sampled. The number of EGL cells/area and thenumber of ISEL-, c-Jun-, and PCNA-labeled nuclei in theEGL/area were counted. For each parameter, the areavalues were summed for each section, and a mean value/section was determined. For each ISEL-stained section,an apoptotic index (Al) was expressed as the percentageof ISEL-labeled cells/number of EGL cells, and a mean Alwas calculated for each genotype and age group. C-Junand PCNA labeling indexes were similarly evaluated.Statistical analysis was performed using one-way analy-sis of variance (ANOVA) and post hoc Scheffe F-test.

EM-ISELThe EM-ISEL technique has been recently developed byus to localize DNA strand breaks at the ultrastructurallevel.26 Thin sections were collected on Formvar-coatednickel grids, rinsed in TdT buffer, and incubated with thelabeling mix (1 U of TdT (Boehringer Mannheim) and 0.5nmol/L digoxigenin-11-dUTP (Boehringer Mannheim) in100 Al of TdT buffer) for 2 to 10 minutes at 370C. Thereaction was terminated by rinsing in 2X SSC. Grids werethen rinsed in TBS, incubated in 10% normal goat serumin TBS for 30 minutes, and placed overnight at 40C inanti-digoxigenin goat immunoglobulins coupled with10-nm colloidal gold (BioCell, Cardiff, UK), diluted 1/50 inTBS. Grids were extensively washed in TBS and lightlystained with 2% uranyl acetate. Control of the reactionwas performed by omitting TdT from the labeling mix.

Biochemical Analysis ofDNADNA fragmentation was analyzed using a modification ofthe DNA isolation, end-labeling, and autoradiographyprocedure reported by Tilly and Hsueh.31 Briefly, lysisbuffer (0.3 mol/L Tris/HCI (pH 8.0), 0.2 mol/L sucrose, 0.1mol/L NaCI, and 0.01 mol/L EDTA) was added to snap-frozen tissues (-40:1 volume), the tissue homogenizedby loose dounce, SDS added to a final concentration of1%, the mixture incubated at 650C for 60 minutes, 8 mol/Lpotassium acetate added to the cells (final concentration,1.3 mol/L), and the solution gently mixed and allowed tostand on ice for 30 minutes. Protein and some high mo-lecular weight DNA was pelleted by centrifugation(- 12,000 rpm in a microfuge), the supernatant was trans-ferred to new tubes, and DNA (in supernatant) was ex-tracted with phenol/chloroform/isoamyl alcohol (25:24:1)and with chloroform/isoamyl alcohol (24:1). DNA waspelleted by adding ammonium acetate (1/10) and ethylalcohol (1/2.5) and resuspended in 300 ,tl of 1X TE. Afteraddition of 3 ,Ag of DNAse-free RNAse, the mixture wasincubated at 370C for 60 minutes, phenol extraction andprecipitation were repeated, the final DNA pellet was

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Figure 1. Distribution of the apoptotic index (Al) in cerebellar EGL of wv/wvand +/+ mice from P1 to P21 (ANOVA: P < 0.05 at P1 and P < 0.01 at P7and P14). The AI was calculated on ISEL-stained sections (see text forexplanation of the procedure). A similar trend was seen for c-Jun and PCNAlabeling indexes.

washed with 70% ethyl alcohol, and the DNA was resus-pended in 1X TE (0.5 ,tg/j,l). Isolated DNA was end-labeled with [a32P]dideoxy ATP, separated electro-phoretically in an acrylamide gel, and detected byautoradiography of the gel after drying.

Results

Cerebellum+/+ Mice

Scattered ISEL-labeled nuclei were found in the outerportion of the EGL between P1 and P14 (the EGL disap-pears after P14 in +/+ mice), reflecting developmentallyprogrammed cell death (PCD) via apoptosis32 (Figure 1).In adjacent sections, scattered pyknotic and fragmentednuclei from the same areas also showed strong c-Junand PCNA immunoreactivity. PCNA also stained nuclei ofproliferating granule cell precursors of the EGL. By EM,degenerating cells showed typical apoptotic changes,such as chromatin condensation along the nuclear mem-brane, increased electron density of the cytoplasm, andfragmentation into apoptotic bodies with preservation oforganelle and membrane integrity. By EM-ISEL, intensegold labeling, indicative of DNA fragmentation, wasfound in all apoptotic nuclei. Apoptotic PCs were notfound; only a faint immunoreactivity for c-Jun was presentin the nuclei of most PCs.

wvAvv Mice

In the EGL, ISEL-labeled nuclei were found at all agesstudied, with a far greater frequency than in +/+ mice(Figure 1). Most labeled nuclei were located in the prox-imity of the PC layer (Figure 2a). In this location, theyoften appeared grouped together to form clusters closeto, or even surrounding, PCs. All of the clustered cellsshowed advanced apoptotic changes, such as nuclearpyknosis and fragmentation into apoptotic bodies (Figure2, b and c). Each cluster was formed by a variablenumber of aggregated apoptotic nuclei, ranging between3 and 14 labeled structures. The number of apoptoticcells was highest between P7 and P21. Clusters wereespecially abundant at P7, when they accounted for over

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1632 Migheli et alAJP December 1997, Vol. 151, No. 6

dFigure 2. a to c: ISEL in the external germinal layer (EGL) of weavercerebellum at P7. a: Most labeled cells form clusters in the proximity of the Purkinje cell layer.Magnification, X 200. b to c: Detail of clusters at P7. All labeled nuclei have apoptotic morphology; in C, an unstained Purkinje cell (arrow) is surrounded by labelednuclei (EGL layer on bottom). Magnification, X 1000. d: PCNA immunoreactivity in weaver EGL at P7. a: Light staining is seen in most EGL cells, indicating theirproliferative status. Strong staining is seen in apoptotic cells. Magnification, X 1000.

80% of labeled cells. At P14 and P21, clustered cellsamounted to nearly 50% of labeling. Strong immunore-activity for c-Jun and PCNA was present in both isolatedand clustered apoptotic nuclei, with the same distributionas ISEL staining (Figures 2d and 3a). The labeling indexfor c-Jun and PCNA did not differ from the apoptoticindex evaluated by ISEL (Figure 1). By EM, degeneratingcells showed various apoptotic changes and DNA frag-mentation by EM-ISEL, without differences between iso-lated and clustered cells.

ISEL labeling was also found in the disorganized PClayer. Although most of the labeling occurred in pyknoticnuclei of granule cell precursors, labeling was also foundin cells of non-granule-cell origin. 1) Rare PCs showedlight ISEL labeling in their nucleus (Figure 3b). Thesecells were infrequently noted at all ages studied andnever exceeded 0.3% of all PCs in the section. 2) Occa-sionally, ISEL-labeled PCs were also found with ISELreactivity in the atrophic dendritic tree, suggesting leak-age of fragmented DNA from the nucleus26 (Figure 3c).3) ISEL-positive large nuclei devoid of cytoplasm werealso occasionally found. As they were larger than the

nuclei of apoptotic granule cell precursors, they mightbelong to PCs undergoing the final stages of apoptosis,although a precise assessment of the cell type was notpossible. The staining pattern of c-Jun was similar to thatof ISEL, except that the number of c-Jun-immunoreactivePCs (clearly identifiable as such) was approximately 10-fold higher (2.2 to 3.5% of all PCs) than that of ISEL-labeled PCs. C-Jun-immunoreactive PCs typically had adistorted nuclear profile and condensed cytoplasm (Fig-ure 2d). PCNA immunoreactivity was never found in PCs.

Substantia NigraThe investigation was focused on P14 and P21 mice, asloss of DA neurons in weaver becomes only detectable atP1433 and amounts to nearly 50% at P20 and P21.3334

+/+ Mice

Rare ISEL-, c-Jun-, and PCNA-positive apoptotic cells,never exceeding four per section of substantia nigra,were found at both ages, as evidence of developmental

Cell Death in weaver Mice 1633AJP December 1997, Vol. 151, No. 6

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Figure 3. a: c-Jun immunoreactivity in weaver EGL at P7. All apoptotic cells are strongly reactive and tend to form clusters. Magnification, X400. b: Diffuse ISELin the nucleus of a Purkinje cell in weaverEGL at P7. An adjacent Purkinje cell (arrow) is unstained. Magnification, X 1000. C: ISEL fluorescence labeling of weavercerebellum at P7. In addition to clustered apoptotic cells in the EGL, a Purkinje cell shows labeling of both the nucleus and the dendritic tree. Magnification, X400.d: c-Jun immunoreactivity in two Purkinje cells in weaver cerebellum at P7. In addition to the strong nuclear immunoreactivity, the cytoplasm of the two cellsappears condensed and diffusely eosinophilic, as it occurs during the end stages of apoptosis.48 Magnification, X 1000.

PCD.35 All stained nuclei were pyknotic or fragmented.By EM, rare cells with typical apoptotic morphology werefound in both P14 and P21 mice. By EM-ISEL, intensegold labeling was found in their nuclei.

wvANv Mice

The distribution and amount of ISEL-, c-Jun-, andPCNA-positive apoptotic cells did not differ from +/+mice. At the EM level, apoptotic cells were also occasion-ally noted. A far more frequent observation at both ageswas that of degenerating cells showing nonapoptoticfeatures, which were never encountered in +/+ mice.The following changes were noted. First, some cellsshowed dilatation of cisterns of the Golgi apparatus andendoplasmic reticulum (ER). Occasionally, large vacu-oles were seen in continuity with normal ER cisterns orcontaining membranous material. The integrity of cellmembranes and organelles appeared to be maintained,and the synaptic contacts were well preserved. The nu-cleus showed normal chromatin distribution and oftenhad an evident nucleolus (Figure 4, a and b). Second,

other cells showed a more advanced pattern of degen-eration. The nucleus was shrunken, yet the chromatinwas not clumped or marginated. The cytoplasm wasalmost completely occupied by large and small roundvacuoles, which often appeared to coalesce and createlarge void intracytoplasmic spaces. Organelles andplasma membranes appeared intact. In addition, severalmultimembranous bodies were noted. These structureswere made of aggregates of fibrillary, granular, andmembranous material, strongly suggesting that a pro-cess of autophagy was taking place (Figure 4, c and d).

By EM-ISEL, apoptotic cells of wv/wv mice were simi-larly labeled as in +/+ mice (Figure 5a). On the contrary,in the degenerating cells showing vacuolar and autoph-agic changes, nuclei showed no labeling (Figure 5b),indicating that no DNA fragmentation had occurred.

Biochemical Analysis ofDNAWe investigated whether the increased cell death inwv/wv cerebellum is associated with increased DNA frag-

1634 Migheli et alAJP December 1997, Vol. 151, No. 6

Figure 4. Ultrastructural features of nonapoptotic cell death in weaver suLbstantia nigra. a: A cell with initial degenerative changes shows discrete dilatation ofcisterns of the Golgi apparatus and endoplasmic reticulum; the nucleus is indented but does not show condensation of chromatin and has an evident nucleolus.Magnification, X6800. b: Detail of dilated cisterns; mitochondria (arrow) appear intact. Magnification, X 19,500. C: A cell in a more advanced stage of degeneration.The nucleus is shruLnken but shows no chromatin margination. Small and large cytoplasmic vacuoles occupy most of the cytoplasm. In addition, severalmembranous bodies (arrows) can he seen in the cytoplasm. Magnification, X6800. d: Detail of a membranous body. Note the presence of filamentous structuresand of remnants of organelles, indicating an autophagic process. Magnification, X46,300.

Cell Death in weaver Mice 1635AJP December 1997, Vol. 151, No. 6

Figure 5. EM-ISEL in weaver substantia nigra. a: Strong gold labeling, indicating the presence of DNA fragmentation, is seen in the nucleus of an apoptotic cell.Magnification, X34,300. b: On the contrary, only background labeling (arrows) is present in the nucleus of a nonapoptotic cell showing vacuolar degeneration.Magnification, X 58,200.

mentation (ie, laddering) as would be expected for anapoptotic process. We expected that, at any one time,the fraction of total cells undergoing such fragmentationwould be small, and thus we used a modified end-label-ing and autoradiography procedure31 for these studies(Figure 6). Although background smear was present inthe channels from brain tissue, distinct DNA fragmentsreminiscent of the DNA laddering of apoptosis appearedto be present in the cerebellum in a gene-dose-depen-dent fashion (ie, the bands were most evident in wv/wvcerebellum). Similar bands, if present, were not distinct incerebrum. The midbrain (substantia nigra) was difficult toanalyze in this regard. Similar DNA bands may be faintlypresent in this area, but a wv-dose-related phenomenonwas not documented (not shown).

DiscussionThis study demonstrates that diverse cell death pathwaysare activated in the two main targets of the weaver gene,ie, cerebellum and substantia nigra. In particular, weprovide evidence that 1) in cerebellar EGL, an apoptoticprogram with overexpression of c-Jun and PCNA is ac-tive in normal mice and dramatically enhanced in weaver,2) in weaver EGL, the apoptotic program affects granulecell precursors in clusters, 3) weaver PCs are also af-fected, albeit rarely, by apoptotic changes and c-Junactivation, and 4) the degeneration of weaver DA neuronsdoes not involve DNA fragmentation or increased expres-sion of c-Jun and PCNA and is morphologically different

from both necrotic and apoptotic cell death. Furthermore,the combined vacuolar and autophagic changes seen byEM suggest the presence of a hitherto unrecognizedmechanism of cell destruction. The key observations arediscussed below.

In the developing mouse cerebellum, PCD via apopto-sis operates in the EGL during the first two postnatalweeks to adjust the final number of granule cells.32 Themolecular regulation of this physiological death processhas not been previously investigated. Molecules that par-ticipate in apoptotic cell death are commonly divided intoregulatory and effector molecules36; the first mediate thedeath process, whereas the second are directly involvedin the demise of the cell. In this report we have focusedour attention on two regulatory molecules that are knownto mediate neuronal apoptosis. The immediate-earlygene c-jun has been recognized as a necessary gene forapoptosis induced by growth factor deprivation.27'28 Onthe other hand, sustained high levels of several cell cycleregulatory proteins, including PCNA, are found duringapoptosis,29,30'37 and the death process can be blockedwhen cell cycle protein expression is inhibited.38 Thepresent observation of c-Jun and PCNA accumulationstrongly suggests that both proteins are directly involvedin the developmentally regulated apoptotic death of EGLcells in +/+ mice.

In the cerebellum of weaver mutants, recent ISEL andEM data have shown that most EGL cells die via apopto-sis during the first two postnatal weeks.17-19 Thus, thetiming of pathological apoptosis shows a remarkable

1636 Migheli et alAJP December 1997, Vol. 151, No. 6

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Figure 6. Biochemical analysis of DNA from mice of different genotypes. Anautoradiograph of DNA extracted from the tissues indicated was end labeledand resolved electrophoretically. Lane 1, marker, 100-bp ladder; lane 2,+/+ cerebellum (age PlO); lane 3, wv/+ cerebellum (age PlO); lane 4,wv/wvcerebellum (age P10); lane 5, +/+ cerebellum (age P5); lane 6, wv/+cerebellum (age P5); lane 7, wv/wv cerebellum (age P5); lane 8, +/+cerebrum (age P10); lane 9, wv/wv cerebrum (age P10); lane 10, thymus(positive apoptosis control, cells cultured ovemight, 1/100 dilution); lane 11,thymus (as in lane 10 but 1/50 dilution).

overlapping with physiological apoptosis, suggestingthat the two processes might have a common geneticregulation. Here we show that the levels of c-Jun areconsistently elevated in weaver EGL throughout the pe-riod of nerve cell degeneration. This observation extendspreliminary data obtained on P7 mice only.17 It is likelythat the chronic depolarization induced by GIRK2 dys-function is directly responsible for c-Jun up-regulation, asc-jun expression increases during apoptosis in potassi-um/serum-deprived (ie, chronically depolarized) cerebel-lar granule cells.39 In addition, we show that also PCNA isconstantly overexpressed in weaver EGL cells undergo-ing apoptosis. Taken together, these observations sug-gest that the massive cell death of weaver EGL cellsrepresents an exaggeration of the physiological apopto-tic program.

Another novel observation of this study was the pres-ence of apoptotic granule cell precursors in clusters.These were found only in weaver mice and showed thesame morphology and genetic regulation as isolated ap-optotic cells. Clusters of apoptotic cells have been re-

ported to occur in the spinal cord and optic stalk and atthe fusion of cerebral hemispheres during normal neuro-genesis (reviewed in Ref. 40) and have been interpretedas evidence of morphogenetic cell death. Interestingly,they are absent when the apoptotic program is geneti-cally disrupted.40 Therefore, the presence of clusters inweaver cerebellum appears as another proof of alteredexpression of the developmental apoptotic program.

The laminar organization of the PC layer is severelyaltered in the adult weaver cerebellum, and quantitative

analyses have shown that PCs are also reduced in num-ber.22'23 The present observations show, for the first time,direct evidence of PC involvement in the apoptotic cas-cade, with the demonstration of ISEL positivity and in-creased c-Jun expression. The rarity of ISEL-labeled PCsis not unexpected, because of the relatively low numberof PCs and the rapidity of the apoptotic phenomenon (2to 5 minutes for chromatin condensation and cell frag-mentation and 3 hours for apoptotic body phagocytosisand digestion).41 The 10-fold higher frequency of c-Jun-positive PCs compared with ISEL-labeled ones is likely todepend on the earlier activation of c-Jun in the apoptoticcascade, compared with the DNA fragmentation pro-cess.27'42 The negativity of PCNA immunostaining is bestexplained by the post-mitotic nature of PCs. Whether thedeath process in PCs is directly triggered by the faultyweaver gene or is a secondary event needs to be furtherinvestigated. Although several lines of evidence suggestthat PC abnormalities are due to a direct effect of theweaver gene,22'43 at this time it is not clear whether thegene and the protein are expressed in PCs.2-5The substantia nigra is a known site of apoptotic cell

death during both physiological35 and pathological44'45development. The present data obtained in +/+ micedemonstrate that the molecular regulation of develop-mental PCD via apoptosis involves an increased expres-sion of c-Jun and PCNA. In the substantia nigra of weavermice, 35% of DA neurons are lost between P7 and P1433and nearly 50% by P20 to P21. The possibility that,as in the EGL, death of weaver DA neurons represents anenhancement of developmentally regulated apoptosis isunlikely, as the period of maximal expression of develop-mental PCD in the substantia nigra occurs at an earliertime than the onset of cell loss in weaver.35 Even moreimportant, we show here that the number of ISEL-, c-Jun-,and PCNA-positive neurons observed in weaver is thesame as in controls, an observation that suggests that thedevelopmental program of apoptosis in the substantianigra is unaffected by the GIRK2 dysfunction. In agree-ment with this conclusion, the present and previous20'21ultrastructural findings demonstrate that most degenerat-ing weaver DA neurons show extensive vacuolar changesof cytoplasm rather than apoptotic changes. In addition,we report here for the first time the presence of strikingmembrane and organelle preservation and the cytoplas-mic accumulation of several multimembranous bodies.The latter change indicates an ongoing process of auto-phagy, ie, sequestration of intracellular components forsubsequent degradation into secondary lysosomes.46'47To our knowledge, a combination of autophagic andvacuolar changes has not been previously reported tooccur in developmental or pathological cell death.46

The degeneration affecting weaver substantia nigra islikely to represent not only a morphological alternative toapoptosis but also a process with a profoundly differentgenetic regulation. In fact, it does not involve increasedexpression of c-Jun and PCNA. Moreover, our observa-tions using the ISEL technique at the EM level clearlyshow that DNA fragmentation is restricted to DA cellsundergoing apoptosis and is absent in the vacuolatedcells, thus providing morphological evidence that a typ-

Cell Death in weaver Mice 1637AJP December 1997, Vol. 151, No. 6

ical biochemical change of apoptosis, ie, the endonu-cleolytic cleavage of DNA, is not involved in the degen-erative process.The nature of cell death in weaver substantia nigra is

unclear. Although the vacuolar changes might be relatedto a passive process of osmotic lysis due to failure ofionic pumps (ie, necrosis),48 the combined presence ofautophagic changes and the integrity of organelles andmembranes rule out this possibility and suggest thatsome type of active cell death alternative to apoptosis istaking place. The degenerative changes of DA neuronsmight be directly related to the chronic depolarizationinduced by GIRK2 channel dysfunction. In fact, slowexcitotoxic injury induces the formation of vacuolarchanges that are not accompanied by membrane disin-tegration or apoptotic fragmentation of DNA.49 On theother hand, an association between membrane depolar-ization and the autophagic changes that occur in certaintypes of developmental PCD has been reported.50 Stud-ies on cultured DA neurons are needed to confirm thispossibility.

It has been hypothesized that the degree of differenti-ation of target cells might play a key role in the inductionof an apoptotic versus a nonapoptotic death pathway.21The weaver EGL cells start dying by apoptosis shortlyafter their last cell division, before differentiating, migrat-ing to form the internal granule cell layer, and connectingwith PCs.51 Indeed, the time of exit from the cell cyclemay be considered as a period of increased susceptibil-ity to apoptosis, presumably because cell cycle genes,which may also induce apoptosis,29'3037 38 are stillturned on. On the contrary, in the substantia nigra, theactivity of the weaver gene is lethal only after DA neuronshave been generated, migrated, and settled22,52-54 andare therefore fully differentiated. However, this explana-tion seems unsatisfactory. In fact, apoptosis is not aunique feature of developmental neuronal death, as it hasalso been described in several human neurodegenera-tive diseases such as Alzheimer disease,55 amyotrophiclateral sclerosis,56 and even in the substantia nigra inParkinson disease.57 An alternative possibility is that spe-cific features of substantia nigra neurons, different fromtheir degree of maturation, may favor the activation of anonapoptotic pathway. For example, DA neurons appearto be highly vulnerable to oxidant stress, as both theoxidative metabolism of DA and the iron contained inneuromelanin granules have the potential to generatefree radicals.58 Interestingly, vacuolar changes similar tothose of weaver DA neurons and attributable to increasedintracellular oxidant stress have been described in trans-genic mice expressing mutant Cu, Zn superoxide dis-mutase.59 Furthermore, autophagic degeneration hasbeen recently reported to occur in DA neurons in patientswith Parkinson disease.60 Additional factors such as theinteraction between the various GIRK subunits andGIRK2 isoforms61 may further modify the neuronal sus-ceptibility in the various central nervous system areas.The present findings might be relevant for understandingthe nature of neuronal death in human neurodegenerativediseases. In these conditions, the importance of apopto-sis has been questioned,25 whereas a key role of other

mechanisms, eg, autophagy, in the nerve cell degener-ation, has been emphasized.62

AcknowledgmentsWe thank Anna Dutto, Constance Alyea, Deborah Lucas,Rose Funkhouser, Yue Feng, Carmen Stauss, and Taty-ana Verina for technical help.

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