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From the Department of Neuroscience,*
Laboratory of
Neuropathology, University of Turin, Turin, Italy; and the Department
of Pathology and Laboratory Medicine,
Laboratory of Cellular and Molecular Neuropathology, Indiana University
School of Medicine, Indianapolis, Indiana
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Introduction |
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Why EGL cells are selectively prone to undergoing massive apoptosis is unclear. Based on similarities in timing and genetic regulation between girk2wv-dependent cell death and naturally occurring cell death in the developing cerebellum,16,18 it is likely that a disruption of selective developmental features of EGL cells caused by GIRK2 channel malfunction may lead to an increased susceptibility to apoptosis. It has been hypothesized that the weaver gene acts on EGL cells after their exit from cell cycle and before their migration from the EGL;19 accordingly, failure of neuronal migration and axonogenesis would be the primary consequences of the mutation.1,19-23 EGL cells would thus be prevented from responding to appropriate environmental cues, a situation that may eventually lead to apoptosis.24,25
In this paper we have considered an alternative hypothesis, namely, that the girk2wv gene mutation produces abnormalities of control of the cell cycle in EGL cells. This abnormality may be of fundamental importance for the onset of neurodegeneration and apoptosis and may precede further developmental events such as migration and axonogenesis. Cell proliferation and apoptosis are closely related phenomena, as they share several regulatory mechanisms, and any perturbation of the cell cycle control may greatly influence the cell's susceptibility to apoptosis.26,27 For example, increased expression of cell cycle genes and proteins is found during apoptosis28-31 and rescue from death occurs if expression of cell cycle proteins is inhibited.31-34 Furthermore, apoptosis-related proteins have been recently shown to modulate cell cycle protein activation during apoptosis.31
To clarify whether abnormalities of the cell cycle regulation are present in the weaver cerebellum, we have studied the expression of the proliferating cell nuclear antigen (PCNA), a DNA polymerase subunit which is required for S-phase DNA synthesis and is widely expressed during the cell cycle from late G1 to late G2/early M phase.35 To define more precisely any perturbation of cell cycle progression, we have investigated the expression of cyclin D (a marker of early and mid-G1 phase), cyclin-dependent kinase (Cdk) -4 (involved in G1-to-S progression but expressed throughout the cell cycle), cyclin A (a marker of G2-to-M transition),36 and the Cdk inhibitor p27/kip1 (a negative regulator of G1 progression).37 Finally, data related to expression of cell cycle proteins have been compared with those obtained using the in situ end-labeling (ISEL) technique for detecting DNA damage and apoptosis-related DNA fragmentation.38
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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weaver mutant and wild-type mice were obtained from a colony established at Indiana University Medical Center from heterozygous weaver mice that originated from Jackson Laboratory (Bar Harbor, ME). Mutant and wild-type mice are maintained on a B6CBA-Aw-J/A hybrid stock. Genotype analysis was performed as described.39 Twenty-four mice were used for morphological studies, eight each of three genotypes (+/+, wv/+, wv/wv) were divided into four age groups (P7, P11, P14, P21). For biochemical studies, eight mice, four each from the homozygous genotypes +/+ and wv/wv, were divided into two age groups, P7 and P14. We did not study mice younger than P7, because until this age active proliferation occurs throughout the entire thickness of the EGL in all three genotypes,19 and differences in the expression of proliferation markers would have been negligible.
Tissue Preparation for Morphology Studies
Mice were anesthetized with sodium pentobarbital (50 mg/kg i.p.) and perfused transcardially with 10 ml of normal saline and subsequently with 60–120 ml of 4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.2. Brains were removed and postfixed in the same fixative for 30–60 minutes. Tissues were dehydrated in graded ethanols, cleared in xylene, and embedded in paraffin. Midsagittal sections of cerebrum and cerebellum and transverse sections of midbrain were cut at 6 µm and mounted on poly-L-lysine-coated slides.
Antibodies
Anti-PCNA is a mouse monoclonal antibody against rat PCNA (clone PC10, Dako, Glostrup, Denmark) that reacts with PCNA from yeast, insects, and vertebrates. Anti-cyclin D1 is a rabbit polyclonal antibody against mouse cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA) that is also reactive, to a lesser extent, with cyclin D2. Anti-Cdk4 is a rabbit polyclonal antibody against mouse Cdk4 (Santa Cruz). Anti-cyclin A is a rabbit polyclonal antibody against mouse cyclin A (Santa Cruz). Anti-p27 is a rabbit polyclonal antibody against human p27 (Santa Cruz) that is also mouse and rat reactive.
Recognition of dopaminergic neurons in the substantia nigra was accomplished using an antiserum raised in rabbits against bovine tyrosine hydroxylase (Eugene Tech, Allendale, NJ). Whenever feasible, specificity of the immune reaction was tested by incubating the antibodies with the respective immunizing peptides before immunostaining.
Immunohistochemistry
Deparaffinized sections were soaked in 3% hydrogen peroxide to block endogenous peroxidase activity. Primary antibodies were applied overnight at 4°C at the following dilutions: anti-PCNA, 1/30,000; anti-cyclin D and -Cdk4, 1/2000; anti-cyclin A and -p27, 1/200. Before immunostaining, the sections were placed in 0.01 mol/L citrate buffer, pH 6, and heated in a microwave oven (Whirlpool) at 750 W until boiling and then at 350 W for 15 minutes. Immune reactions were revealed with the streptavidin biotin complex technique, except for PCNA, which was revealed with the peroxidase-antiperoxidase technique. Cobalt chloride-intensified diaminobenzidine was used as peroxidase substrate. Sections were counterstained with hematoxylin, dehydrated, and mounted in Permount.
In all experiments, the immunoreactivity of proliferating cells in the periventricular matrix served as an internal control of the reaction.
Western Blot Analysis
Cerebella from P7 and P14 +/+ and wv/wv mice were snap-frozen and homogenized at 4°C by loose dounce in 200 µl of RIPA buffer (20 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 0.1% sodium dodecyl sulfate, 1%Triton X-100, 5 mmol/L EDTA) in the presence of a protease inhibitor cocktail. The homogenates were cleared by centrifugation for 15 minutes at 15,000 rpm, sonicated for 15 seconds, and total protein in the extracts was determined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed by loading equal protein amounts (25 µg) for each sample into 12% acrylamide/bis-acrylamide gel.40 The proteins were transferred to nitro-cellulose and blots were incubated with the primary antibodies. Immune complexes were visualized by the enhanced chemiluminescence Western blotting analysis system (Amersham Pharmacia Biotech).
To quantify cell cycle protein immunoreactivities, films were scanned using a Scanjet 4C (Hewlett-Packard, Tokyo) scanner. Densitometric analysis was performed using Image ProPlus (Media Cybernetics, Silver Spring, MD) software. Protein levels were expressed as relative optical density measurements, determined by comparing the density of the immunoreactive bands in the same blot.
ISEL
ISEL was performed as previously described.16 Briefly, deparaffinized sections were treated with 0.5–2.5 µg/ml of proteinase K (Sigma) in Tris-buffered saline (TBS) for 15 minutes at room temperature. After rinsing in terminal deoxynucleotidyl transferase (TdT) buffer (25 mmol/L Tris/HCl, 200 mmol/L sodium cacodylate, 5 mmol/L cobalt chloride), the sections were incubated with the labeling mix (20 U of TdT (Boehringer Mannheim) and 1 nmol of fluorescein-11-dUTP (Boehringer Mannheim) in 100 µl of TdT buffer) for 120 minutes at 37°C. After rinsing in 300 mmol/L sodium chloride/30 mmol/L sodium citrate, the sections were incubated with anti-fluorescein sheep antibody conjugated with peroxidase (Boehringer Mannheim), diluted 1/500 in TBS, for 30 minutes at 37°C. The reaction was revealed with cobalt chloride-intensified DAB. Sections were counterstained with hematoxylin. Control of the reaction was performed by omitting TdT from the labeling mix.
Quantification of Mitotic Cells
Cell counts were made from 5 to 8 cerebellar sections for each animal, using an oil-immersion objective at 1000x magnification. On each section, six areas (0.02 mm2 each) containing the whole thickness of the EGL were chosen, so that the anterior-to-posterior axis of the cerebellum was equally sampled. The number of EGL cells per area and the number of mitotic figures per area were counted. The area values were added together and a mean value/section was determined. For each section, a mitotic index (MI) was expressed as the percentage of mitotic cells/number of EGL cells, and a mean MI was calculated for each genotype and age group. Statistical analysis was performed using one-way analysis of variance and a Student's t-test.
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PCNA
In all preparations studied, the immunoreactivity for PCNA was exclusively located in the nucleus. In the EGL, the staining appeared most frequently as diffuse or less frequently as punctate, without a clear difference in the proportion of the two patterns among the three genotypes. In the subependymal matrix, the diffuse pattern was predominant, whereas a punctate pattern was less frequently seen. Differences in the pattern of staining among the genotypes are described below.
In +/+ mice at P7, the EGL was 6 to 10 cells thick and could be grossly
divided into a superficial zone, formed by undifferentiated round
cells, and a deeper zone, formed by elongated cells about to migrate
toward the internal granule cell layer.41
Mitoses and rare
pyknotic nuclei were mainly found in the outer EGL. Strong PCNA
immunoreactivity was found in nuclei of the superficial zone, with a
progressive reduction of the labeling in the deeper zone (Figure 1A)
. Cells at the interface between EGL
and Purkinje cell layer were PCNA-negative. In the Purkinje and
internal granule cell layers, numerous cells, that we interpreted as
proliferating astrocytes on the basis of previous
studies,30,41
were PCNA-positive. At P11, the EGL was
reduced to 2–3 layers of PCNA-positive cells, while most migrating
cells across the developing molecular layer and mature granule cells
were PCNA-negative. At P14, the EGL was reduced to a thin, almost
single cell layer, while the granule cell layer was already well
developed. Most EGL cells were still PCNA-positive (Figure 1D)
,
indicating their proliferative status, in agreement with previous
autoradiography data.41
At P21, the EGL had completely
disappeared, while the granule cell layer had reached its full
thickness and contained occasional PCNA-positive glial cell. (Figure 1G)
.
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. Two well
demarcated zones were still present at P11 and to a minor extent at P14
(Figure 1E)
. No PCNA-labeled granule cells were found in the
hypercellular molecular layer or in the granule cell layer. At P21, the
EGL had largely disappeared along the cerebellar surface except for
occasional patches of 3–4 layers of cells. In these areas, PCNA
immunoreactivity was completely absent, indicating that these cells had
already exited the cell cycle (Figure 1H)
.
In wv/wv mice at P7, the EGL was primarily formed by round,
undifferentiated cells.1
Mitotic figures were scattered in
the whole EGL, whereas clusters of pyknotic nuclei16,19
were mostly located in the inner portion of the EGL. By PCNA
immunohistochemistry, virtually all cells throughout the entire EGL
were strongly stained (Figure 1C)
. Progressive reduction in thickness
of the EGL occurred in P11 and P14 mice; however, PCNA immunoreactivity
was still present in all cells of the EGL (Figure 1F)
. At P21, the EGL
was composed of 1–3 layers of cells. Contrary to wv/+ mice,
cells of the EGL in wv/wv mice were still
PCNA-immunoreactive, indicating that these cells had never exited the
cell cycle (Figure 1I)
.
Other Cell Cycle Markers
An immunostaining pattern consistent with that described for PCNA
was found with the antibody to Cdk4; as a matter of fact, most cells in
the PCNA-labeled compartments were also Cdk4-positive. Antibodies to
cyclin D and cyclin A stained a major subset of cells in these
compartments. The specificities in the pattern of expression for each
marker are likely due to the different temporal appearance of the
proteins during the cell cycle: Cdk4 and PCNA levels remain elevated
for most of the cell cycle, whereas cyclin D and cyclin A have a short
half-life.36
At P7-P11, immunoreactive cells in +/+ mice
(Figure 2, A and D)
and in
wv/+ mice (Figure 2, B and E)
were found in the outer
proliferative zone of the EGL, while the inner postproliferative zone
was free of labeling. On the contrary, labeled cells in
wv/wv mice were distributed throughout the entire EGL
(Figure 2, C and F)
. At P14, labeled cells were rare in +/+ mice and
limited to the outermost zone of the EGL in wv/+ mice, while
they were numerous in wv/wv mice. At P21, all cells forming
patches in the EGL of wv/+ mice were unlabeled (Figure 3A)
, whereas the majority of EGL cells in
wv/wv mice were strongly labeled (Figure 3B)
.
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. In wv/wv mice, all cells
of the EGL were constantly p27-negative at all ages, further proof that
these cells had never exited the cell cycle (Figure 2I)
. Differences in
the staining pattern were particularly impressive at P21. The p27
labeling was diffusely present in the internal granule cell layer of
+/+ and wv/+ mice and in the patches of residual EGL in
wv/+ mice (Figure 3D)
. In contrast, the EGL cells of
wv/wv mice remained p27-negative (Figure 3E)
. Western Blot Analysis
The expression of PCNA and p27 was evaluated on frozen samples of
whole cerebellum from +/+ and wv/wv mice at P7 and P14
(Figure 4)
. Immunoblotting for PCNA
revealed in all cases a band of 36 kd. At P7, PCNA was present in both
+/+ and wv/wv mice, with a four-fold increase in the latter.
At P14, PCNA was virtually absent in +/+ mice, while it was still
detectable in wv/wv mice, although its levels were reduced
to 10% of those of P7 wv/wv mice. Therefore, results from
Western blot analysis of PCNA expression showed a close correlation
with those of immunohistochemistry.
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Evaluation of the Mitotic Index
The percentage of mitotic cells in the EGL was evaluated on H&E
sections from +/+, wv/+, and wv/wv mice at P7,
P11, P14, and P21. Despite the massive activation of cell cycle
proteins in wv/wv EGL, no change in the mitotic index was
found among the genotypes at all ages studied (Figure 5)
.
|
Cell death in the EGL was examined by ISEL staining as well as by
immunohistochemistry for cell cycle-related proteins on P7-P21 mice of
all three genotypes. ISEL staining was always limited to nuclei with
evident apoptotic features (nuclear pyknosis, fragmentation into
apoptotic bodies). In +/+ and wv/+ mice, scattered (less
than 2% of total EGL cells) ISEL-labeled nuclei were found at P7,
extremely rare at P14, and absent at P21. In wv/wv mice,
ISEL-labeled nuclei were found at all ages and amounted to nearly 10%
of all EGL cells. Most apoptotic nuclei were located in clusters in the
proximity of the Purkinje cell layer (Figure 3C)
.
On sections adjacent to ISEL-stained ones, we investigated whether cell
cycle markers were also expressed by cells showing morphological
changes of apoptosis, a likely reflection of the involvement of cell
cycle proteins to the final stage of the apoptotic cascade. The pattern
of PCNA immunoreactivity in the apoptotic cells closely matched that of
ISEL, revealing a distinct labeling of most apoptotic nuclei in all
genotypes (Figure 1, A and C)
. The intensity of PCNA staining in the
apoptotic cells was constantly higher than that of surrounding
nonapoptotic cells. On the contrary, we could not detect any staining
of apoptotic nuclei using the antibodies to the other cell cycle
markers.
Substantia Nigra
Activation of cell cycle genes occurs during apoptosis of
postmitotic neurons as an abortive attempt to reenter the cell
cycle.26,28,30,32-34
We investigated whether postmitotic
dopaminergic neurons, which are a major target of girk2wv
action, showed aberrant expression of cell cycle markers during the
period of maximal cell loss. Sections of midbrain were double
immunostained for tyrosine hydroxylase and cell cycle markers. However,
no staining of dopaminergic neurons could be detected with any of the
proliferation markers tested. Only occasional ISEL-labeled,
PCNA-positive apoptotic nuclei (1–2/section) were found, with no
differences among the three genotypes (Figure 3F)
. This apoptosis
is a likely evidence of developmentally programmed cell death, as
reported previously.16,42
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Developmental Changes in weaver Cerebellum
In earlier studies, the development of the EGL in weaver mutant mouse had been examined by means of autoradiography20 and mitotic index evaluation.19 These studies showed that the mitotic activity in P1-P8 weaver cerebellum was unchanged in comparison to wild-type mice, leading to the hypothesis that postproliferative events such as failure of neuronal migration and of neurite extension might be the primary consequences of the weaver mutation.1,19-22 After identification of the mutation, the same hypothesis was still supported by Maricich et al,23 who suggested that the girk2 gene might be necessary for proper migration of neurons during development.
Cell Cycle Abnormalities in weaver Cerebellum
Although failure of neuronal migration and axonogenesis may indeed induce an apoptotic response,24 we reasoned that perhaps some earlier event related to regulation of the cell cycle might be disrupted by the GIRK2 malfunction, leading to massive apoptosis. In normal conditions, the cell cycle represents a sequence of highly ordered processes that result in the duplication of a cell. Cell cycle proteins are sequentially activated and inactivated to move the cell through a series of checkpoints,36 and any perturbation of this sequence may lead to growth arrest and death through activation of apoptosis.26,28
Whereas the present evaluation of the mitotic index in mice up to P21 confirms that the weaver mutation apparently does not perturb cellular proliferation, analysis of cell cycle events reveals striking changes in the cerebellum of wv/wv mice as compared to +/+ and wv/+ animals. In wild-type and heterozygous mice, cell cycle proteins are up-regulated in proliferating cells located in the outer zone of the EGL, whereas migrating cells of the inner zone are no longer immunoreactive. These results are in line with previous autoradiography data showing that normal granule cell precursors cease DNA synthesis before differentiating and starting their migration.41 A recent report on PCNA and cyclin D1 expression in the developing rat cerebellum also reached similar conclusions.43
In contrast, several cell cycle abnormalities are present in homozygous weaver mice. First, marked up-regulation of cell cycle proteins occurs throughout the entire EGL at all ages studied, suggesting that weaver EGL cells cannot switch off the cell cycle. This hypothesis is further supported by the observation that p27, a cell-intrinsic timer that arrests the cell cycle and initiates differentiation,37 is not expressed in the weaver EGL. Second, the mitotic activity in the EGL is unchanged, suggesting that persistent activation of cell cycle machinery is unrelated to proliferative events. Third, continuous activation of cell cycle proteins appears to be a prolonged phenomenon, as weaver EGL cells are known to die 2 to 4 days after being generated.20 Fourth, continuous cell cycle activation in wv/wv mice is not due merely to migration delay or to a position effect on cells within the EGL, but is rather an early consequence of the altered GIRK2 channel function. As a matter of fact, EGL cells in wv/+ mice have a delay in migration but exit the cell cycle normally, as shown by the complete absence of cell cycle marker expression and by the strong p27 immunostaining in the cell patches at P21.
Cell Cycle Abnormalities and Apoptosis
Abnormalities in the cell cycle machinery may directly favor the activation of apoptosis.26,27 Furthermore, inability to switch off the cell cycle in wv/wv mice affects cells that are receiving at the same time developmental cues to stop proliferating and start migration/differentiation, as suggested by the observation that neurotrophin and neurotrophin receptor expression is unchanged in the weaver cerebellum.44 Thus, weaver EGL cells would at the same time receive conflicting signals for proliferation and growth arrest/differentiation, a situation that has been postulated to promote apoptosis in neurons.28
In the present model, PCNA was strongly expressed in apoptotic cells as a likely reflection of its other activity in nucleotide excision repair of damaged DNA.45 Lack of staining of apoptotic cells with the other cell cycle markers may be due to the short half-life of these proteins;36 alternatively, it may indicate that they are necessary for the preparation phase, but not for the execution phase of apoptosis.28,30
Ion Channel Malfunction and Cell Cycle Abnormalities
The mechanism through which regulation of the cell cycle is altered by the GIRK2wv channel activity is at present only a matter of speculation. Cell cycle deregulation is unlikely to be a direct consequence of chronic depolarization, as the latter event usually arrests cell cycle progression and promotes differentiation.46,47 Moreover, chronically depolarized cerebellar granule cells show an overall decrease of cell cycle-related proteins before undergoing apoptosis.48 On the other hand, changes in voltage-dependent ion currents occur during the cell cycle, suggesting a close interdependence between the two events.49 Abnormal permeability of the mutated channel to calcium ions might be important, since calcium fluxes are known to modulate progression throughout the cell cycle.50 In vitro studies should better clarify this point.
Lack of Cell Cycle Abnormalities in weaver Substantia Nigra
Dopaminergic neurons in the weaver substantia nigra die through a nonapoptotic mechanism of programmed cell death.16,17 Here we show that this neuronal population does not express cell cycle proteins during the period of maximal neurodegeneration, a finding that has some interesting implications. At the time of degeneration, dopaminergic neurons are postmitotic, fully differentiated cells that have already made synaptic connections with the striatal target cells. Lack of cell cycle protein expression rules out the presence of an abortive mitotic attempt, as has been shown during death of postmitotic neurons in several experimental paradigms in vitro28,32-34 and in vivo,30 as well as in human neurodegenerative disorders.51 Lack of cell cycle reactivation in weaver dopaminergic neurons might simply depend on the nonapoptotic nature of the death process, although cell cycle-independent pathways of neuronal apoptosis have been also described.34 The present observations suggest that the susceptibility to undergo apoptotic versus nonapoptotic cell death might greatly depend on the degree of maturation of a neuron.52 This fact may be relevant for the pathogenesis of neurodegenerative diseases, in which the role of apoptosis has been questioned.53
Conclusion
In conclusion, our data point to a severe alteration of cell cycle regulation in wv/wv EGL cells, a situation that highly increases their susceptibility to undergo massive apoptosis. We suggest that cell cycle perturbation, rather than failure of axonogenesis and migration, is the primary consequence of the mutated GIRK2 channel activity in cerebellum. These observations extend our knowledge on mechanisms of neuronal degeneration and may help in understanding the pathogenesis of human neurological disorders characterized by defective ion channel functioning.54-56
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Acknowledgements |
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Footnotes |
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Supported in part by PHS grants P01 NS 27613 and R01 NS14426 and by European Community Copernicus program grant CIPA-CT93–0210.
Accepted for publication April 11, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-insensitive weaver K+ channels. Science 1996, 272:1950-1953[Abstract]
. Nature 1987, 326:515-517[Medline]
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