Purkinje neuron degeneration in nervous (nr) mutant mice is mediated by a metabolic pathway involving excess tissue plasminogen activator

Abstract

Purkinje neurons (PNs), the central cells in cerebellar circuitry and function, constitute a vulnerable population in many human genetic, malignant, hypoxic, and toxic diseases. In the nervous (nr) mutant mouse, the majority of PNs die in the fourth to fifth postnatal weeks, but the responsible molecules are unknown. We first disclose a remarkable increase in mRNA expression and protein concentration in the nr cerebellum of tissue plasminogen activator (tPA), a gene closely linked to the mapped but as-yet-uncloned nr locus. Evidence that excessive tPA triggers nr PN death was obtained with organotypic slice cultures expressing the nr PN phenotype, in which an inhibitor of tPA led to increased nr PN survival. An antagonist of protein kinase C, a downstream component in the tPA pathway, also increased nr PN survival. Additional downstream targets in the tPA pathway (the mitochondrial voltage-dependent anion channel, brain-derived neurotrophic factor, and neurotrophin 3) were also abnormal, in parallel with the alterations in PN mitochondrial morphology, dendritic growth, and synaptogenesis that culminate in nr PN death and motor incoordination. We thus propose a molecular pathway by which the excessive tPA in nr cerebellum mediates PN degeneration.

Cerebellar Purkinje neuron (PN) degeneration resulting in impaired motor coordination appears to be the final common pathway for a number of genetic, malignant, hypoxic, and toxic disorders (1). The nervous (nr) autosomal recessive mutation, although the first described inherited mouse model of PN degeneration (2), has not yet been cloned, nor has its molecular mode of action been determined. Mitochondria in the nr PN begin to enlarge and round up on postnatal day 9 (P9), with every PN so affected by P15 (3), but with no histochemical abnormalities recognizable by the methods available when these striking abnormalities were analyzed (4). Some mitochondria undergo an unusual type of degeneration in which the outer membrane partially or completely dissolves, occasionally accompanied by focal interruptions of the inner membrane (5). Between P21 and P35, the majority of the PNs degenerate. Motor hyperactivity and ataxia become evident early in the course of PN degeneration. The PN loss in nr mice is not random, especially in the early stages, selectively involving the lateral hemispheres and certain parasagittal bands in the vermis (6). Although a few classes of neurons other than PNs transiently show the same mitochondrial abnormalities, no other neurons actually degenerate in significant numbers, except for retinal photoreceptor cells, which become abnormal and die on a different schedule than PNs (7).

The nr gene was mapped to a 1.4-cM region of mouse chromosome 8, between the D8Rck1 and D8Mit3 markers (8). This region is now known to contain >10 candidate genes, including tissue plasminogen activator (tPA), an extracellular serine protease, that is used clinically in the treatment of acute occlusive arterial lesions in the brain (9, 10) but carries the risk that it can modify neuronal structure and initiate a proteolytic cascade that ultimately mediates degeneration of neurons after CNS injury (11, 12). tPA also can exert other profound and varied neurobiological effects (10, 13), some of which can be counteracted by a naturally produced inhibitor, neuroserpin (14, 15).

To explore molecular mechanisms resulting in the nr PN death, we examined candidate gene coding regions and quantified gene products. Our major finding is that tPA is increased 10-fold in the nr mutant cerebellum and is responsible for PN death either directly or possibly through its effect on downstream modulators: voltage-dependent anion channel (VDAC) and two neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3). These downstream targets of the tPA/plasmin proteolytic system, participating in PN mitochondrial function, dendritic growth, and synaptogenesis, respectively, are abnormal in postnatal developing nr cerebellum at the time the mutant phenotype becomes established.

Results and Discussion

tPA Is Markedly Excessive in nr Cerebellum.

Cerebellar PNs, not quantified previously on the BALB/cByJ congenic background, were stained immunohistochemically with the selective protein marker calbindin and were found to be normal in number and distribution in nr mouse cerebellum up to approximately P20, but, by approximately P35, depletion of PNs was severe and selective in the lateral hemispheres (>70% loss) (Fig. 1A and C), and calbindin protein was significantly decreased (Fig. 1B). Astrocytes (glial fibrillary acidic protein+) were increased in adult nr cerebellar cortex and white matter (Fig. 1 B and D), a common reaction in neurodegenerative settings. Expression of CD68 and activated caspase 3 in young (P14–P20) and adult nr cerebella by immunohistochemistry (data not shown) and Western blot (Fig. 1B) indicated that nr PN death is likely triggered by a molecular mechanism independent of either destructive microglial activation or caspase 3-mediated apoptosis. Like nr mice on the original BALB/cGr strain (2), adult BALB/cByJ congenic nr mice also displayed extensive loss of retinal photoreceptor cells (Fig. 1E). Adult nr mice showed moderate ataxia and poor rotarod performance compared with wild-type (wt) controls (Fig. 1F).

Fig. 1.

Phenotypes of nr mice on the BALB/cByJ congenic background. (A) Cerebellar sections of adult (P60) nr mice were stained with anti-calbindin (calb; red) antibody for PNs and with DAPI (dapi; blue) for cell nuclei (same DAPI staining also used in Figs. 2, 4, and 5). PN number was markedly reduced in the lateral hemispheres of adult mutants. GL, granular layer; ML, molecular layer. Arrows point to the monolayer of PN cell bodies. (B) Western blots show decreased calbindin (calb) protein and increased glial fibrillary acidic protein (gfap) in adult (P60) nr mouse cerebellum but not in young (P16) cerebellum. No change was found in levels of CD68 (cd68) and activated caspase 3 (casp3). (C) PN numbers in cerebellar lateral hemispheres of adult wt and nr mice. Values represent percentage of wt PN number ± SD (n = 6; ∗∗, P < 0.01). (D) Cerebellar sections stained with anti-glial fibrillary acidic protein (gfap; red) antibody show astrocytes to be increased extensively in adult nr cerebellar cortex and white matter (WM) (controls not shown). (E) Eye sections stained with toluidine blue show a marked depletion of cells in the photoreceptor (ph) and bipolar (bi) layers in adult (P60) nr retinas. (F) Rotarod test shows reduced motor function of adult nr mice. Values represent means ± SD of time on the rotating rod (n = 6–8 for each group of three independent tests; ∗∗, P < 0.01).

In an attempt to identify the nr gene, we measured coding regions of candidate genes known through mouse genome mapping to be located in a 1.4-cM segment of mouse chromosome 8, where the nr gene lies (ref. 8 and Fig. 2A). Amino acid substitutions found in Defer-rs1 (P13L and C14A), tPA (A260G), Sfrp1 (A28S and A245R), and Vdac3 (an M insertion at position 40 in cerebellum but not in liver) merely reflect polymorphisms in the coding sequences. We then measured transcriptional activities of candidate genes in this chromosomal segment. To our surprise, RT-PCR revealed that tPA mRNA expression was remarkably up-regulated in cerebellum and testis of young and adult nr mice (Fig. 2B). Also, tPA protein levels were greatly increased in young and adult nr cerebella, as shown in Western blots (Fig. 2C), and in the molecular and granular layers, as shown by immunofluorescence staining (Fig. 2D). To check for tissue specificity, we quantified tPA proteins in wt and nr cerebrum, cerebellum, and liver. In wt mice, the order of tPA protein levels was liver > cerebrum > cerebellum, whereas, in nr mutants, a 1,000% increase was found in cerebellum, and 50–100% increases were found in cerebrum and liver (Fig. 2E).

Fig. 2.

Excessive tPA in nr cerebellum. (A) A schematic diagram locating 12 candidate genes for nr in a segment of mouse chromosome 8. CE, centromeric direction; TE, telomeric direction. (B) Expression of tPA mRNA detected by RT-PCR was remarkably up-regulated, >10-fold, in both young (P16) and adult (P90) nr cerebellum and was up-regulated ≈5-fold in young testis and ≈7-fold in adult testis. (C) Western blots show that levels of tPA protein were extremely elevated in young (P16, ≈10-fold) and adult (P90, >10-fold) nr cerebella. (D) Cerebellar sections stained with anti-tPA (tPA; red) antibody show tPA levels in the molecular and granular layers to be obviously higher in young nr cerebellum than in wt controls. GL, granular layer; ML, molecular layer. (E) Quantification of tPA protein detected by Western blotting in adult liver, cerebrum, and cerebellum. Values represent tPA relative levels (% of wt cerebrum ± SD, n = 4). Comparison between wt and nr: ∗, P < 0.05; ∗∗, P < 0.01.

The best-known action of tPA is to cleave single-chain enzymatically inactive plasminogen to the two-chain serine protease plasmin which, in turn, can cleave several extracellular substrates (10). tPA itself also has direct neurobiological roles. It is a presynaptic component in some cell types (13) and is thought to influence synaptogenesis, long-term potentiation, and synaptic alterations in disease states (10). The action of tPA at synapses is based on its binding to the NMDA receptor, which increases calcium influx and augments the severity of NMDA-mediated excitotoxic lesions (16). Binding to an NMDA subunit also controls ethanol withdrawal seizures (17).

Excessive tPA Is Responsible for nr Cerebellar PN Death in Vitro.

We next adopted a cerebellar organotypic slice culture system (18, 19) to explore whether excess tPA has a role in nr PN death. The wt cerebellar cortex in slice cultures retained essentially normal lamination (data not shown).

tPA is an agonist of protein kinase C (PKC) (20), and inhibition of PKC can diminish PN death in organotypic cultures initiated at P3 (18). We found that a PKC antagonist, Gö 6976 (2 μM), greatly inhibited PN death in nr cerebellar slice cultures initiated at P9 (P < 0.05) (Fig. 3A). We then treated nr cerebellar slices with a tPA-specific inhibitor, tPA-STOP. At low (0.04 μM) and intermediate (0.2 μM) doses, tPA-STOP promoted nr PN survival (Fig. 3B). Interestingly, and somewhat counterintuitively, when wt cerebellar slices were similarly treated with increasing doses of tPA-STOP (0, 0.04, 0.2, and 1 μM), a dose-dependent reduction in wt PN survival was detected (Fig. 3B). These apparently conflicting results appear to reflect both the pivotal role of normal levels of tPA activity in normal PN viability and the very different baseline levels of tPA activity in wt vs. nr cerebella. Depression of tPA activity to below the value found in wt mice is harmful to PN survival, whereas a decrease from the abnormally high values toward normal in the inhibitor-treated nr cerebellum is protective. By analyzing enzymatic activity of tPA and counting PN number in tPA-supplemented and tPA inhibitor-treated cerebellar slices, we established a curvilinear regressive equation, Y = a + b₁log X + b₂log X², that describes the logarithmic–parabolic relationship between tPA activity (X) and PN viability (Y) in cerebellar slice cultures (Fig. 3B).

Fig. 3.

Excessive tPA is responsible for nr PN death in vitro. (A) The PKC antagonist Gö 6976 protected PNs in cerebellar slice cultures. (a) Gö 6976 significantly increased PN (calb; red) survival in wt and nr cerebellar slices. (b) PN number and distribution in wt and nr slice cultures, each without (−) or with (+) Gö 6976, were classified by inspection of stained cultures into types I–IV, as described in ref. 19 and Materials and Methods. Comparisons are pooled from two independent experiments: P < 0.05 for wt − Gö 6976 vs. wt + Gö 6976 and P < 0.01 for nr − Gö 6976 vs. nr + Gö 6976. (B) Relationship between tPA activity and PN viability in cerebellar slice cultures. (a) Dose–response curves with the tPA inhibitor tPA-STOP. All doses (0.04, 0.2, and 1 μM) of tPA-STOP increased PN death in wt slices but promoted PN survival in nr slices. (b) A logarithmic–parabolic relationship between tPA activity and PN viability in cerebellar slice cultures was fitted with a curvilinear regressive equation.

tPA gene expression is induced during cerebellar development at the stage when granule cells migrate inward from the external granular layer (21). This major developmental event is retarded in tPA-deficient mice (22). Lack of tPA activity also impairs cerebellar motor learning (23), perhaps reflecting a more general role of tPA in activity-dependent synaptic plasticity (24). Consistent with our in vitro study of tPA activity and PN survival described above, our in vivo study, correlating high cerebellar tPA with motor impairment in the nr mutant mouse and coupled with data reported by Seeds et al. (23), also suggests that either too high or too low tPA may impair motor function.

Unusual VDAC and PN Mitochondria Coexist with Excessive tPA.

To further explore how excess tPA triggers nr PN death, we measured a downstream target, VDAC. Isoforms 1–3 of VDAC, encoded by three genes (25), one of which (Vdac3) lies near tPA and nr on chromosome 8 (Fig. 2A), together form large voltage-gated pores in the mitochondrial outer membrane (26). These pores not only provide a pathway for the passive in-and-out diffusion of metabolites such as ATP but also may have a role in synaptic transmission (27) and apoptotic cell death (28, 29). The tPA/plasmin system contains five binding segments called “kringles,” one of which (kringle 5) can specifically bind brain mitochondrial VDAC to induce partial closure of the VDAC channel (30) and thus interfere with mitochondria-related regulation of intracellular calcium and pH (31). We found that, in P16 nr cerebellum, before PN death and coincident with the excess tPA, the concentrations of VDAC protein and cytochrome c oxidase subunit IV, a key respiratory chain component in the mitochondrial inner membrane, were high in PN somata but decreased in PN dendrites (data not shown). The exceptionally enlarged, rounded mitochondria in P16 nr PNs also confirmed the earlier results that had been obtained in BALB/cGr coisogenic mice (2, 3). These changes may cause alterations in ion fluxes and cytochrome c distribution that are incompatible with cell survival. However, more substantial evidence is to be sought about whether nr PN mitochondria are actually abnormal in function; they may be “supernormal,” trying to counteract the disease process and reverting to a normal morphology in those PNs that survive through the “death window” time period.

Abnormal Neurotrophins and PN Dendrites Occur with Excessive tPA.

We examined two downstream neurotrophin targets of the tPA/plasmin proteolytic system: BDNF and NT3. An effect of tPA on conversion of precursors to active neurotrophins is thought to account for tPA-mediated synaptic plasticity (32) and neuronal survival (33). In young (P14) nr mutant cerebellum, coincident with the extremely high tPA level, total BDNF [precursor of BDNF (proBDNF) plus mature BDNF (mBDNF)] was significantly reduced (Fig. 4A), despite the increased protease cleavage of proBDNF into mature BDNF (Fig. 4D). These data reflect an increased degradation of BDNF and its precursor in the mutant cerebellum. In the PNs themselves, we found an obviously decreased NT3 expression in P14 nr PN somata compared with wt controls, both by immunohistochemistry (Fig. 4B) and Western blotting (Fig. 4D). Tyrosine receptor kinase B (trkB) at the interface between the external granular and molecular layers and tyrosine receptor kinase C (trkC) in PN somata were unaltered (Fig. 4 C and D). The neurotrophin family participates in regulation of neuronal morphogenesis and maintenance (34). In BDNF^−/− mice, PN dendrites are abnormal, and paired-pulse facilitation, a form of short-term plasticity in parallel fiber–PN synapses, is significantly decreased (35). The growth and maturation of PN dendritic arbors may be influenced by NT3 activation of trkC receptors distributed within developing dendrites (36). PNs are also reported to experience a critical switch from BDNF dependence to NT3 dependence during early postnatal development (37).

Fig. 4.

Neurotrophin changes in P14 nr cerebellum. (A) Cerebellar sections were costained with anti-calbindin (calb; red) and anti-BDNF (bdnf; green) antibodies. BDNF was expressed mainly in the zone (dotted ellipse) between the external granular layer (EGL) and molecular layer (ML) of young wt cerebellum but was markedly decreased in young nr cerebellum. (B) Cerebellar sections were costained with anti-calbindin (calb; red) and anti-NT3 (nt3; green) antibodies. NT3 was expressed mainly in PN somata (ellipse) in young wt cerebellum but was almost absent in young nr cerebellum. (C) No changes in neurotrophin receptors (only wt data shown). (a) Cerebellar sections were costained with anti-calbindin (calb; red) and anti-trkB (green) antibodies. trkB was expressed mainly in the zone (dotted ellipse) between the external granular and molecular layers of young wt and nr cerebella. (b) Cerebellar sections were costained with anti-calbindin (calb; red) and anti-trkC (green) antibodies. trkC was prominent in PNs (ellipse) of young wt and nr cerebella. (D) Neurotrophins and receptors. Western blots show an increased protease cleavage of proBDNF (30 kDa) into mature BDNF (mBDNF) (13 kDa) and decreased levels of total BDNF, proBDNF, and NT3 in young nr cerebella compared with wt controls. No changes were found in trkB and trkC levels in young nr cerebella.

We next examined P14 nr PN dendrites and synaptic markers that might be affected by abnormal neurotrophin concentrations. PN dendritic branchlets and postsynaptic density protein 95 (Psd 95) were significantly reduced in the molecular layer of young nr cerebellum (Fig. 5A). Psd 95 is normally prominent in the postsynaptic sites on young PN dendrites receiving inputs from climbing and parallel fibers and is thought to participate in rapid formation and remodeling of postsynaptic densities (38). In contrast, a relatively normal distribution of synapsin I, a presynaptic (afferent axon) marker, was observed in the molecular layer of young nr cerebellum (Fig. 5A). Thus, the nr mutation appears to affect components of young PNs exclusively, sparing other neuronal types in the cerebellar cortex.

Fig. 5.

Changes in PN dendrites, synapses, and axons in nr cerebella with excessive tPA. (A) P16 cerebellar sections. (a) nr PNs somata were present, but their dendrites (calb; red) were reduced in the molecular layer (ML). (b) Cerebellar sections stained with anti-Psd 95 antibody (psd95; red) show decreased expression of the PN dendrite postsynaptic marker in the molecular layer. (c) A relatively normal distribution of a presynaptic axonal marker in the molecular layer of the nr cerebellum in a section stained with anti-synapsin I (syn; red) antibody. GL, granular layer. (B) Adult cerebellar sections. (a) Psd 95 immunostaining (psd95; red) shows abnormal postsynaptic connections (above yellow curve) between basket cell axons and PN somata in nr cerebellum compared with wt control even in areas such as this, where PNs persisted in the mutant cerebellum. (b) wt PNs extended long axons (calb; red) through white matter (WM) toward the deep nuclei, but few axons were seen in the nr cerebellum, even in areas where PNs persisted.

In adult wt mouse cerebellum, Psd 95 was detected mainly in postsynaptic sites on PN somata, where basket cell axons terminate. In contrast, in adult nr cerebellum, even at sites where there were residual PNs, Psd 95 had totally disappeared (Fig. 5B). Calbindin-immunostained adult wt cerebellum clearly showed PN axons with normal trajectories extending through the internal granular layer and white matter to synapses on neurons in the cerebellar deep nuclei, whereas, in adult nr mice, even where PN somata persist, immunostained axons are rare (Fig. 5B).

Excessive tPA may mediate the nr cerebellar phenotypes through at least two types of downstream modulators, VDAC and neurotrophins, as diagramed in Fig. 6. Moreover, the tPA action on PN survival may not be limited to the nr mutant case. When a double mutant was created from GluRδ2^Lc mutant (GluRδ2^Lc/+) and tPA knockout mice (tPA^−/−), the elimination of tPA was found to delay the cerebellar PN death expected from the dominant GluRδ2^Lc/+ mutation (39). A closer examination of tPA and neuroserpin levels, as well as other components upstream and downstream from tPA, at various ages in different PN mouse mutants and human PN-selective diseases is clearly warranted.

Fig. 6.

Schematic of a proposed molecular mechanism leading to nr PN death. Arrows suggest signaling pathways through which excessive tPA in the nr cerebellum, through direct action or by acting on downstream targets VDAC and BDNF, can mediate nr PN degeneration. A dashed long arrow (right) represents as-yet-unrecognized pathways.

In conclusion, the present study discloses an underlying molecular mechanism: excess tPA triggering neuronal death at late developmental stages, which provide insights into crucial roles of tPA in CNS disorders and clues toward therapeutic strategies.

Materials and Methods

Animals.

The nr mutation originally occurred in the BALB/cGr strain (2), and we have maintained it congenic with the almost identical BALB/cByJ strain for >15 backcross generations. The nr mice were identified in segregating litters by hyperactivity and ataxia after P21, which was confirmed by histological examination of the retina (7). At P14–P20, PN mitochondria were checked in 1-μm Epon sections, as described below. Use of animals was in accordance with National Institutes of Health-approved institutional guidelines established by Harvard Medical School. All efforts were made to minimize the number of animals used and animal suffering.

Antibodies.

All antibodies were obtained from Abcam, Inc. (Cambridge, MA), Chemicon, Santa Cruz Biotechnology, or Sigma. Primary antibodies were each titrated for optimum staining: 1:100–200 dilutions for immunohistochemistry and 1:500–1,000 dilutions for Western blotting.

Histological Analysis and Immunofluorescence Staining.

Tissues were fixed by perfusion of deeply anesthetized mice through the heart with freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Cerebellar coronal cryosections (10 μm) were stained with hematoxylin/eosin or neutral red. Postosmicated, Epon-embedded 1-μm cerebellar sections were stained with alkaline toluidine blue to visualize mitochondria in PNs. Glycol methacrylate-embedded 3-μm sections of mouse eyes stained with toluidine blue were scored for retinal photoreceptor cell degeneration (7). For immunostaining, slide-mounted sections from slice cultures or cerebella were incubated in a blocking solution of 3% goat serum and 0.3% Triton X-100 at room temperature for 30 min and then reacted with specific primary antibodies at 4°C for 16–48 h. Slides were washed three times and stained by secondary antibodies conjugated with fluorescent FITC or Cy3 at 4°C overnight. After washing, the section was sealed with mounting medium containing DAPI to stain cell nuclei and visualized by fluorescence (Nikon Eclipse E600) and/or confocal (Zeiss Axiovert 100M) microscopy.

Behavioral Test.

Adult P60 mice were trained, three trials per day for 3 days, to hold onto a rotating rod (Economex Instruments, Columbus, OH) at 10, 20, and 30 rpm for the first, second, and third days, respectively. Then motor coordination and sustainability were assessed by the time to falling off the rod rotating at 30 rpm, averaged from three successive trials.

RT-PCR and Nucleotide Sequencing.

Two-step RT-PCR was performed to maximize uniformity of PCR templates for all reactions. Up to 2 μg of total RNA from each sample was reverse transcribed into the first-strand cDNA in 20 μl with oligo(dT) primers and SuperScript first-strand synthesis system (Invitrogen). PCR amplification of coding sequences of selected genes was performed with gene-specific oligonucleotide primers and ReddyMix PCR Master Mix (ABgene, Surrey, U.K.). PCR products were verified with agarose gel and cleaned up with ExoSAP-IT (USB Corp.). The coding sequences were measured by the Beth Israel Deaconess Medical Center Sequencing Facility. Sequence analysis was performed with gene runner and blast software.

Western Blot.

Denatured proteins were separated with SDS/12% PAGE (Bio-Rad) and transferred from gel to a nitrocellulose membrane that was then blocked with 5% fat-free milk at room temperature for 30 min and incubated with primary antibodies at 4°C overnight. After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, washed, and then reacted with enhanced chemiluminescence Western blot detection reagent (Amersham Pharmacia) for 2 min. The membrane was covered with plastic wrap, and a high-performance autoradiography film was taped onto the wrapped membrane in a dark room for 2–30 sec and then developed to visualize the bound antibody.

Cerebellar Organotypic Slice Cultures.

The method for culturing organotypic slices has been described by Samoilova et al. (40). Sagittal 400-μm slices obtained from P9 mouse cerebella were cultured in 30-mm dishes containing sterilized porous membrane inserts (Millipore) and 1 ml culture medium composed of 50% Eagle's minimal essential medium with glutamine, 25% Hanks' balanced salt solution, and 25% horse serum with 20 mM Hepes buffer and 6.5 mg/ml d-glucose. Slice cultures were kept at 35°C in 5% CO₂ for 17 days, and the medium was exchanged (50% of volume) three times per week. Some slices were treated with a PKC antagonist (Gö 6976) (Calbiochem) or a tPA inhibitor (tPA-STOP) (American Diagnostica, Greenwich, CT). The slices were evaluated semiquantitatively after calbindin immunostaining and DAPI counterstaining in terms of PN number and distribution with the following scale (19): type I, no clusters with >100 PNs; type II, one cluster of 100–200 PNs; type III, two clusters of 100–200 PNs or one cluster of 200–300 PNs; and type IV, at least three clusters of 100 PNs singly or grouped into one or more clusters totaling ≥300 PNs.

Fibrinolytic Assay for tPA Activity.

Cerebellar slice cultures were lysed in 0.1 M Tris·HCl (pH 8.1)/0.1% Tween 80/0.25% Triton X-100 and incubated with a plasmin-sensitive chromogenic substrate (S-2251; Chromogenix, Molndal, Sweden) and 0.5 μM plasminogen at 25°C. The ability of tPA to convert plasminogen to plasmin was monitored by measuring the absorbance at 405 nm at different time points with a microplate reader (Bio-Tek, Burlington, VT). Total protein content was determined from an aliquot of each sample with the bicinchoninic acid protein assay kit (Pierce), and tPA activities in each sample were normalized. Relative activities of tPA in different samples were based on the tPA activity of untreated wt cerebellar slices.

Quantification and Statistic Analysis.

The number and proportion of calbindin-stained PNs were counted in slice cultures or in a series of representative serially cut tissue sections. quantity i software was used for the quantitative analyses of cDNA and protein bands on gel or film. Comparison between groups, either paired or unpaired as required, was performed with (i) the Student t test for the measurement data, (ii) the χ² test for enumeration data, (iii) linear regression analysis for correlation data, and (iv) the ridit analysis for ranked data. Results are presented as means ± SD, and differences were considered significant at P < 0.05 or very significant at P < 0.01.

Abbreviations

BDNF

brain-derived neurotrophic factor

NT3

neurotrophin 3

nr

nervous mutation

PKC

protein kinase C

PN

Purkinje neuron

Pn

postnatal day n

proBDNF

precursor to BDNF

Psd 95

postsynaptic density protein 95

tPA

tissue plasminogen activator

trkB

tyrosine receptor kinase B

trkC

tyrosine receptor kinase C

VDAC

voltage-dependent anion channel

wt

wild-type.