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BDNF accelerates gene expression in cultured cerebellar granule neurons
Developmental Brain Research 105 Ž1998. 277–286
Research report
BDNF accelerates gene expression in cultured cerebellar granule neurons Xi Lin, Hong Cui, Robert F. Bulleit
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Department of Pharmacology, UniÕersity of Maryland School of Medicine, Baltimore, MD 21201, USA Accepted 21 October 1997
Abstract This study reports that in purified cultures of postnatal cerebellar granule cells, BDNF significantly accelerated GABA A receptor a 6 subunit ŽGABA A a 6. mRNA expression, a marker for terminally differentiated cerebellar granule neurons, and also accelerated p21cip1 expression. p21cip1 is a general cyclin-dependent kinase ŽCdk. inhibitor that can inhibit progression through the cell cycle. Alternatively, the expression of p27 kip1, another Cdk inhibitor closely related to p21cip1, is not modified by BDNF. In cultured granule cells, the increase in p21cip1 expression induced by BDNF occurred after dividing granule cells had left the cell cycle and thus was not required to direct granule neuron precursors out of the cell cycle. p21cip1 may have an alternative function during granule neuron terminal differentiation, separate from its ability to regulate cell cycle exit. This report shows that, in vitro, BDNF accelerates granule cell gene expression and may thus modulate cerebellar granule cell differentiation. q 1998 Elsevier Science B.V. Keywords: BDNF; Neurotrophin; IGF-I; Differentiation; Gene expression; Mouse
1. Introduction Brain-derived neurotrophic factor ŽBDNF. is a member of the neurotrophin family of trophic factors. Expression of BDNF and its high affinity cognate receptor ŽTrkB. occurs in various regions of the developing and adult brain w7,14,43,48x. This pattern of expression suggests a number of potential roles for BDNF in neuronal survival and differentiation. As a neurotrophic factor, BDNF can promote the survival of various types of CNS neurons w2,24,24,26,28x and may also exert an influence on neuronal differentiation w2,25,26x. Expression of both BDNF and TrkB occurs in developing cerebellar granule cells w17,41,51,57,63x. In culture, BDNF can promote granule cell survival w17,32,50,63x and also enhance neurite extension w17,51x. In BDNFŽyry . null mutant mice, postnatal cerebellar granule cell development was delayed with a persistence of the external granule cell layer ŽEGL. into the late postnatal period w29x. Thus, BDNF might regulate the appropriate timing of cerebellar granule cell differentiation. )
Corresponding author. Department of Pharmacology, University of Maryland School of Medicine, 655 W. Baltimore St., RM 4-018 Baltim o re , M D 2 1 2 0 1 . F a x : q 1 -4 1 0 -7 0 6 -0 0 3 2 ; E -m a il: [email protected] 0165-3806r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 3 8 0 6 Ž 9 7 . 0 0 1 9 3 - 4
One of BDNF’s functions might be to modulating the rate at which granule neurons differentiate. This modulation might include regulating the expression of gene involved in granule neuron terminal differentiation. Recent identification of a number of cyclin-dependent kinase inhibitors, including p21cip1, provides insights into how cells might stop dividing and begin terminal differentiation w20,22,34,61x. The cell cycle is controlled by a complex interaction of the cyclin proteins and the cyclin-dependent kinases ŽCdks. w13,53,54x. Cdk inhibitors like p21cip1 could serve to coordinate withdrawal from the cell cycle with terminal differentiation. During muscle development p21cip1 may inhibit cell cycle progression as well as directly control differentiation w19,22,45,55x. In the CNS p21cip1 expression occurs in both developing and mature neurons w5,10,45x and thus might also play a role in neuronal differentiation. In this study we investigated the effects of BDNF on the expression of p21cip1 in pure cerebellar granule cell cultures. We simultaneously examined GABA A receptor a 6 subunit ŽGABA A a 6. expression. GABA A a 6 expression serves as a marker for granule neuron terminal differentiation w31,33x. Addition of BDNF to granule cell cultures significantly enhanced p21cip1 expression and dramatically accelerated GABA A a 6 expression suggesting that BDNF can modulate the timing of cerebellar granule neu-
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ron differentiation in vitro. BDNF’s influence on p21 expression occurred after granule cells had stopped dividing suggesting this increase was not needed to direct cell out of the cell cycle. BDNF appears to accelerate gene expression associated with granule neuron terminal differentiation. 2. Materials and methods 2.1. Granule cell culture Cerebellar granule cell cultures were prepared from postnatal CD-1 mice. We generated Percoll-purified granule cell cultures using procedures described by Hatten w23x with minor modifications. Cerebella from postnatal day 7 mice were incubated in 1% trypsin ŽSigma, type III. and 0.1% DNase I ŽBoehringer Mannheim, Indianapolis, IN. in calcium and magnesium-free Tyrode’s solution ŽCMFTyrode’s. for 3 min. We dissociated cells by repeated passage through a small bore pipet in MEM ŽGibcorBRL. containing 0.1% DNase I and 0.2% glucose. Granule cells were separated from larger cell types by centrifugation through a 35%r60% percoll gradient. The granule cell fraction was collected at the 35%r60% percoll interface. To further purify the granule cell fraction, the cells were subjected to three rounds of 30 min plating on poly-Llysine-coated tissue culture dishes in minimal medium ŽMM. wMEM supplemented with 5 mgrml glucose, 0.292 mgrml glutamine, 50 unitsrml penicillin and 50 m grml streptomycinx containing 10% FBS. Granule cells can be washed off the plates while the glia remain attached. This method yielded a granule cell purity of 95–98%. We determined purity by counting the number of small sized phase bright cells characteristic of granule cells. This purity is consistent with previous estimates w23x. This pure population of granule cells was resuspended in MM without serum and cultured at a density of 1 = 10 7 cells on 35 mm uncoated petri dishes in the presence of various combinations of IGF-I Ž5 ngrml., BDNF Ž10 ngrml. or K252a Ž50 nM.. 2.2. RNA blot analysis RNA blot analysis was performed as described previously w6x. Briefly, total RNA was isolated by acid guanidinium thiocyanate–phenol–chloroform extraction w9x. RNA was fractionated by formaldehyde agarose gel electrophoresis and transferred by capillary blotting to nylon membranes w49x. RNA blots were prehybridized for 2–4 h at 428C in H buffer w5.6 = SSPE Ž0.84 M NaCl, 64 mM Na 2 PO4 , 6 mM EDTA., 50% formamide, 5 = Denhardt’s solution Ž0.1% ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin., 1% SDS and 200 m grml herring sperm DNAx. The hybridization was continued in H buffer containing 1–3 = 10 6 cpmrml of probe Žspecific activity of 1–2 = 10 9 cpmrm g. for 48 h at 428C. Blots were
washed in 0.1 = SSC, 1% SDS at 658C and exposed to X-ray film ŽDuPont.. GABA A a 6 cDNA probe used in the hybridization reactions were described previously w39x. Random primed p21cip1 and p27 kip1 probes were synthesized from cDNAs generated by PCR. We designed the PCR primers according to the published mouse cDNA sequences of p21cip1 and p27 kip1 w11,58x. DNA isolated from an adult mouse brain cDNA library was used as template for the PCR reaction. PCR products of expected sizes were isolated and their identities were confirmed by restriction enzyme analysis. We did densitometric analysis on the autoradiographs of RNA bolts ŽMolecular Dynamic w .. The optical density of autoradiographic signals was normalized to the level of 18S RNA. The level of 18S RNA was determined by densitometry of the negative image of ethidium bromide stained gels. 2.3. Immunoblot analysis Purified cerebellar granule cells were cultured in minimal medium supplemented with 5 ngrml of IGF-I or 10 ngrml of BDNF for 3 days. Cells were lysed in SDS– PAGE sample buffer Ž9.0% SDS, 15% glycerol, 6% bmercaptoethanol, 0.186 M tris pH 6.7, and 0.5% bromophenol.. The cell lysate was subjected to SDS–PAGE, and the proteins transferred to a nitrocellulose Membrane ŽNitroBind, MSI. by electrobloting ŽTrans-Blot Semi-Dry Electrophoretic Transfer Cell, BIO-RAD.. Protein blots were incubated in blocking buffer Ž10 mM Tris, 2.5 mM EDTA, 50 mM NaCl, 0.1% Tween 20, 3% nonfat dry milk. for 3 h at room temperature. These blots were then sequentially incubated for 1 h with a p21cip1 rabbit polyclonal antibody ŽSanta Cruz. diluted in blocking buffer at 1 m grml, 3 h with washing buffer Ž10 mM Tris, 2.5 mM EDTA, 50 mM NaCl, 0.1% Tween 20. and 1 h with horseradish peroxidase conjugated anti-rabbit second antibody ŽSanta Cruz.. Immunoreactivity was visualized using a chemiluminescent reagent ŽECL Western Blotting reagent, Amersham.. 2.4. Determination of Õiable cell number Viable cell number was determined as described by Volonte et al. w59x. Granule cells were lysed in a detergent-containing solution Ž0.5% ethylhexadecyldimethylammonium bromide, 0.28% acetic acid, 0.5% Triton X-100, 3 mM NaCl, 2 mM MgCl 2 , in PBS pH 7.4.. Under these conditions viable nuclei remained intact and could be easily distinguished from the small, broken or phase bright apoptotic nuclei. Viable nuclei were counted using a hemacytometer. This method was comparable to the MTT survival assay or a LDH assay in measuring cell survival in granule cell cultures w59x. We determined viable nuclei counts from three separate cultures for each treatment and presented the data as the percentage of initially plated cells. Statistical significance of the difference between treatments was evaluated by the Student’s t-test. Nuclei
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counts were consistent with the degree of cell death estimated from observation of culture morphology or the number of propidium iodide stained cells. 2.5. Measurement of DNA synthesis Granule cells were cultured for 24 or 48 h in MM containing 5 ngrml IGF-I or 10 ngrml BDNF. 2.5 m cirml of 3 H-thymidine was added to the culture medium 2 h before the end of culture. In control experiments, we cultured granule cells for 2 h in MM alone containing 2.5 m cirml of 3 H-thymidine to determine the initial amount of 3 H-thymidine incorporation. In all experiments cultured granule cells were removed from the plates by washing in PBS. The cells were lysed by the addition of four volumes of water and the DNA was precipitated with an equal volume of 20% TCA. Incorporation of 3 H-thymidine was determined by scintillation counting of TCA precipitated pellets.
3. Results Previous studies showed that p21cip1 mRNA expression occurs in the adult mouse brain w45x. We found that, in adult mice, p21cip1 mRNA is predominantly expressed in the olfactory bulb with low but detectable levels in other
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regions of the brain including the cerebellum ŽFig. 1A.. However, during cerebellar development, p21cip1 mRNA is transiently elevated between postnatal days 12 and 18 ŽFig. 1B.. During this time a large number of granule cell precursors undergo terminal differentiation which includes the expression of the GABA A receptor a 6 subunit ŽGABA A a 6. w31,33x. During in vivo cerebellar development, GABA A a 6 mRNA is detected at P9 and gradually increased reaching a plateau at P15, which is maintained in the adult ŽFig. 1B.. Since GABA A a 6 is expressed exclusively in cerebellar granule neurons w31,33x this increase is likely to result from an increase in the number of differentiating granule neurons. The increase of p21cip1 mRNA appeared to occur shortly after the induction of GABA A a 6. The expression of p21cip1 is likely to occur in many cell types w12,27,45,56x. Thus, we can not definitively know whether the change in p21cip1 expression in cerebellar tissue is related solely to a change in its expression in granule cells. However, in experiments using pure granule cell cultures the temporal pattern of p21cip1 RNA expression is similar to the expression in postnatal cerebellar tissue ŽFig. 3. suggesting that the in vivo change in p21cip1 expression might in part reflect changes in differentiating granule cells. We also examined the mRNA expression of another Cdk inhibitor, p27 kip1, which is closely related to p21cip1. RNA levels for p27 kip1 also seemed to be regulated during postnatal cerebellar development with highest lev-
Fig. 1. p21cip1 mRNA expression occurs in the adult mouse brain and the developing postnatal cerebellum. Total RNA Ž; 20 m g. was isolated from different brain regions ŽA., or from the cerebellum at different days of embryonic ŽE18. or postnatal ŽP0-21. development ŽB.. RNA blot analysis was performed using specific probes for p21cip1 , p27 kip1 and GABA A a 6 mRNAs. Ethidium bromide staining of 18S rRNA was used as an indication of the level of RNA loaded on gels.
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els of expression occurring between postnatal days 6 and 15 ŽFig. 1B.. The overlapping but distinct expression patterns of p21cip1 and p27 kip1 during cerebellar development suggest that these two closely related Cdk inhibitors are differentially regulated. During in vivo cerebellar development the level of BDNF mRNA begins to increase at about the same time GABA A a 6 and p21cip1 RNAs are increasing w57x. We examined whether BDNF could regulate the expression of these genes in cultures of purified cerebellar granule cells from postnatal day 7 mice. In order to minimize the complexity of the culture medium, we used a minimal medium ŽMM. wminimal essential medium supplemented with 5 mgrml glucose, 0.292 mgrml glutamine, 50 unitsrml penicillin and 50 m grml streptomycinx. In MM containing either IGF-I Ž5 ngrml. or BDNF Ž10 ngrml., granule cells can not only survive but also differentiate, as indicated by the neurite extension ŽFig. 2.. Granule cells cultured for 3 days formed aggregates with extended processes. The majority of cells and processes had smooth surfaces with no blebbing and we observed few pyknotic cells indicating the cultures remained relatively healthy ŽFig. 2.. Our previous studies suggest that IGF-I is a survival factor for developing granule cells but did not
Fig. 2. Cultures of purified granule cells maintained in MM containing either IGF-I or BDNF appear healthy and differentiate morphologically. Phase contrast photographs of pure granule cell cultures maintained for 3 days in minimal medium containing either 10 ngrml BDNF ŽA. or 5 ngrml IGF-I ŽB.. Arrow heads indicate pyknotic cells. Scale bar s 20 m m.
Fig. 3. Expression of GABA A a 6, p21cip1 and p27 kip1 mRNAs in granule cell cultures maintained in IGF-I. Total RNA Ž ;15 m g. was isolated from pure granule cell cultures maintained in 5 ngrml IGF-I following 0–6 days in culture. RNA blot analysis was performed using cDNA probes for p21cip1 , p27 kip1 and GABA A a 6 mRNAs. Ethidium bromide staining of 18S rRNA was used to indicate the level of RNA loaded on gels.
seem to play an instructive role in granule cell differentiation w37,38x. Granule cells could differentiate even in the absence of IGF-I as indicated by the expression of GABA A a 6 even though these cell were in the process of dying w37x. GABA A a 6 RNA expression increased between 3 and 6 days of culture. The addition of IGF-I Ž40 ngrml. did not modify this pattern of expression w37x. We observed a similar temporal expression pattern for GABA A a 6 using 5 ngrml IGF-I ŽFig. 3.. We also observed that the pattern of expression of GABA A a 6, p21cip1 and p27 kip1 mRNAs in pure granule cell cultures maintained only in IGF-I Ž5 ngrml. was similar to that during in vivo cerebellar development ŽFigs. 1 and 3.. Increasing the concentration of IGF-I to 40 ngrml did not alter the level or pattern of expression of these genes wX. Lin, unpublished observationsx. GABA A a 6 and p21cip1 mRNA expression increased while p27 kip1 mRNA remained at a constant level during the 6 days of culture. It is of note that on postnatal day 7, when these cultures were started, p27 kip1 mRNA was already expressed at a high level in vivo ŽFig. 1B.. Thus, granule cells cultured in the presence of only IGF-I Ž5 ngrml. appeared to retain an in vivo temporal pattern of differentiation as indicated by the pattern of gene expression. We compared the expression of p21cip1 , p27 kip1 and GABA A a 6 in cultured granule cells maintained in either BDNF Ž10 ngrml. or IGF-I Ž5 ngrml.. BDNF dramati-
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cally modified the mRNA expression patterns of GABA A a 6 and p21cip1 ŽFig. 4.. In cells maintained in BDNF, GABA A a 6 mRNA was detected on the first day of culture, whereas it was not detectable until day 3 in
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IGF-I treated granule cells ŽFig. 4A., suggesting that BDNF dramatically accelerated granule cell terminal differentiation. BDNF also greatly enhanced the expression of p21cip1 mRNA at day 2 and 3 of culture ŽFig. 4B.. In contrast, the mRNA for p27 kip1 stayed at a constant level and was not changed in the presence of BDNF ŽFig. 4C.. This pattern of gene expression was also observed in granule cell cultures co-treated with IGF-I and BDNF ŽFig. 5B.. Thus, under these culture conditions, BDNF seemed to accelerate granule cell terminal differentiation and enhanced p21cip1 mRNA expression. These experiments using pure granule cells cultured in minimal medium also indicated that BDNF may act directly on granule cells. If an increase in p21cip1 mRNA expression is to result in a functional change in granule cells than there must also occur a corresponding increase in the level of p21cip1 protein. We examined the level of p21cip1 protein in granule cells cultured for 3 days in the presence of IGF-I or BDNF. Immunoblots using a p21cip1 antiserum showed that BDNF increased the level of p21cip1 protein when compared to cultures maintained in IGF-I alone ŽFig. 4D.. Thus, BDNF treatment results in an increased level of expression of both p21cip1 mRNA and protein. BDNF’s effects are presumably mediated by binding with its high affinity cognate receptor TrkB, a receptor tyrosine kinase w30,52x. To demonstrate the specificity of BDNF’s action through the TrkB signalling pathway, we used a selective inhibitor for Trk tyrosine kinases, K252a, which has been shown in cerebellar granule cell cultures to inhibit BDNF’s action w17,35x. The addition of K252a Ž50 nM. to granule cell cultures specifically eliminated the survival promoting effects of BDNF Ž10 ngrml. without effecting the survival promoting action of IGF-I Ž5 ngrml. ŽFig. 5A.. The survival of cells cultured in BDNF plus K252a was similar to the cells maintained in MM alone and was significantly less than BDNF or IGF-I treated cells ŽFig. 5A.. The survival of cells maintained in IGF-I plus K252a did not differ from cultures maintained in IGF-I alone ŽFig. 5A.. This observation was consistent
Fig. 4. BDNF accelerates GABA A a 6 mRNA expression, and specifically enhances p21cip1 mRNA expression. Total RNA Ž ;15 m g. was isolated from granule cells maintained in either 5 ngrml IGF-1 or 10 ngrml BDNF following 0–3 days in culture. Presented is RNA blot analysis using cDNA probes for GABA A a 6 ŽA., p21cip1 ŽB., and p27 kip1 ŽC. mRNAs. Ethidium bromide staining of 18S rRNA was used to control for variations in the level of RNA loaded on gels. Data is expressed as relative optical density of autoradiographic hybridization signals normalized to the level of 18S rRNA in 3 separate experiments. The level of 18S rRNA was determined by densitometry of the negative image of ethidium bromide stained gels. The asterisk Ž ) . indicates a statistically significant difference in mean hybridization values between BDNF and IGF-I treated cultures, P - 0.05. Also presented is an immunoblot using a p21cip1 specific antibody to identify p21cip1 protein ŽD.. Cell lysates from an equal number of cells, treated with either IGF-I Ž5 ngrml. or BDNF Ž10 ngrml. for 3 days, were separated by SDS–PAGE prior to preparing immunoblots.
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with the selectivity of K252a for Trk tyrosine kinase receptors. These experiments also show that IGF-I alone and BDNF alone have a similar ability to promote survival. However their ability to modify differentiation of GABA A a 6 and p21cip1 expression are different. Suggesting that in general survival promotion alone is not enough to accelerate expression of these genes. To examine whether the effect of BDNF on the expression patterns of GABA A a 6 and p21cip1 could be blocked by K252a, we used IGF-I to maintain the survival of
BDNF treated granule cells when there were exposed to K252a. In the presence of both IGF-I and BDNF, the mRNA expression patterns of GABA A a 6 and p21cip1 were similar to those in BDNF alone ŽFigs. 4 and 5B.. The effect of BDNF on this RNA expression was eliminated by K252a ŽFig. 5B,C.. Moreover, the expression patterns of both GABA A a 6 and p21cip1 in granule cells exposed to K252a in the presence of IGF-I were similar to cells exposed only to IGF-I ŽFig. 5B compared to Fig. 4.. This observation also suggested that BDNF, endogenously produced by granule cells w51x and acting through Trk receptors, was not required for the observed expression of GABA A a 6 and p21cip1 in granule cells maintained in MM containing only IGF-I. To determine whether the increase in p21cip1 expression induced by BDNF might direct granule cells to stop proliferating we evaluated the degree of proliferation in BDNF and IGF-I treated cultures. Previous studies indicate that IGF-I, above 10 ngrml, can stimulate proliferation of granule cell precursors while BDNF did not stimulate proliferation w16,17x. To evaluate the level of proliferation in these cultures we measured 3 H-thymidine incorporation. We exposed cultured granule cells to IGF-I Ž5 ngrml. or BDNF Ž10 ngrml. for 24 and 48 h and added 3 H-thymidine for the last 2 h of culture. The amount of incorporation in these cultures was compared to the initial incorporation over the first 2 h of culture before the addition of BDNF or IGF-I. We measured a significant amount of incorporation after labeling cells for the first 2 h of culture ŽTable 1, control.. This incorporation represents a measure of the number of granule cells in the cell cycle at the start of culture. Bromodeoxyuridine labeling experiments suggest that 30–40% granule cells are in the cell cycle at this time of culture wH. Cui and R. Bulleit unpublished observationsx. If 3 H-thymidine was added for 2 h at the end of a 24 h culture period then we observed a significant reduction in the amount of thymidine incorporation compared to the initial incorporation over the first 2 h of culture, 98%
Fig. 5. K252a selectively blocks BDNF’s effects on granule neuron survival and GABA A a 6 and p21cip1 mRNA expression. Granule cells were cultured in minimal medium containing 5 ngrml IGF-I plus or minus BDNF Ž10 ngrml. andror K252a Ž50 nM. as indicated. The percent surviving granule neurons was determine after 3 days of culture ŽA.. The asterisk Ž ) . indicates a statistically significant difference in mean percent survival between treated cultures and cultures maintained in MM alone, P - 0.01. Total RNA Ž ;15 m g. was isolated following 0–3 days in culture. Presented is RNA blot analysis using cDNA probes for GABA A a 6 and p21cip1 mRNAs ŽB.. Ethidium bromide staining of 18S rRNA was used to indicate the level of RNA loaded on gels. Also presented is the relative optical density of autoradiographic hybridization signals for p21cip1 mRNA ŽC.. The optical density was normalized to the level of 18S rRNA in 3 separate experiments. The level of 18S rRNA was determined by densitometry of the negative image of ethidium bromide stained gels. The asterisk Ž ) . indicates a statistically significant difference in mean hybridization values between IGF-IqBDNF ŽIB. and IGF-IqBDNFqK252A ŽIBK. treated cultures, P - 0.05.
X. Lin et al.r DeÕelopmental Brain Research 105 (1998) 277–286 Table 1 Incorporation of 3 H-thymidine into cultured cerebellar granule cells Culture treatment a
Control 5 ngrml IGF-I b 5 ngrml IGF-I b 10 ngrml BDNF b 10 ngrml BDNF b
Time in culture Žh.
CPM
% Control
2 24 48 24 48
11204"1220 236"29 52"4 342"36 98.9
y 2.1 0.5 3.1 0.9
a
Granule cells were cultured in minimal medium ŽMM. alone without trophic factors. About 2.5 m cirml of 3 H-thymidine was added at the start of culture and 2 h later 3 H-Thymidine incorporation was measured. b Granule cells were cultured in MM containing IGF-I or BDNF for 24 or 48 h 2.5 m cirml of 3 H-thymidine was added for the last 2 h of culture. 3 H-Thymidine incorporation was measured following this labeling period.
reduction in IGF-I and 97% reduction in BDNF ŽTable 1.. By 48 h of culture there was greater than a 99% reduction in thymidine incorporation ŽTable 1.. If the cell cycle time of proliferating granule cells is 18–24 h w15,42,44x, then these results suggest that most of the granule cells that are dividing at the start of culture do not reenter the cell cycle. Thus, most granule neuron precursors stopped proliferating by the first 24 h of culture Žgreater than 97%. in the presence of either IGF-I Ž5 ngrml. or BDNF Ž10 ngrml.. This is consistent with previous experiments indicating that low concentrations of IGF-I Ž1–5 ngrml. or BDNF Ž10 ngrml. do not substantially support granule cell proliferation in vitro w16,17x, wX. Lin, unpublished observationsx. The results indicate that 97–98% of the cells stopped proliferating before we observed an increase in p21cip1 RNA expression which occurred 48–72 h after the start of culture ŽFig. 4..
4. Discussion Neurogenesis in the central nervous system requires the precise temporal and spatial regulation of cell proliferation and differentiation. This regulation is dependent on both genetic and epigenetic cues. The experiments presented in this study suggest that epigenetic cues, specifically BDNF, can modulate differentiation of postnatal cerebellar granule cells. In comparison with IGF-I, BDNF dramatically accelerated the expression of granule cell terminal differentiation marker GABA A a 6 mRNA ŽFig. 4.. Previous studies suggest that even high concentrations of IGF-I Ž40 ngrml. did not accelerate GABA A a 6 expression w37x. Thus, BDNF appears to modify the timing of granule cell differentiation in vitro. Interestingly, the expression of the cell cycle inhibitor p21cip1 was also accelerated by BDNF treatment. The increase in p21cip1 expression occurred with a similar time course as the increase in expression of the terminal differentiation marker GABA A a 6. This increased expression occurred after the majority of cultured granule cells stopped proliferating ŽTable 1.. Thus, BDNF appeared to
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accelerate granule neuron terminal differentiation after granule neuron precursors had already withdrawn from the cell cycle. The observation that these cultures are 98% pure granule cells ŽSection 2. w23x and that GABA A a 6 expression occur exclusively in mature cerebellar granule neurons w31,33x suggest that BDNF may directly modulate gene expression in maturing granule neurons. It has been reported that postnatal granule neurons express functional BDNF receptors ŽTrkB. w17,51x. However, its is also possible that BDNF is acting indirectly by inducing a small minority of another cell type in the culture to produce factors which in turn modulate granule neuron gene expression. In either case BDNF can ultimately accelerate the maturation of cerebellar granule neurons in vitro. The observations that the increase in p21cip1 occurs after granule cell stop proliferating might suggest that the increase in p21cip1 expression is not required to direct granule neuron precursors out of the cell cycle but occurs as granule neurons initiate terminal differentiation. An increase in p21cip1 and GABA A a 6 RNA expression may be part of a granule neuron differentiation program that is specified prior to the time we isolate cell for culture w1,37x. Activation of Trk receptors by BDNF does not appear to be required for the ultimate expression of the program. Even in the absence of added BDNF or in the presence of the Trk inhibitor K252a, granule cells differentiated and expressed GABA A a 6 and p21cip1 ŽFigs. 3 and 5.. However, we cannot completely eliminate the possibility that BDNF or other trophic factors, produced by granule neurons themselves and acting through non Trk receptors such as the low affinity nerve growth factor receptor Žp75., are required for this program’s expression. We have previously observed that in the absence of all exogenously added trophic factors this program of gene expression appears to still function even though these cells are in the process of dying w37x. Thus, survival signals themselves are not require for this programs expression. This differentiation program may be regulated by granule cell intrinsic mechanisms. Although, exogenous factors may not be required for this program’s ultimate expression they appear to modulate the timing of its expression. BDNF can accelerate the expression of terminal differentiation markers. This modulation of gene expression may be a direct action of BDNF on the granule neuron differentiation program or alternatively an indirect result of BDNF’s survival promoting action. We can not entirely dissociate these two actions of BDNF. However, IGF-I which promotes survival to a similar degree as BDNF ŽFig. 5. does not accelerate gene expression. Thus, a general survival action is not enough to modulate this gene expression. BDNF may alternatively have a different survival promoting mechanism that includes a modulation of gene expression associated with granule neuron terminal differentiation. The same intracellular signal transduction cascade may mediate both the survival and differentiation promoting actions of BDNF and may differ from IGF-I survival promoting cascade.
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Ultimately BDNF can accelerate the maturation of cerebellar granule neurons in vitro. The phenotype of BDNF knock-out mice also suggests that BDNF may regulate the timing of granule cell development in vivo w29x. At postnatal day 17 the EGL is still present in BDNF Žyry . mutant mice while by this time in wild type mice the EGL is completely absent. However, the elimination of the EGL occurs by postnatal day 23 in mutant mice suggesting that cerebellar granule neuron development was delayed but not prevented. Several lines of evidence suggested that p21cip1 may contribute to cell cycle arrest during terminal differentiation of multiple cell types w12,27,45,56x. However, in the present study we observed an increase in p21cip1 expression that occurred after dividing granule cells left the cell cycle. This temporal distinction between the cessation of cell proliferation and the increase in p21cip1 expression would suggest that an increase in p21cip1 is not required for directing dividing granule cells out of the cell cycle. Thus p21cip1 must have an alternative function during this period of granule neuron differentiation. One function may be to maintain differentiating granule neurons in a non-proliferative state. In most proliferating cells the progression through the cell cycle is controlled by the activities of cyclin-dependent kinases ŽCdks.. Cdks are a family of serinerthreonine kinases which have almost no kinase activity in monomeric form and require interaction with members of the cyclin family of proteins for kinase activation. A number of polypeptides have been identified, which inhibit Cdk activity by direct association with cyclin–Cdk complexes. p21cip1 is the prototypic Cdk inhibitor which can inhibit G1 cyclin–Cdks in a stoichiometric fashion w22,62x. A low amount of p21cip1 is present in active cyclin–Cdk complexes and may potentially modulate their activity w62x. However, higher amounts of p21cip1 function as a potent inhibitor and arrests cells in the G1 phase of the cell cycle w21x. Recent experiments also suggest that p21cip1 can inhibit DNA synthesis via the binding of its’ carboxy-terminal region with the DNA polymerase d processivity factor, PCNA Žproliferating cell nuclear antigen.w8,36,40x. Thus, an increase in p21cip1 expression might provide and assurance that early differentiating granule neurons do not inappropriately reentering the cell cycle and begin DNA synthesis. Alternatively, p21cip1 could have a function separate from cell cycle control. Recent experiments suggest that p21cip1 may protect differentiating myocytes from apoptotic cell death w60x. A similar function may occur in differentiating neurons w46x. Thus, an increase in p21cip1 may be involved in BDNF’s survival promoting effect. However, this mechanism would differ from IGF-I’s survival promoting mechanism. IGF-I does not increase p21cip1 expression but maintains granule cell survival to a similar degree as BDNF ŽFig. 5.. During postnatal cerebellar development a transient but marked increase of p21cip1 mRNA was observed between postnatal day 12 and 18 ŽFig. 1B.. This is a period of
cerebellar development when many granule cells are undergoing terminal differentiation w3,4x. The related Cdk inhibitor p27 kip1 shares a common amino-terminal domain of 60 amino acids with p21cip1 w47,58x. This domain is responsible for interacting with cyclin–Cdk complexes and inhibiting their kinase activity w18,21,40x. However, their carboxy terminal regions are different and may specify independent and specialized functions. In the cerebellum p27 kip1 mRNA expression also occurs in the adult and is transiently increased during postnatal cerebellar development ŽFig. 1A.. The expression of p21cip1 and p27 kip1 mRNAs in the postnatal cerebellum is temporally distinct but overlapping, indicating a potential difference in their function during cerebellar development. The expression of these two related Cdk inhibitors also appears to be differentially regulated by epigenetic cues. BDNF modifies the level of p21cip1 mRNA but not p27 kip1 mRNA under our culture conditions ŽFig. 4.. However, under these conditions the level of expression of p27 kip1 appears to have peaked by the start of culture. Thus, BDNF may not be able to enhance its expression further. At an earlier point in development BDNF might regulate p27 kip1 expression. This regulation would depend on the presence of BDNF receptors on early developing granule cells. Previous experiments suggest that early proliferating granule cells may not express BDNF’s cognate receptor, TrkB w17x.
Acknowledgements We thank D. Frost and D. Burt for critical reading of the manuscript. This work was in part supported by the Bressler Research Fund of the University of Maryland at Baltimore.
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Research report
BDNF accelerates gene expression in cultured cerebellar granule neurons Xi Lin, Hong Cui, Robert F. Bulleit
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Department of Pharmacology, UniÕersity of Maryland School of Medicine, Baltimore, MD 21201, USA Accepted 21 October 1997
Abstract This study reports that in purified cultures of postnatal cerebellar granule cells, BDNF significantly accelerated GABA A receptor a 6 subunit ŽGABA A a 6. mRNA expression, a marker for terminally differentiated cerebellar granule neurons, and also accelerated p21cip1 expression. p21cip1 is a general cyclin-dependent kinase ŽCdk. inhibitor that can inhibit progression through the cell cycle. Alternatively, the expression of p27 kip1, another Cdk inhibitor closely related to p21cip1, is not modified by BDNF. In cultured granule cells, the increase in p21cip1 expression induced by BDNF occurred after dividing granule cells had left the cell cycle and thus was not required to direct granule neuron precursors out of the cell cycle. p21cip1 may have an alternative function during granule neuron terminal differentiation, separate from its ability to regulate cell cycle exit. This report shows that, in vitro, BDNF accelerates granule cell gene expression and may thus modulate cerebellar granule cell differentiation. q 1998 Elsevier Science B.V. Keywords: BDNF; Neurotrophin; IGF-I; Differentiation; Gene expression; Mouse
1. Introduction Brain-derived neurotrophic factor ŽBDNF. is a member of the neurotrophin family of trophic factors. Expression of BDNF and its high affinity cognate receptor ŽTrkB. occurs in various regions of the developing and adult brain w7,14,43,48x. This pattern of expression suggests a number of potential roles for BDNF in neuronal survival and differentiation. As a neurotrophic factor, BDNF can promote the survival of various types of CNS neurons w2,24,24,26,28x and may also exert an influence on neuronal differentiation w2,25,26x. Expression of both BDNF and TrkB occurs in developing cerebellar granule cells w17,41,51,57,63x. In culture, BDNF can promote granule cell survival w17,32,50,63x and also enhance neurite extension w17,51x. In BDNFŽyry . null mutant mice, postnatal cerebellar granule cell development was delayed with a persistence of the external granule cell layer ŽEGL. into the late postnatal period w29x. Thus, BDNF might regulate the appropriate timing of cerebellar granule cell differentiation. )
Corresponding author. Department of Pharmacology, University of Maryland School of Medicine, 655 W. Baltimore St., RM 4-018 Baltim o re , M D 2 1 2 0 1 . F a x : q 1 -4 1 0 -7 0 6 -0 0 3 2 ; E -m a il: [email protected] 0165-3806r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 3 8 0 6 Ž 9 7 . 0 0 1 9 3 - 4
One of BDNF’s functions might be to modulating the rate at which granule neurons differentiate. This modulation might include regulating the expression of gene involved in granule neuron terminal differentiation. Recent identification of a number of cyclin-dependent kinase inhibitors, including p21cip1, provides insights into how cells might stop dividing and begin terminal differentiation w20,22,34,61x. The cell cycle is controlled by a complex interaction of the cyclin proteins and the cyclin-dependent kinases ŽCdks. w13,53,54x. Cdk inhibitors like p21cip1 could serve to coordinate withdrawal from the cell cycle with terminal differentiation. During muscle development p21cip1 may inhibit cell cycle progression as well as directly control differentiation w19,22,45,55x. In the CNS p21cip1 expression occurs in both developing and mature neurons w5,10,45x and thus might also play a role in neuronal differentiation. In this study we investigated the effects of BDNF on the expression of p21cip1 in pure cerebellar granule cell cultures. We simultaneously examined GABA A receptor a 6 subunit ŽGABA A a 6. expression. GABA A a 6 expression serves as a marker for granule neuron terminal differentiation w31,33x. Addition of BDNF to granule cell cultures significantly enhanced p21cip1 expression and dramatically accelerated GABA A a 6 expression suggesting that BDNF can modulate the timing of cerebellar granule neu-
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ron differentiation in vitro. BDNF’s influence on p21 expression occurred after granule cells had stopped dividing suggesting this increase was not needed to direct cell out of the cell cycle. BDNF appears to accelerate gene expression associated with granule neuron terminal differentiation. 2. Materials and methods 2.1. Granule cell culture Cerebellar granule cell cultures were prepared from postnatal CD-1 mice. We generated Percoll-purified granule cell cultures using procedures described by Hatten w23x with minor modifications. Cerebella from postnatal day 7 mice were incubated in 1% trypsin ŽSigma, type III. and 0.1% DNase I ŽBoehringer Mannheim, Indianapolis, IN. in calcium and magnesium-free Tyrode’s solution ŽCMFTyrode’s. for 3 min. We dissociated cells by repeated passage through a small bore pipet in MEM ŽGibcorBRL. containing 0.1% DNase I and 0.2% glucose. Granule cells were separated from larger cell types by centrifugation through a 35%r60% percoll gradient. The granule cell fraction was collected at the 35%r60% percoll interface. To further purify the granule cell fraction, the cells were subjected to three rounds of 30 min plating on poly-Llysine-coated tissue culture dishes in minimal medium ŽMM. wMEM supplemented with 5 mgrml glucose, 0.292 mgrml glutamine, 50 unitsrml penicillin and 50 m grml streptomycinx containing 10% FBS. Granule cells can be washed off the plates while the glia remain attached. This method yielded a granule cell purity of 95–98%. We determined purity by counting the number of small sized phase bright cells characteristic of granule cells. This purity is consistent with previous estimates w23x. This pure population of granule cells was resuspended in MM without serum and cultured at a density of 1 = 10 7 cells on 35 mm uncoated petri dishes in the presence of various combinations of IGF-I Ž5 ngrml., BDNF Ž10 ngrml. or K252a Ž50 nM.. 2.2. RNA blot analysis RNA blot analysis was performed as described previously w6x. Briefly, total RNA was isolated by acid guanidinium thiocyanate–phenol–chloroform extraction w9x. RNA was fractionated by formaldehyde agarose gel electrophoresis and transferred by capillary blotting to nylon membranes w49x. RNA blots were prehybridized for 2–4 h at 428C in H buffer w5.6 = SSPE Ž0.84 M NaCl, 64 mM Na 2 PO4 , 6 mM EDTA., 50% formamide, 5 = Denhardt’s solution Ž0.1% ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin., 1% SDS and 200 m grml herring sperm DNAx. The hybridization was continued in H buffer containing 1–3 = 10 6 cpmrml of probe Žspecific activity of 1–2 = 10 9 cpmrm g. for 48 h at 428C. Blots were
washed in 0.1 = SSC, 1% SDS at 658C and exposed to X-ray film ŽDuPont.. GABA A a 6 cDNA probe used in the hybridization reactions were described previously w39x. Random primed p21cip1 and p27 kip1 probes were synthesized from cDNAs generated by PCR. We designed the PCR primers according to the published mouse cDNA sequences of p21cip1 and p27 kip1 w11,58x. DNA isolated from an adult mouse brain cDNA library was used as template for the PCR reaction. PCR products of expected sizes were isolated and their identities were confirmed by restriction enzyme analysis. We did densitometric analysis on the autoradiographs of RNA bolts ŽMolecular Dynamic w .. The optical density of autoradiographic signals was normalized to the level of 18S RNA. The level of 18S RNA was determined by densitometry of the negative image of ethidium bromide stained gels. 2.3. Immunoblot analysis Purified cerebellar granule cells were cultured in minimal medium supplemented with 5 ngrml of IGF-I or 10 ngrml of BDNF for 3 days. Cells were lysed in SDS– PAGE sample buffer Ž9.0% SDS, 15% glycerol, 6% bmercaptoethanol, 0.186 M tris pH 6.7, and 0.5% bromophenol.. The cell lysate was subjected to SDS–PAGE, and the proteins transferred to a nitrocellulose Membrane ŽNitroBind, MSI. by electrobloting ŽTrans-Blot Semi-Dry Electrophoretic Transfer Cell, BIO-RAD.. Protein blots were incubated in blocking buffer Ž10 mM Tris, 2.5 mM EDTA, 50 mM NaCl, 0.1% Tween 20, 3% nonfat dry milk. for 3 h at room temperature. These blots were then sequentially incubated for 1 h with a p21cip1 rabbit polyclonal antibody ŽSanta Cruz. diluted in blocking buffer at 1 m grml, 3 h with washing buffer Ž10 mM Tris, 2.5 mM EDTA, 50 mM NaCl, 0.1% Tween 20. and 1 h with horseradish peroxidase conjugated anti-rabbit second antibody ŽSanta Cruz.. Immunoreactivity was visualized using a chemiluminescent reagent ŽECL Western Blotting reagent, Amersham.. 2.4. Determination of Õiable cell number Viable cell number was determined as described by Volonte et al. w59x. Granule cells were lysed in a detergent-containing solution Ž0.5% ethylhexadecyldimethylammonium bromide, 0.28% acetic acid, 0.5% Triton X-100, 3 mM NaCl, 2 mM MgCl 2 , in PBS pH 7.4.. Under these conditions viable nuclei remained intact and could be easily distinguished from the small, broken or phase bright apoptotic nuclei. Viable nuclei were counted using a hemacytometer. This method was comparable to the MTT survival assay or a LDH assay in measuring cell survival in granule cell cultures w59x. We determined viable nuclei counts from three separate cultures for each treatment and presented the data as the percentage of initially plated cells. Statistical significance of the difference between treatments was evaluated by the Student’s t-test. Nuclei
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counts were consistent with the degree of cell death estimated from observation of culture morphology or the number of propidium iodide stained cells. 2.5. Measurement of DNA synthesis Granule cells were cultured for 24 or 48 h in MM containing 5 ngrml IGF-I or 10 ngrml BDNF. 2.5 m cirml of 3 H-thymidine was added to the culture medium 2 h before the end of culture. In control experiments, we cultured granule cells for 2 h in MM alone containing 2.5 m cirml of 3 H-thymidine to determine the initial amount of 3 H-thymidine incorporation. In all experiments cultured granule cells were removed from the plates by washing in PBS. The cells were lysed by the addition of four volumes of water and the DNA was precipitated with an equal volume of 20% TCA. Incorporation of 3 H-thymidine was determined by scintillation counting of TCA precipitated pellets.
3. Results Previous studies showed that p21cip1 mRNA expression occurs in the adult mouse brain w45x. We found that, in adult mice, p21cip1 mRNA is predominantly expressed in the olfactory bulb with low but detectable levels in other
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regions of the brain including the cerebellum ŽFig. 1A.. However, during cerebellar development, p21cip1 mRNA is transiently elevated between postnatal days 12 and 18 ŽFig. 1B.. During this time a large number of granule cell precursors undergo terminal differentiation which includes the expression of the GABA A receptor a 6 subunit ŽGABA A a 6. w31,33x. During in vivo cerebellar development, GABA A a 6 mRNA is detected at P9 and gradually increased reaching a plateau at P15, which is maintained in the adult ŽFig. 1B.. Since GABA A a 6 is expressed exclusively in cerebellar granule neurons w31,33x this increase is likely to result from an increase in the number of differentiating granule neurons. The increase of p21cip1 mRNA appeared to occur shortly after the induction of GABA A a 6. The expression of p21cip1 is likely to occur in many cell types w12,27,45,56x. Thus, we can not definitively know whether the change in p21cip1 expression in cerebellar tissue is related solely to a change in its expression in granule cells. However, in experiments using pure granule cell cultures the temporal pattern of p21cip1 RNA expression is similar to the expression in postnatal cerebellar tissue ŽFig. 3. suggesting that the in vivo change in p21cip1 expression might in part reflect changes in differentiating granule cells. We also examined the mRNA expression of another Cdk inhibitor, p27 kip1, which is closely related to p21cip1. RNA levels for p27 kip1 also seemed to be regulated during postnatal cerebellar development with highest lev-
Fig. 1. p21cip1 mRNA expression occurs in the adult mouse brain and the developing postnatal cerebellum. Total RNA Ž; 20 m g. was isolated from different brain regions ŽA., or from the cerebellum at different days of embryonic ŽE18. or postnatal ŽP0-21. development ŽB.. RNA blot analysis was performed using specific probes for p21cip1 , p27 kip1 and GABA A a 6 mRNAs. Ethidium bromide staining of 18S rRNA was used as an indication of the level of RNA loaded on gels.
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els of expression occurring between postnatal days 6 and 15 ŽFig. 1B.. The overlapping but distinct expression patterns of p21cip1 and p27 kip1 during cerebellar development suggest that these two closely related Cdk inhibitors are differentially regulated. During in vivo cerebellar development the level of BDNF mRNA begins to increase at about the same time GABA A a 6 and p21cip1 RNAs are increasing w57x. We examined whether BDNF could regulate the expression of these genes in cultures of purified cerebellar granule cells from postnatal day 7 mice. In order to minimize the complexity of the culture medium, we used a minimal medium ŽMM. wminimal essential medium supplemented with 5 mgrml glucose, 0.292 mgrml glutamine, 50 unitsrml penicillin and 50 m grml streptomycinx. In MM containing either IGF-I Ž5 ngrml. or BDNF Ž10 ngrml., granule cells can not only survive but also differentiate, as indicated by the neurite extension ŽFig. 2.. Granule cells cultured for 3 days formed aggregates with extended processes. The majority of cells and processes had smooth surfaces with no blebbing and we observed few pyknotic cells indicating the cultures remained relatively healthy ŽFig. 2.. Our previous studies suggest that IGF-I is a survival factor for developing granule cells but did not
Fig. 2. Cultures of purified granule cells maintained in MM containing either IGF-I or BDNF appear healthy and differentiate morphologically. Phase contrast photographs of pure granule cell cultures maintained for 3 days in minimal medium containing either 10 ngrml BDNF ŽA. or 5 ngrml IGF-I ŽB.. Arrow heads indicate pyknotic cells. Scale bar s 20 m m.
Fig. 3. Expression of GABA A a 6, p21cip1 and p27 kip1 mRNAs in granule cell cultures maintained in IGF-I. Total RNA Ž ;15 m g. was isolated from pure granule cell cultures maintained in 5 ngrml IGF-I following 0–6 days in culture. RNA blot analysis was performed using cDNA probes for p21cip1 , p27 kip1 and GABA A a 6 mRNAs. Ethidium bromide staining of 18S rRNA was used to indicate the level of RNA loaded on gels.
seem to play an instructive role in granule cell differentiation w37,38x. Granule cells could differentiate even in the absence of IGF-I as indicated by the expression of GABA A a 6 even though these cell were in the process of dying w37x. GABA A a 6 RNA expression increased between 3 and 6 days of culture. The addition of IGF-I Ž40 ngrml. did not modify this pattern of expression w37x. We observed a similar temporal expression pattern for GABA A a 6 using 5 ngrml IGF-I ŽFig. 3.. We also observed that the pattern of expression of GABA A a 6, p21cip1 and p27 kip1 mRNAs in pure granule cell cultures maintained only in IGF-I Ž5 ngrml. was similar to that during in vivo cerebellar development ŽFigs. 1 and 3.. Increasing the concentration of IGF-I to 40 ngrml did not alter the level or pattern of expression of these genes wX. Lin, unpublished observationsx. GABA A a 6 and p21cip1 mRNA expression increased while p27 kip1 mRNA remained at a constant level during the 6 days of culture. It is of note that on postnatal day 7, when these cultures were started, p27 kip1 mRNA was already expressed at a high level in vivo ŽFig. 1B.. Thus, granule cells cultured in the presence of only IGF-I Ž5 ngrml. appeared to retain an in vivo temporal pattern of differentiation as indicated by the pattern of gene expression. We compared the expression of p21cip1 , p27 kip1 and GABA A a 6 in cultured granule cells maintained in either BDNF Ž10 ngrml. or IGF-I Ž5 ngrml.. BDNF dramati-
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cally modified the mRNA expression patterns of GABA A a 6 and p21cip1 ŽFig. 4.. In cells maintained in BDNF, GABA A a 6 mRNA was detected on the first day of culture, whereas it was not detectable until day 3 in
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IGF-I treated granule cells ŽFig. 4A., suggesting that BDNF dramatically accelerated granule cell terminal differentiation. BDNF also greatly enhanced the expression of p21cip1 mRNA at day 2 and 3 of culture ŽFig. 4B.. In contrast, the mRNA for p27 kip1 stayed at a constant level and was not changed in the presence of BDNF ŽFig. 4C.. This pattern of gene expression was also observed in granule cell cultures co-treated with IGF-I and BDNF ŽFig. 5B.. Thus, under these culture conditions, BDNF seemed to accelerate granule cell terminal differentiation and enhanced p21cip1 mRNA expression. These experiments using pure granule cells cultured in minimal medium also indicated that BDNF may act directly on granule cells. If an increase in p21cip1 mRNA expression is to result in a functional change in granule cells than there must also occur a corresponding increase in the level of p21cip1 protein. We examined the level of p21cip1 protein in granule cells cultured for 3 days in the presence of IGF-I or BDNF. Immunoblots using a p21cip1 antiserum showed that BDNF increased the level of p21cip1 protein when compared to cultures maintained in IGF-I alone ŽFig. 4D.. Thus, BDNF treatment results in an increased level of expression of both p21cip1 mRNA and protein. BDNF’s effects are presumably mediated by binding with its high affinity cognate receptor TrkB, a receptor tyrosine kinase w30,52x. To demonstrate the specificity of BDNF’s action through the TrkB signalling pathway, we used a selective inhibitor for Trk tyrosine kinases, K252a, which has been shown in cerebellar granule cell cultures to inhibit BDNF’s action w17,35x. The addition of K252a Ž50 nM. to granule cell cultures specifically eliminated the survival promoting effects of BDNF Ž10 ngrml. without effecting the survival promoting action of IGF-I Ž5 ngrml. ŽFig. 5A.. The survival of cells cultured in BDNF plus K252a was similar to the cells maintained in MM alone and was significantly less than BDNF or IGF-I treated cells ŽFig. 5A.. The survival of cells maintained in IGF-I plus K252a did not differ from cultures maintained in IGF-I alone ŽFig. 5A.. This observation was consistent
Fig. 4. BDNF accelerates GABA A a 6 mRNA expression, and specifically enhances p21cip1 mRNA expression. Total RNA Ž ;15 m g. was isolated from granule cells maintained in either 5 ngrml IGF-1 or 10 ngrml BDNF following 0–3 days in culture. Presented is RNA blot analysis using cDNA probes for GABA A a 6 ŽA., p21cip1 ŽB., and p27 kip1 ŽC. mRNAs. Ethidium bromide staining of 18S rRNA was used to control for variations in the level of RNA loaded on gels. Data is expressed as relative optical density of autoradiographic hybridization signals normalized to the level of 18S rRNA in 3 separate experiments. The level of 18S rRNA was determined by densitometry of the negative image of ethidium bromide stained gels. The asterisk Ž ) . indicates a statistically significant difference in mean hybridization values between BDNF and IGF-I treated cultures, P - 0.05. Also presented is an immunoblot using a p21cip1 specific antibody to identify p21cip1 protein ŽD.. Cell lysates from an equal number of cells, treated with either IGF-I Ž5 ngrml. or BDNF Ž10 ngrml. for 3 days, were separated by SDS–PAGE prior to preparing immunoblots.
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with the selectivity of K252a for Trk tyrosine kinase receptors. These experiments also show that IGF-I alone and BDNF alone have a similar ability to promote survival. However their ability to modify differentiation of GABA A a 6 and p21cip1 expression are different. Suggesting that in general survival promotion alone is not enough to accelerate expression of these genes. To examine whether the effect of BDNF on the expression patterns of GABA A a 6 and p21cip1 could be blocked by K252a, we used IGF-I to maintain the survival of
BDNF treated granule cells when there were exposed to K252a. In the presence of both IGF-I and BDNF, the mRNA expression patterns of GABA A a 6 and p21cip1 were similar to those in BDNF alone ŽFigs. 4 and 5B.. The effect of BDNF on this RNA expression was eliminated by K252a ŽFig. 5B,C.. Moreover, the expression patterns of both GABA A a 6 and p21cip1 in granule cells exposed to K252a in the presence of IGF-I were similar to cells exposed only to IGF-I ŽFig. 5B compared to Fig. 4.. This observation also suggested that BDNF, endogenously produced by granule cells w51x and acting through Trk receptors, was not required for the observed expression of GABA A a 6 and p21cip1 in granule cells maintained in MM containing only IGF-I. To determine whether the increase in p21cip1 expression induced by BDNF might direct granule cells to stop proliferating we evaluated the degree of proliferation in BDNF and IGF-I treated cultures. Previous studies indicate that IGF-I, above 10 ngrml, can stimulate proliferation of granule cell precursors while BDNF did not stimulate proliferation w16,17x. To evaluate the level of proliferation in these cultures we measured 3 H-thymidine incorporation. We exposed cultured granule cells to IGF-I Ž5 ngrml. or BDNF Ž10 ngrml. for 24 and 48 h and added 3 H-thymidine for the last 2 h of culture. The amount of incorporation in these cultures was compared to the initial incorporation over the first 2 h of culture before the addition of BDNF or IGF-I. We measured a significant amount of incorporation after labeling cells for the first 2 h of culture ŽTable 1, control.. This incorporation represents a measure of the number of granule cells in the cell cycle at the start of culture. Bromodeoxyuridine labeling experiments suggest that 30–40% granule cells are in the cell cycle at this time of culture wH. Cui and R. Bulleit unpublished observationsx. If 3 H-thymidine was added for 2 h at the end of a 24 h culture period then we observed a significant reduction in the amount of thymidine incorporation compared to the initial incorporation over the first 2 h of culture, 98%
Fig. 5. K252a selectively blocks BDNF’s effects on granule neuron survival and GABA A a 6 and p21cip1 mRNA expression. Granule cells were cultured in minimal medium containing 5 ngrml IGF-I plus or minus BDNF Ž10 ngrml. andror K252a Ž50 nM. as indicated. The percent surviving granule neurons was determine after 3 days of culture ŽA.. The asterisk Ž ) . indicates a statistically significant difference in mean percent survival between treated cultures and cultures maintained in MM alone, P - 0.01. Total RNA Ž ;15 m g. was isolated following 0–3 days in culture. Presented is RNA blot analysis using cDNA probes for GABA A a 6 and p21cip1 mRNAs ŽB.. Ethidium bromide staining of 18S rRNA was used to indicate the level of RNA loaded on gels. Also presented is the relative optical density of autoradiographic hybridization signals for p21cip1 mRNA ŽC.. The optical density was normalized to the level of 18S rRNA in 3 separate experiments. The level of 18S rRNA was determined by densitometry of the negative image of ethidium bromide stained gels. The asterisk Ž ) . indicates a statistically significant difference in mean hybridization values between IGF-IqBDNF ŽIB. and IGF-IqBDNFqK252A ŽIBK. treated cultures, P - 0.05.
X. Lin et al.r DeÕelopmental Brain Research 105 (1998) 277–286 Table 1 Incorporation of 3 H-thymidine into cultured cerebellar granule cells Culture treatment a
Control 5 ngrml IGF-I b 5 ngrml IGF-I b 10 ngrml BDNF b 10 ngrml BDNF b
Time in culture Žh.
CPM
% Control
2 24 48 24 48
11204"1220 236"29 52"4 342"36 98.9
y 2.1 0.5 3.1 0.9
a
Granule cells were cultured in minimal medium ŽMM. alone without trophic factors. About 2.5 m cirml of 3 H-thymidine was added at the start of culture and 2 h later 3 H-Thymidine incorporation was measured. b Granule cells were cultured in MM containing IGF-I or BDNF for 24 or 48 h 2.5 m cirml of 3 H-thymidine was added for the last 2 h of culture. 3 H-Thymidine incorporation was measured following this labeling period.
reduction in IGF-I and 97% reduction in BDNF ŽTable 1.. By 48 h of culture there was greater than a 99% reduction in thymidine incorporation ŽTable 1.. If the cell cycle time of proliferating granule cells is 18–24 h w15,42,44x, then these results suggest that most of the granule cells that are dividing at the start of culture do not reenter the cell cycle. Thus, most granule neuron precursors stopped proliferating by the first 24 h of culture Žgreater than 97%. in the presence of either IGF-I Ž5 ngrml. or BDNF Ž10 ngrml.. This is consistent with previous experiments indicating that low concentrations of IGF-I Ž1–5 ngrml. or BDNF Ž10 ngrml. do not substantially support granule cell proliferation in vitro w16,17x, wX. Lin, unpublished observationsx. The results indicate that 97–98% of the cells stopped proliferating before we observed an increase in p21cip1 RNA expression which occurred 48–72 h after the start of culture ŽFig. 4..
4. Discussion Neurogenesis in the central nervous system requires the precise temporal and spatial regulation of cell proliferation and differentiation. This regulation is dependent on both genetic and epigenetic cues. The experiments presented in this study suggest that epigenetic cues, specifically BDNF, can modulate differentiation of postnatal cerebellar granule cells. In comparison with IGF-I, BDNF dramatically accelerated the expression of granule cell terminal differentiation marker GABA A a 6 mRNA ŽFig. 4.. Previous studies suggest that even high concentrations of IGF-I Ž40 ngrml. did not accelerate GABA A a 6 expression w37x. Thus, BDNF appears to modify the timing of granule cell differentiation in vitro. Interestingly, the expression of the cell cycle inhibitor p21cip1 was also accelerated by BDNF treatment. The increase in p21cip1 expression occurred with a similar time course as the increase in expression of the terminal differentiation marker GABA A a 6. This increased expression occurred after the majority of cultured granule cells stopped proliferating ŽTable 1.. Thus, BDNF appeared to
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accelerate granule neuron terminal differentiation after granule neuron precursors had already withdrawn from the cell cycle. The observation that these cultures are 98% pure granule cells ŽSection 2. w23x and that GABA A a 6 expression occur exclusively in mature cerebellar granule neurons w31,33x suggest that BDNF may directly modulate gene expression in maturing granule neurons. It has been reported that postnatal granule neurons express functional BDNF receptors ŽTrkB. w17,51x. However, its is also possible that BDNF is acting indirectly by inducing a small minority of another cell type in the culture to produce factors which in turn modulate granule neuron gene expression. In either case BDNF can ultimately accelerate the maturation of cerebellar granule neurons in vitro. The observations that the increase in p21cip1 occurs after granule cell stop proliferating might suggest that the increase in p21cip1 expression is not required to direct granule neuron precursors out of the cell cycle but occurs as granule neurons initiate terminal differentiation. An increase in p21cip1 and GABA A a 6 RNA expression may be part of a granule neuron differentiation program that is specified prior to the time we isolate cell for culture w1,37x. Activation of Trk receptors by BDNF does not appear to be required for the ultimate expression of the program. Even in the absence of added BDNF or in the presence of the Trk inhibitor K252a, granule cells differentiated and expressed GABA A a 6 and p21cip1 ŽFigs. 3 and 5.. However, we cannot completely eliminate the possibility that BDNF or other trophic factors, produced by granule neurons themselves and acting through non Trk receptors such as the low affinity nerve growth factor receptor Žp75., are required for this program’s expression. We have previously observed that in the absence of all exogenously added trophic factors this program of gene expression appears to still function even though these cells are in the process of dying w37x. Thus, survival signals themselves are not require for this programs expression. This differentiation program may be regulated by granule cell intrinsic mechanisms. Although, exogenous factors may not be required for this program’s ultimate expression they appear to modulate the timing of its expression. BDNF can accelerate the expression of terminal differentiation markers. This modulation of gene expression may be a direct action of BDNF on the granule neuron differentiation program or alternatively an indirect result of BDNF’s survival promoting action. We can not entirely dissociate these two actions of BDNF. However, IGF-I which promotes survival to a similar degree as BDNF ŽFig. 5. does not accelerate gene expression. Thus, a general survival action is not enough to modulate this gene expression. BDNF may alternatively have a different survival promoting mechanism that includes a modulation of gene expression associated with granule neuron terminal differentiation. The same intracellular signal transduction cascade may mediate both the survival and differentiation promoting actions of BDNF and may differ from IGF-I survival promoting cascade.
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Ultimately BDNF can accelerate the maturation of cerebellar granule neurons in vitro. The phenotype of BDNF knock-out mice also suggests that BDNF may regulate the timing of granule cell development in vivo w29x. At postnatal day 17 the EGL is still present in BDNF Žyry . mutant mice while by this time in wild type mice the EGL is completely absent. However, the elimination of the EGL occurs by postnatal day 23 in mutant mice suggesting that cerebellar granule neuron development was delayed but not prevented. Several lines of evidence suggested that p21cip1 may contribute to cell cycle arrest during terminal differentiation of multiple cell types w12,27,45,56x. However, in the present study we observed an increase in p21cip1 expression that occurred after dividing granule cells left the cell cycle. This temporal distinction between the cessation of cell proliferation and the increase in p21cip1 expression would suggest that an increase in p21cip1 is not required for directing dividing granule cells out of the cell cycle. Thus p21cip1 must have an alternative function during this period of granule neuron differentiation. One function may be to maintain differentiating granule neurons in a non-proliferative state. In most proliferating cells the progression through the cell cycle is controlled by the activities of cyclin-dependent kinases ŽCdks.. Cdks are a family of serinerthreonine kinases which have almost no kinase activity in monomeric form and require interaction with members of the cyclin family of proteins for kinase activation. A number of polypeptides have been identified, which inhibit Cdk activity by direct association with cyclin–Cdk complexes. p21cip1 is the prototypic Cdk inhibitor which can inhibit G1 cyclin–Cdks in a stoichiometric fashion w22,62x. A low amount of p21cip1 is present in active cyclin–Cdk complexes and may potentially modulate their activity w62x. However, higher amounts of p21cip1 function as a potent inhibitor and arrests cells in the G1 phase of the cell cycle w21x. Recent experiments also suggest that p21cip1 can inhibit DNA synthesis via the binding of its’ carboxy-terminal region with the DNA polymerase d processivity factor, PCNA Žproliferating cell nuclear antigen.w8,36,40x. Thus, an increase in p21cip1 expression might provide and assurance that early differentiating granule neurons do not inappropriately reentering the cell cycle and begin DNA synthesis. Alternatively, p21cip1 could have a function separate from cell cycle control. Recent experiments suggest that p21cip1 may protect differentiating myocytes from apoptotic cell death w60x. A similar function may occur in differentiating neurons w46x. Thus, an increase in p21cip1 may be involved in BDNF’s survival promoting effect. However, this mechanism would differ from IGF-I’s survival promoting mechanism. IGF-I does not increase p21cip1 expression but maintains granule cell survival to a similar degree as BDNF ŽFig. 5.. During postnatal cerebellar development a transient but marked increase of p21cip1 mRNA was observed between postnatal day 12 and 18 ŽFig. 1B.. This is a period of
cerebellar development when many granule cells are undergoing terminal differentiation w3,4x. The related Cdk inhibitor p27 kip1 shares a common amino-terminal domain of 60 amino acids with p21cip1 w47,58x. This domain is responsible for interacting with cyclin–Cdk complexes and inhibiting their kinase activity w18,21,40x. However, their carboxy terminal regions are different and may specify independent and specialized functions. In the cerebellum p27 kip1 mRNA expression also occurs in the adult and is transiently increased during postnatal cerebellar development ŽFig. 1A.. The expression of p21cip1 and p27 kip1 mRNAs in the postnatal cerebellum is temporally distinct but overlapping, indicating a potential difference in their function during cerebellar development. The expression of these two related Cdk inhibitors also appears to be differentially regulated by epigenetic cues. BDNF modifies the level of p21cip1 mRNA but not p27 kip1 mRNA under our culture conditions ŽFig. 4.. However, under these conditions the level of expression of p27 kip1 appears to have peaked by the start of culture. Thus, BDNF may not be able to enhance its expression further. At an earlier point in development BDNF might regulate p27 kip1 expression. This regulation would depend on the presence of BDNF receptors on early developing granule cells. Previous experiments suggest that early proliferating granule cells may not express BDNF’s cognate receptor, TrkB w17x.
Acknowledgements We thank D. Frost and D. Burt for critical reading of the manuscript. This work was in part supported by the Bressler Research Fund of the University of Maryland at Baltimore.
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