Calcium binding protein (calbindin-D28k) gene expression in the developing and aging mouse cerebellum

Molecular Brain Research, 8 (1990) 283-290 Elsevier

283

BRESM 70235

Calcium binding protein (calbindin-D28k) gene expression in the developing and aging mouse cerebellum Anthony M. Iacopino 1, William B. Rhoten e and Sylvia Christakos 1 ~Department of Biochemistry and Molecular Biology and 2Departmentof Anatomy, Universityof Medicine and Dentistry of New Jersey~New Jersey Medical School, Newark, NJ 07103-2714 (U.S.A.) (Accepted 24 April 1990) Key words: Development; Aging; Cerebellum; Purkinje cell; Calbindin-D2sk; Immunocytochemistry; Hybridization cytochemistry; Mouse

Calbindin-D2s k (CaBPzsk) protein and gene expression were examined in the mouse cerebellum during development and aging utilizing slot and Northern blot hybridization analyses for mRNA levels, Western blot analysis and radioimmunoassay (RIA) for protein levels, and by in situ studies using immunocytochemistry and hybridization cytochemistry on prepared tissue sections. Samples were obtained and analyzed from C57BL/6J mice aged day of birth and postnatal weeks 1, 2, 4, 8, and 120. A specific cDNA and antibody for CaBP2sk were utilized in these studies. Analysis of mRNA levels showed a steady rise in CaBP2sk mRNA from birth to a peak at postnatal week (3.4-fold increase) and then a decline to steady-state levels at postnatal weeks 4 and 8 (47% reduction of peak level) followed by a reduction of CaBP2sk mRNA to birth levels at postnatal week 120. The specificity of the changes observed was tested by reprobing blots with fl-actin cDNA. Analysis of CaBP2sk protein levels by both Western blot and RIA showed a similar pattern. In situ analysis of CaBP2sk mRNA levels, based on hybridization signal (silver grains per cell), demonstrated a rise in cellular CaBP2sk mRNA levels which peaked at postnatal week 2 (416.9 + 52.1) and then declined to steady-state levels by postnatal weeks 4 and 8 (267.4 + 35.8). Cellular CaBP28k mRNA levels exhibited a dramatic reduction in the aged cerebellum (postnatal week 120; 78.3 + 16.0). The levels of cellular CaBP2sk mRNA corresponded to the intensity of immunoreactive CaBP2sk localized by immunocytochemistry. The results are consistent with the hypothesis that CaBP2sk may play a critical role in Purkinje cell maturation and maintenance. Decreased CaBP2sk gene expression in the aged cerebellum is not simply a consequence of cell loss (remaining viable cells exhibit reduced CaBP2sk gene expression). Thus, CaBP2sk may have a neuroprotective role and maintaining the concentration of CaBP2s k in Purkinje cells may be important for their survival. INTRODUCTION T h e r e has been much interest and speculation concerning the physiological role of calbindin-Dz8 k (CaBP2sk) in the central nervous system. CaBP28 k belongs to a family of calcium-binding proteins including caimodulin and troponin C 7"32"46. CaBP28 k has also been f o u n d in other tissues such as intestine and kidney w h e r e it has been shown to be vitamin D-dependent 17'35'38'41. Brain CaBP28 k represents one of the most abundant brain proteins (as much as 2% of the total protein in some brain areas) and, unlike CaBP28 k of intestine and kidney, is unresponsive to vitamin D administration 47. Brain CaBP28 k is a phylogenetically conserved protein which has been reported to be present in avian, reptilian, and c e p h a l o p o d brain. This has led to speculation that CaBP28 k in the brain serves a very fundamental role. Considering the calcium-binding ability of CaBP28k, this speculation has included the hypotheses that the protein acts as a t r o p h i c factor during neuronal development ~5 and as an intraneuronal calcium buffer 9"44.

The time course and characteristics of mouse cerebellar development are known 6'4°. Studies have shown that CaBP28 k expression is limited to the Purkinje cells in the mammalian cerebellum 2,15'25'28. This suggests that the protein may be involved in the development and maintenance of these cells. Indeed, some groups have advocated the use of CaBP28 k as a marker for Purkinje cell maturation 4s. The present study seeks in part to ascertain the temporal relationship between levels of CaBP28 k gene expression and major stages of Purkinje cell/cerebellar development. It has been recognized that neuronal degeneration and cell loss are part of the normal aging process and that the cerebellum is particularly affected (Purkinje cell loss) 42. During aging, there are changes in neuronal metabolism and an elevation of intraneuronal free calcium levels in the mammalian central nervous system. This leads to a significant change in the fieuronal Ca 2÷ environment 19' 31,49. Thus, it is likely that several key aspects of neuronal function will be affected (for example, maintenance of the cytoskeleton, synaptic transmission, and calcium-

Correspondence: S. Christakos, Department of Biochemistry and Molecular Biology, UMDNJ/New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103-2714, U.S.A. 0169-328X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

284 mediated

e n z y m e s y s t e m s ) 3"13'16'2°'33. T h e p r e s e n t s t u d y

a l s o s e e k s t o a s c e r t a i n w h e t h e r t h e r e is a n y r e l a t i o n s h i p b e t w e e n C a B P 2 s k g e n e e x p r e s s i o n in t h e a g e d c e r e b e l l u m a n d t h e a g e - r e l a t e d P u r k i n j e cell loss w h i c h is c h a r a c t e r istic o f t h i s b r a i n r e g i o n . M A T E R I A L S AND METHODS

Animals Mice (C57BL/6J) were obtained from Taconic Farms (Germantown, NY). Males aged day of birth and postnatal weeks 1, 2, 4, 8, and 120 were used in the study. Protein isolation Upon dissection, cerebellar samples were homogenized (20% wt./vol.) in cold phosphate buffered saline and centrifuged at 38,000 g for 30 min. Clear supernatant was harvested and stored at -20 °C. Total protein content was determined by the method of Lowry 29. RNA isolation Cerebelli were removed immediately upon sacrifice under sterile, RNase-free conditions and stored at -80 °C. Total RNA was prepared using the guanidinium thiocyanate/phenol-chloroform procedure of Chomczynski and Sacchi s. The integrity of the RNA was visualized by electrophoresis on 1.2% denaturing agarose gels via staining with ethidium bromide. Poly(A) ÷ mRNA was prepared via oligo(dT) chromatographyk Radioimmunoassay (RIA) Calbindin protein content was determined by use of the radioimmunoassay (RIA) for CaBPzs k developed in this laboratory 39. Iodination of rat CaBP2s k with 1251 involved the utilization of the Iodagen procedure 3°. CaBPzs k content is expressed as/~g CaBPzsk/ mg total protein and all RIA results represent data from 5 different animal samples per experimental group. Western blot Triplicate Western blots were performed for each experimental group (3 different animal samples using equivalent protein per experimental group) in order to confirm the RIA results. Standard Western blot procedures were modified as previously described 21 and used in conjunction with our specific antibody to CaBP28 k (ref. 39) and [125I]protein A. Results were analyzed using scanning densitometry. Slot blot Total R N A was used for the slot blot hybridization analysis of CaBPz8 k m R N A as previously described 47. The analysis was performed using a specific cDNA probe for CaBP2s k (a 1.2 kilobase (kb) mouse calbindin-Dzsk cDNA from the EcoRI site of plB176 (ref. 50) which was 32p-labeled by the oligonucleotide labeling procedure using the 'Prime-Time' oligo labeling system from IBI, New Haven, CT). Three different input amounts of tissue RNA were used to estimate the concentration of CaBP28 k mRNA. Standards using 100-fold range of brain RNA were used to determine linearity. After probing with CaBP28 k cDNA the slots were rehybridized with fl-actin cDNA. The relative optical density for each slot probed with CaBP28 k cDNA was divided by the relative optical density value obtained after probing with the control probe to normalize for sample variation in the amount of RNA dotted. Results are reported as arbitrary units of signal intensity. Specificity of changes in CaBP28 k mRNA levels was also tested by reprobing of blots with fl-actin cDNA. A 2.1 kb chicken fl-actin cDNA insert from the HindIIl site of pBR322 (ref. 11) was used. Results are reported as mean + S.E.M. and represent data from 5 different animal samples per experimental group. Northern blot Duplicate Northern blots 43 were performed for each experimental

group (2 different animal samples per experimental group) in order to confirm the slot blot results utilizing the Northern blot protocol for CaBP28 k mRNA previously described 47. All filters were rehybridized to [32p]fl-actin cDNA which was used as a control probe to detect specificity/RNA transfer problems and possible unequal loading of RNA on the gel.

Tissue preparation for in situ studies Mice were sacrificed, the cerebelli were removed, and either snap-frozen on dry ice and stored at -80 °C (hybridization cytochemistry) or immediately Bouin's-fixed (immunocytochemistry). Fresh-frozen tissue was embedded in OCT compound (Miles, Inc., Elkhart, IN) and sectioned at 10/tm in a cryostat at -20 °C. Sections were affixed to acid-cleaned, DEPC-treated microscope slides coated with poly-L-lysine and stored at -70 °C until use. Bouin'sfixed tissue was paraffin embedded, sectioned at 5/~m, affixed to acid-cleaned, poly-L-lysine-coated slides, and stored at room temperature until use. Hybridization cytochemistry In situ hybridization on tissue sections was carried out as described in detail previously 36. Briefly, slides were quickly brought to room temperature with a stream of cool air (hairdryer) and fixed for 5 min in fresh 3% paraformaldehyde (in 0.1 M phosphatebuffered saline with 0.2% DEPC, pH = 7.2). Slides were washed briefly in phosphate-buffered saline (PBS), 2x sodium citrate/ sodium chloride (SSC) pH = 7.0, and treated with acetic anhydride/ triethanolamine pH = 8.0 for 10 min. The slides were then rinsed in 2x SSC, PBS, and treated with 0.1 M glycine in 0.1 M Tris buffer pH = 7.0 for 20 min. Slides were rinsed in 2x SSC, dehydrated in ethanol (50-100% graded series), and allowed to air-dry on a rack. Radiolabeled probe was then applied to tissue sections which were covered with siliconized glass cover slips and sealed with rubber cement. Slides were then incubated in moist chambers inside heat-sealed pouches overnight at 45 °C. After incubation, coverslips were removed in 2 x SSC, slides were rinsed in 2× SSC, and washed in 50% deionized formamide in 2× SSC at 55 °C for 30 min (2 changes). The slides were briefly rinsed in 2x SSC and incubated in RNase A (100 ~g/ml) in 2x SSC at 37 °C for 30 min. The slides were then rinsed in 2x SSC, washed in 50% deionized formamide in 2× SSC at 55 °C for 20 min (2 changes), rinsed in 2x SSC, and washed overnight with 0.05% Triton X-100 in 2× SSC on a rotary shaker. The following day, slides were rinsed briefly in 0.05% Triton X-100 in 2x SSC, 300 mM ammonium acetate, and a graded series of ammonium acetate/ ethanol (300 mM ammonium acetate in 50-95% ethanol). Slides were then allowed to air-dry on a rack. A utoradiography Slides for hybridization cytochemistry were dipped in NTB3 emulsion (Eastman Kodak) via an automated dipping machine (V. Avarlaid). These slides were then stored at 4 °C in light-tight boxes which were heat-sealed in plastic pouches containing dessicant. Exposure was for 3-4 weeks. Slides were developed for 2 min with Kodak D19 diluted 1:1, counterstained with hematoxylin/eosin, and analyzed with a digitizing/image processing system (SEMPER, Iris). Analysis was based on calculation of CaBP~k mRNA levels (silver grains per cell) after background correction (taking into account the number of grains over nonhybridizing areas and the number of grains per cell in the sections exposed to sense (control) probe). lmmunocytochemistry Slides were de-waxed by incubation in xylene for 30 min (3 changes) and rehydrated in a graded series of ethanol (95% to water). Slides were treated with H20 2 (0.3%) in PBS for 15 min. Slides were then incubated for 60 min in a humid chamber with dilutions of primary antiserum (rabbit) against rat renal CaBP28 k (1 : 1,(X)0; 1: 10,000; 1: 100,000) and with CaBP2s k antiserum exposed to CaBP2s k (absorption controls). Slides were then washed with PBS and primary antibody was detected using Biotinylated donkey

285 anti-rabbit serum and streptavidin conjugated to horseradish peroxidase (Amersham Corp., Arlington Heights, IL). Analysis consisted of comparison of intensity of CaBP28k immunoeytochemical localization with quantitation of CaBP2sk mRNA levels via hybridization cytochemistry.

applied to the sections at a concentration of 5.0 pg per section. RESULTS

Analysis of CaBP28k protein levels

Radiolabeled RNA probes for hybridization cytochemistry Single-stranded RNA probes labeled with 35S were constructed from a linearized CaBPzak cDNA template using a riboprobe kit (Promega Corp., Madison, WI). The CaBP2sk cDNA (a 1.2 kb sequence in the EcoRI site of plBI76) was linearized with the appropriate restriction endonuclease. Digestion with Pvut followed by transcription with T7 RNA polymerase in the presence of [35S]UTP generated antisense (experimental) probe. Digestion with XbaI followed by transcription with SP6 RNA polymerase generated sense (control) probe. The control probe served as a test for specificity of hybridization as well as a method of background correction. Following RNA probe synthesis, the cDNA template was digested with DNAse and the RNA probes were phenol-chloroform/ isoamyl alcohol-extracted. The RNA probes were ethanol-precipitated and suspended in hybridization buffer (40% deionized formamide, 10% dextran sulfate, 10 mM dithiothreitol, 1 mg/ml yeast tRNA, 1 mg/ml sheared salmon sperm DNA, 1x Denhardt's solution, and 4x SSC) to the same activity. RNA probes were

Cerebellar Calbindin-D2ak LeveEs (Devel./Age)

Analysis of calbindin-D2ak (CaBP28k) p r o t e i n levels in the C57BL/6J m o u s e c e r e b e l l u m during d e v e l o p m e n t and aging was p e r f o r m e d by r a d i o i m m u n o a s s a y and W e s t e r n blot analysis. CaBP28 k levels were m e a s u r e d at d a y of birth and postnatal weeks 1, 2, 4, 8, and 120. T h e results are shown in Fig. 1. T h e level of p r o t e i n m e a s u r e d by R I A rises from d a y of birth (8.4 _+ 0.3 Mg/mg total

Cerebellar e-

CaBPzek mRNA Levels ( D e v e l . / A g e )

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Fig. 1. Changes in calbindin-D2sk (CaBP2sk) levels in the mouse cerebellum during development and aging. RIA results (above) are a graphic representation of data gathered from 5 different animals and illustrate CaBP2sk levels ~g/mg total protein; mean + S.E.M.) at various time points from day of birth (B) through postnatal weeks 1, 2, 4, 8, and 120 (1W, 2W, 4W, 8W, and 120W). Representative Western blot (below) confirms the result. Lanes 1-6 were loaded with 100 Mg of total protein and represent time points from day of birth (lane 1) through postnatal weeks 1, 2, 4, 8, and 120 (lanes 2-6).

120W

t0 5 I

2

3

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Fig. 2. Changes in CaBP2sk mRNA levels in the mouse cerebellum during development and aging. Slot blot hybridization results (above) are a graphic representation of data gathered from 5 different animals and illustrate CaBP2sk mRNA levels (arbitrary units; mean + S.E.M. after normalization with fl-actin control) at various time points from day of birth (B) through postnatal weeks 1, 2, 4, g, and 120 (1W, 2W, 4W, 8W, and 120W). Representative autoradiographs (below) showing 3 of the animals (lanes 1-3) from which the results were generated. Three amounts of total RNA were utilized for each time point (20, 10, 5 Mg) from day of birth (B) through postnatal weeks 1, 2, 4, 8, and 120 (1W, 2W, 4W, 8W, and 120W).

286 1

2

3

4

5

6

Northern Blot

TABLE I In situ determination of calbindin-D28 , gene expression in mouse cerebellar Purkinje cells Hybridization signal expressed as grains/cell (mean _+ S.E.M.). The number of cells analyzed for each time point was 50.

CoBP28k

/3-actin Fig. 3. Changes in CaBP2s k mRNA levels in the mouse cerebellum during development and aging. Representative Northern blot hybridization results illustrating the same pattern observed in Fig. 2. Lanes I-6 were loaded with 8/~g poly(A) ÷ mRNA and represent time points from day of birth (lane 1) through postnatal weeks 1, 2, 4, 8, and 120 (lanes 2, 3, 4, 5, and 6, respectively). Blots were stripped and reprobed with fl-actin cDNA as a check of specificity/ loading discrepancies.

Age (weeks)

Hybridization signal

2 8 120

416.9 + 52.1 267.4 + 35.8 78.3 + 16.0

protein) to a peak at postnatal week 2 (15.4 + 0.8 Hg/mg total protein) which represents a two-fold increase. The CaBPE8 k level then drops to a steady-state level at postnatal weeks 4 and 8 (13.0 + 0.7 Hg/mg total protein) and then falls off dramatically at postnatal week 120 (5.7 + 0.2 Hg/mg total protein) to a level below that which is present at birth. The Western blot analyses confirmed this result.

Fig. 4. Hybridization cytochemistry demonstrating changes in Purkinje cell CaBP28 k mRNA during development and aging. A: bright-field view of Purkinje cells (arrows) between the molecular layer (M) and granular layer (G) in a 10-gm-thick section at 19x magnification; B: dark-field view of A; C - E : higher magnification (60x) views of Purkinje cells (arrows) showing silver grains which represent positive hybridization of probe to CaBP28 k mRNA at various time points (C = 2 weeks postnatal; D = 8 weeks postnatal; E = 120 weeks postnatal). Note decrease in silver grains/cell from C through E.

287

Fig. 5. Immunocytochemistry demonstrating changes in CaBP28k levels in mouse cerebellar Purkinje cells during development and aging. A: absorption control section counterstained with hematoxyhn/eosin (magnification = 19x). Purkinje cells are not easily discernable between the molecular layer (M) and the granular layer (G); B: adjacent section to (A) exposed to CaBPEak antiserum (1:1,000 dilution). Purkinje cells (arrows) are now obvious; C-E: higher magnification (60 x) views of Purkinje cells (arrows) showing intensity of immunostalning which represents a qualitative estimation of CaBPEsk levels per cell at various time points (C = 2 weeks postnatal; D = 8 weeks postnatal; E = 120 weeks postnatal). Note decrease in the intensity of specific immunostaining from C through E and decrease in the number of immunoreactive Purkinje cells in E.

Analysis of CaBP28k mRNA levels Analysis of CaBP28 k m R N A levels was p e r f o r m e d via slot blot and N o r t h e r n blot analysis following the same f o r m a t as t h e protein m e a s u r e m e n t s . The results of the slot blot analyses are shown in Fig. 2. The level of CaBP28 k m R N A rises from day of birth to a peak at postnatal week 2 (a 3.5-fold increase) and the level then drops to a steady state at postnatal weeks 4 and 8 (a 47% reduction of p e a k level). This is followed by a significant decline in CaBP28 k m R N A at postnatal week 120 (to a level c o m p a r a b l e to the birth level). These results were confirmed by N o r t h e r n blot analysis (Fig. 3).

In situ analysis of CaBP28k gene expression Analysis of CaBP28 k m R N A and protein levels was

performed on prepared tissue sections through hybridization cytochemistry and immunocytochemistry for comparison with previous results. The same experimental format was used. The results of the hybridization cytochemistry (Fig. 4, Table I) were similar to the previous slot blot and Northem blot studies. Quantitation of CaBP28 k m R N A levels (based on silver grains/cell after background correction) showed a rise from birth levels to a peak at postnatal week 2 (416.9 + 52.1 grains/cell). CaBPzsk m R N A levels then declined by postnatal week 8 (267.4 + 35.8 grains/cell) and fell dramatically at postnatal week 120 (78.3 + 16.0 grains/cell). The results of the immunocytochemistry (Fig. 5) show a similar pattern and are in agreement with the previous R I A and Western blot analyses. There is a rise in staining

288 intensity to a peak at postnatal week 2 (Purkinje cell nuclei completely obliterated) and then a decline at postnatal week 8 (nuclei become visible) followed by a greater decrease in staining intensity at postnatal week 120 (nuclei are clearly visible). DISCUSSION At the present time, there is no information available concerning CaBP28 k gene expression in the developing mouse brain although immunocytochemical studies have been done in rats 44'45'48. This study represents the first comprehensive investigation of CaBP28 k gene expression during development and aging in the mouse brain. In addition to utilizing the standard analyses for protein (RIA and Western blot) and message levels (slot and Northern blot hybridizations), this study utilized in situ analyses (immunocytochemistry and hybridization cytochemistry). The in situ studies provided information on CaBP2s k gene expression at the cellular level. It was then possible to ascertain whether reduced levels of CaBP28 k and CaBP28 k mRNA observed in the other analyses were due to cell loss only or whether remaining viable cells actually expressed less protein and mRNA. This was an important and necessary distinction for the conclusions relative to CaBP28 k gene expression which were made. In the mouse, the final mitosis or birthday of the Purkinje cells usually occurs between embryonic days 11-13 ( E l l - E l 3 ) 23. In the rat, CaBP28 k has been seen as early as E14 in cerebellar Purkinje neurons 15. At birth, Purkinje cells are post-mitotic, immature cells which migrate postnatally to their ultimate positions TM. The peak of synapse formation between the parallel fibers and the Purkinje cells occurs at postnatal weeks 2-3 (PN2-3) and by PN4 all synaptic contacts are complete accounting for more than 95% of the synaptic input to Purkinje cells 5"6'14'18"26'27'40. By the end of PN2, all climbing fiber synapses to Purkinje cell dendritic branches are also completed giving rise to the definitive one to one relationship seen in the adult 4°. By the end of PN3, all Purkinje cells are present in their normal location and number 4 and by the beginning of PN5 the mouse cerebellum is thought to be fully mature 4°. The developmental data presented here show an increased gene expression for cerebeUar CaBP28 k from day of birth up to PN2 where peak gene expression occurs. CaBP28 k gene expression then declines to a steady-state level by PN4 and 8. Peak CaBP2s k gene expression is correlated with cessation of migration and peak synapse formation of Purkinje cells. Previous immunocytochemical studies in rats have shown similar results 44'45"48. Thus, the peak of CaBP28 k gene expression is associated with Purkinje cell synaptogenesis.

The role of CaBP2s k in the developing Purkinje cell is open to speculation. Presumably, as the cells mature (lay down mature dendrites and axons and receive increasingly complex synaptic contacts), levels of those proteins which are important in these activities should increase. It has been suggested that CaBP2s k acts by modulating calcium signals either as a buffer or via participation in calcium dependent enzyme-mediated processes 9'45. The onset of synaptogenesis may result in a more complex regulation of calcium fluxes, perhaps requiring increased intraneuronal levels of CaBP28 k. It is also interesting that the peak level of CaBP28k gene expression at PN2 is correlated with the appearance of the neuronal cytoskeleton 15'47. Ultrastructural studies have indicated some localization of CaBPEsk with cytoskeletal components 9. It is well known that the formation and maintenance of the cytoskeleton is calcium-sensitive and excess cytosolic Ca a+ levels can disrupt proper neurofilament and microtubule alignment2°'33. Thus, CaBP28k may have a role in protecting the neuronal cytoskeleton from changes in cytoplasmic Ca 2+ levels. The aging cerebellum has been reported to display significant Purkinje cell loss 12"22'37. The mechanism responsible for the cell loss is unknown. Previous work in our laboratory has shown a decreased CaBPEak gene expression during aging in both the rat and human cerebellum 24. This investigation demonstrates that there is a significant decline of CaBP2sk gene expression in mouse cerebellum between PN8 and 120 as measured by slot/Northern blot hybridization analysis and radioimmunoassay/Western blot analysis. In order to definitively answer the question of whether the decrease in CaBP2sk gene expression could be accounted for by Purkinje cell loss associated with aging or whether Purkinje cells remaining after cell loss truly displayed a decreased CaBP2s k gene expression, we performed in situ studies. Both the hybridization cytochemistry and immunocytochemistry results support our hypothesis that decreased CaBP28 k gene expression precedes cell loss. Analysis of the immunocytochemistry results (cell counts/field) confirmed a 15-20% loss of immunoreactive Purkinje cells in the aged cerebellum (PN120 compared to PNS). However, staining intensity in the viable cells which remained was significantly reduced. Analysis of the hybridization cytochemistry results (grains/cell) showed that, on a cellular level, there was a significant reduction of CaBP28 k mRNA in the viable, aged Purkinje cells (71% decrease, grains/cell, PN120 compared to PNS; Table I). It is possible that the decreased CaBP28 k gene expression in the aged Purkinje cells depletes the cell's ability to buffer cytosolic free calcium rendering it susceptible to the initiation of calcium-mediated irreversible cytotoxic events. Thus, CaBP28k may have a neuroprotective role

289 a n d m a i n t a i n i n g the c o n c e n t r a t i o n of CaBP28k in Purk i n j e cells m a y be critical for their survival.

Acknowledgements. This study was supported by National Institute of Health Grants 1K15DE-027 (to A.M.I.) and DK38961, NS20270 (to S.C.).

REFERENCES

tion, and discipline, Nature, 256 (1985) 586-589. 21 Gona, A., Pendurthi, T.K., Al-Rabiai, S. and Christakos, S., Immunocytochemical localization and immunological characterization of vitamin D-dependent calcium-binding protein in the bullfrog cerebellum, Brain Behav. Evol., 29 (1986) 176-183. 22 Greenough, W.T., McDonald, J.W., Parnisari, R.M. and Cawal, J.E., Environmental conditions modulate degeneration and new dendrite growth in cerebellum of senescent rats, Brain Res., 380 (1986) 136-143. 23 Herrup, K. and Trenkner, E., Regional differences in cytoarchitecture of the weaver cerebellum suggests a new model for weaver gene action, Neuroscience, 23 (1987) 871-885. 24 Iacopino, A.M. and Christakos, S., Specific reduction of calcium binding protein (Calbindin-Dzsk) gene expression in aging and neurodegenerative diseases, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 4078-4082. 25 Jande, S.S., Maler, L. and Lawson, D.E.M., Immunohistochemical mapping of vitamin D-dependent calcium-binding protein in the brain, Nature, 294 (1981) 765-767. 26 Landis, S.C., Ultrastructural changes in the mitochondria of cerebellar Purkinje cells of nervous mutant mice, J. Cell Biol., 57 (1973) 782-797. 27 Langley, O.K., Gombos, G., Hirn, M. and Goridis, C., Distribution of the neural antigen BSP-2 in the cerebellum during development, Int. J. Dev. Neurosci., 1 (1983) 393-401. 28 Legrand, C., Thomasset, M., Parkes, C.O., Clavel, M.C. and Rabie, A., Calcium-binding protein in the developing rat cerebellum: an immunocytochemical study, Cell Tissue Res., 233 (1983) 389-402. 29 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 30 Markwell, M.A.K., Iodination of proteins using the iodagen catalyst, Anal. Biochem., 125 (1982) 427-431. 31 Martinez, A., Victoria, J. and Starustegi, J., Cytosolic free calcium levels increase with age in rat brain synaptosomes, Neurosci. Leg., 88 (1988) 336-341. 32 Means, A.R. and Dedman, J.R., Calmodulin in endocrine cells and its multiple roles in hormone action, Mol. Cell. Endocrinol., 19 (1980) 215-219. 33 Metuzals, J., Pant, H., Gainer, H., Eagles, P.A.M., White, N.S. and Houghton, S., In vitro polymorphisms and phase transitions of the neurofilamentous network isolated from the giant-axon of the squid (loligo pealei L.), Cell Tissue Res., 252 (1988) 249-256. 34 Parmentier, M., Ghysens, M., Rypens, E, Lawson, D.E.M., Pasteels, J.L. and Pochet, R., Calbindin in vertebrate classes: immunohistochemical localization and western blot analysis, Gen. Comp. Endocrinol., 65 (1987) 399-410. 35 Pavlovitch, J.H., Laouari, D., Didierjean, L., Saurat, J.J. and Balsan, S., Skin calcium binding protein: distribution in other tissues. In F.L. Siegel, E. Carafoli, R.H. Kretsinger, D.H. MacLennan and R.H. Wasserman (Eds.), Calcium Binding Proteins: Structure and Function, Elsevier/North Holland, New York, 1980, pp. 417-418. 36 Rhoten, W.B. and Christakos, S., Cellular gene expression for calbindin-D28k in mouse kidney, Anat. Rec., 227 (1990) 145-151. 37 Rogers, J., Zornetzer, S.F., Shoemaker, W.J. and Bloom, EE., Electrophysiology of aging brain: senescent pathology of cerebellum. In S.J. Enna, T. Samorajski and B. Beers (Eds.), Brain Neurotransmitters and Receptors in Aging and Age-Related Disorders: Aging, Vol. 17, Raven, New York, 1981, pp. 81-94. 38 Roth, J., Baetens, D., Norman, A.W. and Garcia-Segura, L.M., Specific neurons in chick central nervous system stain with an antibody against chick intestinal vitamin D-dependent calciumbinding protein, Brain Res., 222 (1981) 452-458.

1 Aviv, H. and Leder, P., Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acidcellulose, Proc. Natl. Acad. Sci. U.S.A., 69 (1972) 1408-1412. 2 Baimbridge, K.G. and Miller, J.J., Immunohistochemicai localization of CaBP28k in the cerebellum, hippocampal formation and olfactory bulb of the rat, Brain Res., 245 (1982) 223-229. 3 Baudier, J. and Cole, R.D., Phosphorylation of tau proteins to a state like that in Alzheimer's brain is catalyzed by a calcium/calmodulin-dependent kinase and modulated by phospholipids, J. Biol. Chem., 262 (1987) 17577-17581. 4 Brion, J.P., Guilleminot, J. and Nunez, J., Dendritic and axonal distribution of the microtubule-associated proteins MAP2 and tau in the cerebellum of the nervous mutant mouse, Brain Res., 44 (1988) 221-232. 5 Burgayne, R.D. and Cambray-Deakin, M.A., The cellular neurobiology of neuronal development: the cerebellar granule cell, Brain Res., 13 (1988) 77-101. 6 Chen, S. and Hillman, D.E., Regulation of granule cell number by a predetermined number of Purkinje cells in development, Brain Res., 45 (1989) 137-147. 7 Cheung, W.Y., Calmodulin plays a pivotal role in cellular regulation, Science, 207 (1980) 19-22. 8 Chomczynski, P. and Sacchi, N., Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem., 162 (1987) 156-159. 9 Christakos, S., Gabrielides, C. and Rhoten, W.B., Vitamin D-dependent calcium binding proteins: chemistry, distribution, functional considerations and molecular biology, Endocrinol. Rev., 10 (1989) 3-26. 10 Christakos, S., Malkowitz, L., Sori, A., Sperduto, A. and Feldman, S.C., Calcium binding protein in squid brain: biochemical similarity to the 28,000 Mr vitamin D dependent calcium binding protein, J. Neurochem., 49 (1987) 1427-1434. 11 Cleveland, D.W., Lopata, M.A., MacDonald, R.J,, Cowan, N.J., Rutter, W.J. and Kirchner, M.W., Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and 7-actin genes using specific cloned cDNA probes, Cell, 20 (1980) 95-105. 12 Corsellis, J.A.N., Some observations on the Purkinje cell population and on brain volume in human aging. In R.D. Terry and S. Gershon (Eds.), Neurobiology of Aging, Vol. 3, Raven, New York, 1976, pp. 205-209. 13 Deary, I.J. and Hendrickson, A.E., Calcium and Alzheimer's disease, Lancet, May 24 (1986) 1219. 14 DeBarry, J., Gombos, G., Klupp, T. and Hamori, J., Alteration of mouse cerebellar circuits following methylazoxymethanol treatment during development: immunohistochemistry of GABAergic elements and electron microscopic study, J. Comp. Neurol., 261 (1987) 253-265. 15 Enderlin, S., Norman, A.W. and Celia, M.R., Ontogeny of the calcium binding protein calbindin-D28k in the rat nervous system, Anat. Embryol., 177 (1987) 15-28. 16 Farber, J.L., Minireview: the role of calcium in cell death, Life Sci., 29 (1981) 1289-1293. 17 Feldman, S. and Christakos, S., Vitamin D-dependent calciumbinding protein in rat brain: biochemical and immunocytochemicai characterization, Endocrinology, 112 (1982) 290-302. 18 Frostholm, A. and Rotter, A., The ontogeny of [3H]-muscimol binding sites in the C57BL/6J mouse cerebellum, Brain Res., 37 (1987) 157-166. 19 Gibson, G.E. and Peterson, C., Calcium and the aging nervous system, Neurobiol. Aging, 8 (1987) 329-331. 20 Gilbert, D.S. and Newby, B.J., Neurofilament disguise, destruc-

290 39 Sonnenberg, J., Pansini, A.R. and Christakos, S., Vitamin D-dependent rat renal calcium-binding protein: development of a radioimmunoassay, tissue distribution, and immunologic identification, Endocrinology, 115 (1984) 640-648. 40 Sotelo, C., Purkinje cell ontogeny: formation and maintenance of spines, Prog. Brain Res., 48 (1978) 149-170. 41 Taylor, A.N., Chick brain calcium binding protein: comparison with intestinal vitamin D-induced calcium binding protein, Arch. Biochem. Biophys., 161 (1974) 100-104. 42 Terry, R.D., Some biological aspects of the aging brain, Mech. Aging Dev., 14 (1980) 191-195. 43 Thomas, P.S., Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 5201-5205. 44 Thomasset, M., Parkes, C.O. and Cuisinier-Gleizes, P., Rat calcium-binding proteins: distribution, development, and vitamin D dependence, Am. J. Phys., E483 (1982) 287-291. 45 Thomasset, M., Rabie, A., Parkes, C.O., Desplan, C., Hanin, D. and Cuisinier-Gleizes, P., Vitamin D-dependent calciumbinding protein in the cerebellum: a marker of the Purkinje cell

development, Dev. Pharmacol. Ther,, 7 (1984) 6-11. 46 Van Eldik, L.J., Zendegui, J.G., Marshak, D.R. and Watterson, D.M., Calcium binding proteins and the molecular basis of calcium action, Int. Rev. Cytol., 77 (1982) 351-357. 47 Varghese, S., Lee, S., Huang, Y. and Christakos, S., Analysis of rat vitamin D-dependent calbindin-D28k gene expression, J. Biol. Chem., 263 (1988) 9776-9784. 48 Wassef, M., Zanetta, J.P., Brehier, A. and Sotelo, C., Transient biochemical compartmentalization of Purkinje cells during early cerebellar development, Dev. Biol., 111 (1985) 129-134. 49 Whitehouse, P.J., Clinical and neurochemical consequences of neuronal loss in the nucleus basalis of Meynert in Parkinson's disease and Alzheimer's disease, Adv. Neurol., 45 (1986) 393-397. 50 Wood, T.L., Kobayashi, Y., Frantz, G., Varghese, S., Christakos, S. and Tobin, A.J., Molecular cloning of mammalian 28,000 M, vitamin D-dependent calcium binding (calbindinD28k): expression of calbindin-D28k RNAs in rodent brain and kidney, DNA, 7 (1988) 585-593.