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Nerve growth factor increases calcium binding protein (Calbindin-D28K) in rat olfactory bulb
Brain Research, 578 (1992) 305-310 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
305
BRES 17655
Nerve growth factor increases calcium binding protein (Calbindin-D28K) in rat olfactory bulb A.M. Iacopino a, S. Christakos a, P.
Modi a and C.A. Altar b
aDepartment of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, Graduate School of Biomedical Sciences, Newark, NJ (USA) and bDepartment of Endocrine Research, Genentech Incorporated, San Francisco, CA (USA) (Accepted 10 December 1991) Key words: Calbindin-D2sK; Immunocytochemistry; Olfactory bulb; Nerve growth factor; Rat; Neurodegeneration
Calbindin-D28~ (CaBP2sK) is a soluble intracellular protein capable of sequestering micromolar concentrations of calcium. The in vivo regulation of CaBP28K by recombinant human nerve growth factor (rhNGF) was studied in adult, male rats. Via Alzet 2002 pumps, each rat received, for 14 days, a lateral ventricle infusion (i.c.v.; n = 5-6/group) of 12/A PBS/day containing 1.0/~g cytochrome C (control) or an equal amount of rhNGE Six other animals received a vehicle or rhNGF infusion into the central neostriatum. CaBP28K was elevated by 75% (P < 0.01) in the olfactory bulb following i.c.v, rhNGF in each of two experiments and was not altered in the temporal cortex, hippocampus, olfactory tubercle, cerebellum, or neostriatum. Direct striatal injections of rhNGF did not alter CaBPzsK in the neostriatum or other regions (including the olfactory bulb). The increases in olfactory bulb CaBPz8r protein levels were verified via Western blot analysis. CaBPEsK immunocytochemistry revealed that 33% of olfactory bulb neurons are immunoreactive for CaBP28K and that the number or proportion of immunoreactive neurons did not change with i.c.v, infusions of rhNGF, suggesting that exogenously delivered rhNGF augments the content of CaBP28K in olfactory bulb neurons that normally express the protein. Endogenous NGF may function as a neuroprotective factor by enhancing the ability of these cells to sequester cytoplasmic calcium and retard calcium-mediated neurodegeneration. INTRODUCTION The 28,000 M r vitamin D - d e p e n d e n t calcium binding protein was discovered in 1966 by Wasserman and Taylor 56. This protein has subsequently b e c o m e known as Calbindin-D2s K or CaBPzsK 55. CaBP28 K belongs to a family of calcium binding proteins which includes calmodulin, parvalbumin, troponin C, and S10054. Since its initial discovery in avian intestine, CaBP28 K has been identified in a variety of species 8'16'43'57 and in many other tissues including kidney 23'52, bone 9, pancreas 38'42, placenta 6, and brain 5A4'27. CaBP:8~ comprises up to 2% of the total soluble protein in some brain areas and is found in most major cell groups and fiber tracts 5'14'27. CaBP2s K immunoreactivity is detected throughout the soma, axon, dendrites, and nerve terminals, but not in glia. It is mainly a cytoplasmic protein, however, it is also associated with cytoskeletal elements 1l. In the brain, CaBP28 K can function as an intraneuronal calcium buffer that prevents accumulation of excessive levels of cytosolic free Ca 2+ 4,35,44, thereby protecting neurons against Ca2+-mediated degeneration 7"~3'~8'24-a6. In contrast to o t h e r tissues, where CaBP2s K gene expression is regulated by vitamin D,
CaBP28~ in the central nervous system is not responsive to vitamin D administration 45'5°. Until very recently, no h o r m o n e or chemical has d e m o n s t r a t e d the ability to regulate CaBP28K gene expression in the brain. Previous results from our l a b o r a t o r y indicate that corticosterone enhances CaBP28K gene expression in the rat hippocampus 25. It is possible that this relationship exists because type I corticosterone receptors and CaBP28K a r e both localized almost exclusively to the h i p p o c a m p a l areas C A x and dentate gyrus ~4'4°. The highest concentrations of nerve growth factor ( N G F ) 22, N G F m R N A 10, and N G F receptors 3"21"41 are located in those brain areas which also contain high amounts of CaBP28K 5'14"27. These brain areas include the cerebellum, olfactory bulb, hippocampus, neocortex, and c a u d a t e - p u t a m e n which can degenerate in A l z h e i m e r ' s disease, H u n t i n g t o n ' s Chorea, and advanced age. Decreased CaBP2s~: 24 and N G F 22'3° gene expression have been observed in these brain areas during aging and neurodegenerative diseases, suggesting that a direct or indirect relationship between N G F and CaBP28K may exist in the brain. A d r e n a l e c t o m y decreases N G F levels ~ and CaBP28K gene expression 25 in the rat hippocampus, further suggesting the possibility of an interaction between
Correspondence: A.M. Iacopino. Present address: Department of Anatomy, Baylor College of Dentistry, Dallas, TX 75246, USA.
306 t h e s e two proteins. I n d e e d , in P C 1 2 cells m a i n t a i n e d invitro, m u r i n e N G F induces the e x p r e s s i o n of genes for calcium binding p r o t e i n s w h o s e s e q u e n c e s are h o m o l o gous to t h o s e of the family of calcium binding p r o t e i n s which includes CaBP28K 34. In o r d e r to ascertain w h e t h e r N G F can effect CaBP28 K p r o t e i n levels in the brain, the p r e s e n t studies e x a m i n e d the effects of e x o g e n o u s adm i n i s t r a t i o n of r e c o m b i n a n t h u m a n N G F ( r h N G F ) on CaBP28K levels and CaBP28 K i m m u n o r e a c t i v i t y in the rat brain. MATERIALS AND METHODS
Animals Male Sprague-Dawley rats (200-220 g body weight) were subcutaneously implanted with osmotic minipumps (2002, 0.5 ~l/h, Alzet Corp., Palo Alto, CA) containing 216 /A of saline vehicle (PBS) with 18/~g cytochrome C or vehicle containing 18/ag of recombinant human NGF (rhNGF). Cytochrome C was used as a control protein because of its size and charge similarity to rhNGE The rhNGF was a generous gift of L.E. Burton (Genentech, Inc.) and has been characterized in prior reports 2"3. Twelve microliters of fluid (1 ~g cytochrome C or 1/~g rhNGF) were infused each day (n = 5-6 animals per group) into the left lateral ventricle via a 28 gauge stainless steel cannula (3280 P Plastics One, Roanoke, VA) that was stereotaxically implanted 0.8 mm posterior to bregma and 1.3 mm lateral to the midline. The cannula penetrated 4.0 mm below the surface of the skull to terminate in the lateral cerebral ventricle. A second series of animals (n = 5 animals per group) received identical vehicle or rhNGF infusions into the right or left caudate-putamen via 5.5 mm cannulas mounted 0.5 mm posterior to bregma and 3.0 mm from the midline suture. Animals were sacrificed 14 days later and brain areas were dissected and either snap frozen at -80°C or immersed in Bouin's solution (75% picric acid, 20% formalin, 5% glacial acetic acid) for immunocytochemical studies.
for 15 min and then incubated in a humid chamber for 60 min with dilutions of primary rabbit antisera generated against rat renal CaBPz8 K. The three dilutions used were 1:1000, 1:10,000 and 1:100,000. Some sections were incubated with CaBPesK antiserum exposed to CaBP28K (absorption controls). These sections were washed with PBS and primary antibody was detected using biotinylated donkey-antirabbit serum and streptavidin conjugated to horseradish peroxidase (Amersham Corp., Arlington Heights, IL). CaBP28 K was localized with peroxidase-antiperoxidase (PAP) and color formation was achieved with 3,3'-diaminobenzidine HC1 (DAB). The details of the PAP technique for CaBP28 K immunocytochemical localization have been described in a prior report 1°. Sections were counterstained with Cresyl violet, cover-slipped, and viewed under a Leitz Dialux 1I photomicroscope. Specificity of the DAB reaction product for CaBP28K was determined by the absence of staining in sections in which antiserum, preabsorbed with CaBP28 K, was substituted for the antiserum to CaBP28K (absorption control). The number of Cresyl violet-positive neurons and the number of CaBP28K-immunoreactive neurons were determined in the olfactory bulbs of rats that received vehicle or rhNGF (n = 5 animals per group). Stained neurons were counted in 3 randomly chosen microscopic fields in each of 5 sections located between 3 and 4 mm from the rostral tip of the olfactory bulb. The data were expressed as mean + S.E.M. of the ratio of CaBP28K-immunopositive cells per total cells observed (Cresyl violet-positive cells) in the microscopic field and tested for significance as in the RIA.
RESULTS CaBP28K levels d e t e r m i n e d via R I A in c e r e b e l l u m , c o r t e x , c a u d a t e - p u t a m e n , and h i p p o c a m p u s w e r e e q u a l to t h o s e r e p o r t e d p r e v i o u s l y 24'25. F o u r t e e n days of i.c.v. infusion with r h N G F e l e v a t e d CaBP28K by 75% in the o l f a c t o r y b u l b but not in the c e r e b e l l u m , cortex, caudate-putamen,
o r h i p p o c a m p u s (Fig. 1). R I A analysis
was r e p e a t e d on animals of a s e c o n d e x p e r i m e n t which i n c l u d e d g r o u p s that r e c e i v e d vehicle o r r h N G F
CaBP28K determination Tissue samples were homogenized (20% wt./vol.) in phosphatebuffered saline (4°C) and centrifuged at 38,000 x g for 30 min. The supernatant was stored at -20°C and total protein content was determined by the method of Lowry 31. CaBP28 K content was determined by radioimmunoassay (RIA) sl, using 12SI-labeled rat CaBP28K33. CaBP28 K was calculated as mean/ag CaBP28K/mg total protein + S.E.M. Group means were tested for significant differences by Student's t-test for two-group comparison or by analysis of variance (ANOVA) and Dunnett's t-test for multiple-group comparisons.
infu-
sions into the c a u d a t e - p u t a m e n in a d d i t i o n to the lateral
Effect of NGF on CaBP2sk Levels in Rat Brain 25 r
lmmunoblot (Western blot)
15
A modified Western blot procedure 2° was performed using supernatants from two randomly chosen vehicle or rhNGF-infused animals from each of the two RIA experiments. Autoradiographs were analyzed using scanning densitometry.
10
lmmunocytochemistry A specific antisera against CaBP28K51 (suitable for CaBP28 K immunocytochemistry in brain) was used to localize the protein in olfactory bulb sections from vehicle and rhNGF-infused animals. Within 5 rain of sacrifice, olfactory bulbs were immersed in Bouin's fixative and, within 4 days, each pair of olfactory bulbs was paraffin-embedded. Tissue sections (10/~m) were generated on a microtome and affixed to poly-L-lysine coated slides. Lipids were removed from the sections in xylene (3 changes, 30 min each) and sections were rehydrated in graded ethanols (95, 75, 70, 50% ethanol) and water. The sections were treated with 0.3% H20 2 in PBS
C| -
CO
Oil -I-
-
÷
--
CS "I-
--
HC ÷
--
AREA +
NGF
Fig. 1. Effect of 14 day i.c.v, infusion of PBS vehicle (-) or 1/~g/ day rhNGF (+) on CaBP28 K levels in rat cerebellum (Ce), olfactory bulb (OB), cortex (Co), caudate-putamen (CS), and hippocampus (HC). RIA values are reported as mean + S.E.M. (n = 5 animals per group) with significant differences (P < 0.01) marked by an asterisk.
307 Effects
of
NGF on CoBPaa K Levels in Rat Brain
CaBP2sr-positive neurons~total neurons in olfactory bulbs of norreal and rhNGF-treated animals
30
2s
The data represents CaBP28K-positive neurons/Cresyl violet stained neurons per microscopic field (data for each animal was obtained by pooling cell counts from 5 separate sections counting 3 separate fields per section generating 15 microscopic fields).
Q.
~ 20 2
e,l
~ (30 o
TABLE I
5
- +
C
CE
-
+
C
o0
-
+
R
L
OT
-
+
R
cs
L
-
+
Control
rhNGF-treated
1 2 3 4 5
1500/4818 1436/4244 1949/5772 1683/5239 1711/4712
1642/4581 1569/4594 1833/5421 1751/5002 1590/4436
RL
HC
Fig. 2. CaBP28K levels in rat brain areas (n = 6 animals per group) after 14 days of i.c.v, or intrastriatal infusions of vehicle (-) or rhNGF (+). Abbreviations are as in Fig. 1 with aT, olfactory tubercle. Bilateral brain areas were pooled and measured after intrastriatal infusions for CE and OB (c). Unilateral areas were measured separately in aT, CS, and HC after intrastriatal infusions (right, R; left, L). Dependent values, significance level, and other parameters are as described in Fig. 1.
ventricle. A g a i n , the only brain area to show a response to r h N G F administration was the olfactory bulb and, again, a 75% elevation in immunoreactive CaBP28 K was obtained (Fig. 2). Infusion of r h N G F into the caudatep u t a m e n had no effect on CaBP28 K levels in the olfactory bulb or o t h e r brain areas, including the caudatep u t a m e n itself. Western blot analysis was p e r f o r m e d to c o r r o b o r a t e
EFFECT OF NGF ON CoBP28k LEVELS (Western Blot Rot Olfactory Bulb)
28,000
Animal
M r-
the R I A results observed for the olfactory bulb. Fig. 3 shows the results for one set of randomly selected vehicle and r h N G F - t r e a t e d animals from one of the two experiments. CaBP28~: immunoreactivity on Western blot analysis was noticeably elevated by r h N G F administration. A l t h o u g h Western blot data are not strictly quantitative, densitometric analysis of autoradiographs revealed that r h N G F - i n f u s e d animals exhibited increases in protein levels ranging from 68-86%. These figures are in a g r e e m e n t with the R I A results. Immunocytochemical analyses were p e r f o r m e d to ascertain whether the increases in CaBP28 K observed in the R I A and Western blot studies were due to increased CaBP28K levels in neurons already expressing the protein, or whether the elevations in CaBP28 K were due to increased numbers of neurons expressing the protein. CaBP28K-positive neurons n u m b e r e d between 80 and 140 p e r microscopic field accounting for 30-40% of the total neurons observed. T h e labeled cells were observed in the glomerular, external plexiform, and internal granular layers of the olfactory bulb. T h e r e was no difference in the n u m b e r of CaBP28K-immunoreactive neurons in the olfactory bulbs of control and r h N G F - t r e a t e d animals. The total n u m b e r of neurons (Cresyl violet stained neurons) p e r microscopic field did not change following r h N G F administration (see Table I), thus there was no change in the p r o p o r t i o n of total neurons in the olfactory bulb that were counterstained with CaBP2s K antisera (Fig. 4), suggesting that the increase in CaBP2s K observed in the olfactory bulb represents an elevation of CaBP28K levels in neurons that normally express the protein.
C NGF Fig. 3. Western blot analysis of CaBP28K levels in rat olfactory bulb. Eight animals (2 vehicle and 2 rhNGF-infused animals from each of the RIA experiments) were compared according to rhNGF status. A representative animal pair is shown (NGF, rhNGF-treated; c, vehicle-treated). Elevation of CaBP28K in the olfactory bulb of rhNGF-treated animals was observed.
DISCUSSION The present results show that CaBP28K levels are relatively high in the rat olfactory bulb and that intraventricular infusions of r h N G F elevate CaBP28 K levels in the
308
Immunocytochemical Chora.cterzation of CaBP28 K Neurons in Rat Olfactory Bulb
Condition Animal# CoBP2/Totol (Mean SEM) Totals Control
NGF
1 2 3 4 5
.31 :~ .02 .34 + .02 .34 :~ .02 .32:1:.01 .36 + .01
.33 ± .02
I 2 3 4
.36 .34 .34 .35 .36
.35 i .01
5
:I: 4:t: • :~
.01 .03 .04 .03 .03
The data is expressedas a ratio of Calbindin-D2~immunopositive cells per total cells observedin the microscopicfield. The ratio for each animal was obtained by pooling cell counts from 5 separatesections counting 3 separate fields per section (15 total fields). Fig. 4. Immunocytochemical characterization of CaBP28K neurons in rat olfactory bulb. Representative photomicrograph showing CaBP28 ~ immunopositive neurons detected by immunocytochemistry in control (A) and NGF-treated animals (B). Results were quantitated by cell counting procedures (below). Results are expressed as means + S.E.M. of the number of CaBP28K immunopositive cells/total cells observed in each microscopic field. For 5 animals in each experimental group (control vs. rhNGF-treated), the relative number of CaBP28K immunopositive neurons did not differ.
bulb but not in 5 other brain areas tested. Administration of rhNGF increased CaBP2s K content, as measured by R I A and Western blot analyses, but not the number of bulb neurons that contained the protein. Thus, the CaBP28 K content per bulb neuron was increased. Immunocytochemical analysis is not quantitative and was utilized to address the question of cell number in rhNGFtreated animals. The R I A and Western blot assays were performed using whole tissue homogenates, thus it was impossible to determine which neuronal cell types within the olfactory bulb contained increased levels of CaBP28K following rhNGF treatment. Analysis of CaBP28 K m R N A in similarly-treated animals could help reveal whether increased protein levels resulted from augmented transcription or decreased degredation. In addition, in situ hybridization studies may reveal whether specific neuronal cell types within the olfactory bulb contain increased levels of CaBP28 K m R N A (i.e. mitral cells vs. all other neurons in the bulb). In any case, the augmentation of olfactory bulb CaBP28 K levels in response to rhNGF treatment is suggestive of a positive relationship between endogenous N G F and CaBP28 K gene expres-
sion. Previous results indicate that CaBP2sK plays a critical role in Ca 2+ homeostasis 4'35'44 and that CaBP28 K gene expression is reduced in aging and neurodegenerative diseases 24. Other studies have shown that excessive neuronal influx of Ca 2÷ is associated with neuronal death induced by excitatory amino acid stimulation 7"13. CaBP28 K may be a critical intracellular calcium buffer that prevents calcium-induced excitatory amino acid-dependent neurotoxicity 4'7'13A8"24-26'35'44. Thus, in addition to the ability of NGF to promote the survival of injured neurons 22'37, NGF may also function as a neuroprotective factor by augmenting calcium sequestration by CaBP28 K. It is well-known that the olfactory system degenerates in patients with Alzheimer's disease 12"39. Perhaps the prominent neurodegeneration of the olfactory bulb seen in Alzheimer's disease 39 represents Ca2÷-me diated neurotoxic sequelae resulting from decreased CaBP28 K levels secondary to a deficiency of human N G F synthesis, binding, or transport. There are two possible mechanisms which may explain the effect of rhNGF on CaBP2s K levels in the rat olfactory bulb. Firstly, augmentation of olfactory bulb CaBP2s K by ventricular delivery of rhNGF may be due to a transynaptic mechanism initiated at a nucleus where i.c.v. NGF is able to gain access and which projects to the olfactory bulb. The horizontal limb of the diagonal band (HDB) is one such area x5'46'47. Cholinergic neurons that innervate the olfactory bulb transport NGF in a retrograde, receptor-dependent manner a. These neurons originate in the HDB and innervate neurons in the periglomerular and glomerular cell layers of the olfactory bulb 32'28. Since the CaBP2sK-positive neurons in the bulb are also observed in these areas 17, the ability of the rhN G F to increase CaBP28 K may be mediated via cholinergic input from the HDB. Thus, transynaptic effects of rhNGF, via intrinsic connections between rhNGF-responsive cells and CaBP28K-positive cells are possible. Experiments with HDB lesions or cholinergic agonist/antagonist drug administration could help to identify such a mechanism. Secondly, NGF receptors are found in the periglomerular cell layer of the olfactory bulb 53. If these cells contain CaBPa8 K, then the rhNGF action could be mediated directly at the receptor level. Clearly, co-localization studies for CaBP28 K and NGF receptors in the olfactory bulb would help resolve these different mechanisms. Direct infusions of rhNGF into the HDB as well as into the olfactory bulb may resolve whether the 75% increase in olfactory bulb CaBP28K is due to local (receptor-mediated) or distal (transynaptic) influences of rhNGF. The biological activity of rhNGF would depend upon binding to high affinity (presumably biologically active) receptors rather than low affinity receptors 2"41'53. The high affinity binding sites in the rat brain have been
309 mapped using a protocol which distinguishes between these two types of receptors 3. It has been shown that each of the brain areas tested contain a high density of N G F receptors 3m and that while most brain regions share both high and low affinity receptors, the caudate/ putamen and neocortex are enriched with high affinity receptors 3. This, it is not likely that the selective effect of r h N G F on CaBP28 K levels in the olfactory bulb would be due to differences in the relative amount of high affinity N G F receptor present in the various brain regions under study. The olfactory bulb is unique among these brain areas as it receives innervation by neurons that are capable of regeneration 36. The sensory neurons of the nasal epithelium and their terminals in the olfactory bulb are in a continual cycle of mortality, development, and maturation. If the effects of r h N G F on CaBP28K levels are more pronounced in neuronal environments in which synaptic contacts are being established, then N G F may alter CaBP2s K in additional regions of developing brains or during reinnervation following neuronal injury. Additionally, CaBP28 K is found in many neuronal populations outside of the olfactory bulb 5'11'14't7'27 and may be regulated in these areas by other neurotrophic factors as well as by corticosteroids 1'25. Besides corticosterone, rhN G F is presently the only agent known to alter CaBP28K. Future research into exogenous manipulation of vitamin-D resistant brain CaBP28 K may lead to therapeutic regimens that can attenuate calcium-mediated neurodegeneration. Such studies may also help elucidate the cal-
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cium-dependency of neurotoxicity in vivo, as has been shown in vitro 4"35'44. The ability of r h N G F to augment CaBP2sK in the olfactory bulb but not in the other brain areas studied is significant, since CaBP28K (present results), N G F m R N A 19'49'5s, N G F protein-like immunoreactivity 29'4s, and receptors for N G F 3'41 are each found in these brain areas. Nevertheless, limited delivery of r h N G F to these parenchymal areas following intraventricular (i.c.v.) infusion may have prevented effects on CaBP2sK. Intraventricular infusions of iodinated antibodies to murine N G F or r h N G F itself (Altar et al., unpublished observations), deliver retrogradely transported material to cholinergic nuclei that project to the neocortex, olfactory bulb, hippocampus, and other brain areas. However, little or no radioactivity is found directly within these or other parenchymal areas, except within the neostriatum where label is found only in cholinergic neurons that border the ventricular injection site (Altar et al., unpublished observations). However, intrastriatal injections of r h N G F also failed to augment striatal CaBP28K, suggesting that the regulation of neostriatal CaBP2sK is unresponsive to r h N G E Thus, the failure of r h N G F given i.c.v, to augment CaBP28K outside of the olfactory bulb is probably not due to limited delivery to other brain areas but to the lack of control of CaBP2sK levels by rhNGF, as shown for the neostriatum. Acknowledgements. This study was supported by National Institute of Health Grants 1K15DE-027 (to A.M.I.) and DK38961, NS20270 (to S.C.).
cium binding protein in bone, Science, 202 (1978) 7027-7030. 10 Christakos, S., Rhoten, W.B. and Feldman, S.C., Rat calbindin-D2sK: purification, quantitation, immunocytochemical localization, and comparative aspects, Met. Enzymol., 139 (1987) 534-551. 11 DiFiglia, M., Christakos, S. and Aronin, N., Ultrastructural localization of immunoreactive calbindin-D2sK in the rat and monkey basal ganglia, including subcellular distribution with colloidal gold labeling, J. Comp. Neurol., 279 (1989) 653-665. 12 Eseri, M.M. and Wilcock, G.K., The olfactory bulbs in Alzheimer's disease, J. Neurol. Neurosurg. Psych., 47 (1984) 56-60. 13 Farber. J.L., Minireview: the role of calcium in cell death, Life Sci., 29 (1981) 1289-1298. 14 Feldman, S.C. and Christakos, S., Vitamin D-dependent calcium binding protein in rat brain: biochemical and immunocytochemical characterization, Endocrinology, 112 (1983) 290299. 15 Fibiger, H.C., The organization and some projections of cholinergic neurons of the mammalian forebrain, Brain Res. Rev., 4 (1982) 327-388. 16 Fullmer, C.S. and Wasserman, R.H., Isolation and partial characterization of intestinal calcium binding protein from cow, pig, horse, guinea pig and chick, Biochem. Biophys. Acta., 393 (1975) 134-140. 17 Garcia-Segura, L.M., Baetens, D., Roth, J., Norman, A.Q. and Orci, L., Immunohistochemical mapping of calcium-binding protein immunoreactivity in the rat central nervous system, Brain Res., 296 (1984) 75-86.
310 18 Gilbert, D.S. and Newby, B.J., Neurofilament disguise, destruction, and discipline, Nature, 256 (1988) 586-590. 19 Goedert, M., Fine, A., Hunt, S.P. and Ullrich, A., Nerve growth factor mRNA in peripheral and central rat tissues and in the human central nervous system: lesion effects in the rat brain and levels in Alzheimer's disease, Mol. Brain Res., 1 (1986) 85-92. 20 Gona, A., Pendurthi, T.K., A1-Rabai, 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) 176-193. 21 Hefti, F. and Mash, D.C., Localization of nerve growth factor receptors in the normal human brain and in Alzheimer's disease, Neurobiol. Aging, 10 (1989) 75-87. 22 Hefti, F. and Weiner, W.J., Nerve growth factor and Alzheimer's disease, Ann. Neurol., 20 (1986) 275-281. 23 Hermsdorf, C.I. and Bronner, E, Vitamin D-dependent calcium binding protein from rat kidney, Biochem. Biophys. Acta., 317 (1975) 172-179. 24 Iacopino, A.M. and Christakos, S., Specific reduction of calcium binding protein (28-kilodalton calbindin-D) gene expression in aging and neurodegenerative diseases, Proc. Natl. Acad. Sci. USA, 87 (1990) 4078-4082. 25 Iacopino, A.M. and Christakos, S., Corticosterone regulates calbindin-D2s K mRNA and protein levels in rat hippocampus, J. Biol. Chem., 265 (1990) 10177-10180. 26 Iacopino, A.M., Rhoten, W.B. and Christakos, S., Calcium binding protein (calbindin-DzsK) gene expression in the developing and aging mouse cerebellum, Mol. Brain Res., 8 (1990) 283-290. 27 Jande, S.S., Maler, L. and Lawson, D.E.M., Immunocytochemical mapping of rat vitamin D-dependent calcium binding protein in brain, Nature, 294 (1981) 765-768. 28 Kasa, P., The cholinergic systems in brain and spinal cord, Prog. Neurobiol., 26 (1986) 211-272. 29 Korsching, S., Auburger, G., Heumann, R., Scott, J. and Thoenen, H., Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation, EMBO J., 4 (1985) 13890-13903. 30 Larkfors, L., Ebendal, T., Whittemore, S.R., Persson, H., Holler, B. and Olson, L., Decreased level of nerve growth factor (NGF) and its messenger RNA in the aged rat brain, Mol. Brain Res., 3 (1987) 55-60. 31 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 32 Macrides, F., Davis, B.J., Youngs, W.M., Nadi, N.S. and Margolis, F.L., Cholinergic and catecholaminergic afferents to the olfactory bulb in the hamster: a neuroanatomical, biochemical, and histochemical investigation, J. Comp. Neurol., 203 (1981) 495-514. 33 Markwell, M.A.K., Iodination of proteins using the iodagen catalyst, Anal. Biochem., 125 (1982) 427-431. 34 Masiakowski, P. and Shooter, E.M., Nerve growth factor induces the genes for two proteins related to a family of calcium binding proteins in PC12 cells, Proc. Natl. Acad. Sci. USA, 85 (1988) 1277-1281. 35 Mattson, M.P., Rychlik, B., Chu, C. and Christakos, S., Evidence for calcium-reducing and excitoprotective roles for the calcium binding protein calbindin-D2s K in cultured hippocampal neurons, Neuron, 6 (1991) 41-51. 36 Meisami, E., Relative contributions of brain and peripheral connections to postnatal growth and cell accretion in the rat olfactory bulb, Brain Res., 394 (1986) 282-291. 37 Montero, C.N. and Hefti, E, Rescue of lesioned septal cholinergic neurons by nerve growth factor: specificity and requirement for chronic treatment, J. Neurosci., 8 (1988) 2985-2999. 38 Morrisey, R.L., Bucci, T.J., Empson, R.N. and Lufkin, E.G., Calcium binding protein: its cellular localization in jejunum, kidney and pancreas, Proc. Soc. Exp. Biol. Med., 149 (1975)
56-66. 39 Ohm, T.G. and Braak, H., Olfactory bulb changes in Alzheimer's disease, Acta. Neuropathol., 73 (1987) 365-369. 40 Reul, J.M.M. and deKloet, E.R., Two receptor systems for corticosterone in rat brain: microdistribution, Endocrinology, 117 (1985) 2505-2511. 41 Riopelle, R.J., Verge, V.M.K. and Richardson, P.M., Distribution and characteristics of nerve growth factor binding on cholinergic neurons of rat and monkey forebrain, Neurochem. Res., 12 (1987) 923-928. 42 Rhoten, W.B. and Christakos, S., Vitamin D-dependent calcium binding protein is present in mammalian beta cells, Diabetes, 32 (1983) 130-134. 43 Rhoten, W.B., Lubit, B. and Christakos, S., Avian and mammalian vitamin D-dependent calcium binding protein in reptilian nephron, Gen. Compar. Endo., 55 (1984) 96-101. 44 Scharfman, H.E. and Schwartzkroin, P.A., Protection of dentate hilar cells from prolonged stimulation by intracellular calcium chelation, Science, 246 (1989) 257-260. 45 Schneeberger, P.R., Norman, A.W. and Heizmann, C.W., Parvalbumin and vitamin D-dependent calcium binding protein (M r 28,000): comparison of their localization in normal and rachitic rats, Neurosci. Lett., 59 (1985) 97-101. 46 Schweitzer, J.B., Nerve grwoth factor receptor-mediated transport from CSF labels cholinergic neurons: direct demonstration by a double-labeling study, Brain Res., 490 (1989) 390-396. 47 Seiler, M. and Schwab, M.E., Specific retrograde transport of nerve growth factor (NGF) from neocortex to nucleus basalis in the rat, Brain Res., 300 (1984) 33-39. 48 Senut, M.C., Lamour, Y., Lee, J., Brachet, E and Dicou, E., Neuronal localization of the nerve growth factor precursor-like immunoreactivity in the rat brain, Int. J. Dev. Neurosci., 8 (1990) 65-80. 49 Shelton, D.L. and Reichardt, L.F., Studies on the expression of beta NGF gene in the central nervous system: level and regional distribution of NGF mRNA suggest that NGF functions as atrophic factor for several neuronal populations, Proc. Natl. Acad. Sci. USA, 83 (1986) 2714-2718. 50 Sonnenberg, J., Luine, V.L., Krey, L. and Christakos, S., 1,25 Dihydroxyvitamin-D 3 treatment results in increased choline acetyltransferase activity in specific brain nuclei, Endocrinology, 118 (1986) 1433-1439. 51 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. 52 Staun, M., Noren, O. and Sjostrom, H., Calcium binding protein from human kidney, Biochem. J., 217 (1984) 229-236. 53 Taniuchi, M.J., Schweitzer, J.B. and Johnson, E.M., Nerve growth factor receptor molecules in the rat brain, Proc. Natl. Acad. Sci. USA, 83 (1986) 4094-4098. 54 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) 1-I0. 55 Wasserman, R.H., Nomenclature of the vitamin-D induced calcium binding proteins. In A.W. Norman, K. Shaefer, H.G. Grigoleit and D.V. Herrath (Eds.), Vitamin D: Chemical Biochemical and Clinical Update, Walter de Gruyter, Berlin, 1985. 321 pp. 56 Wasserman, R.H. and Taylor, A.N., Vitamin D3-induced calcium binding protein in chick intestinal mucosa, Science, 152 (1966) 791-793. 57 Wasserman, R.H. and Taylor, A.N., Evidence for a vitamin D3-induced calcium binding protein in new world primates, Proc. Soc. Exp. Biol. Med., 136 (1971) 25-35. 58 Whittemore, S.R., Ebendal, T., Larkfors, L., Olson, L., Seiger, A., Stromberg, I. and Peterson, H., Developmental and regional expression of nerve growth factor messenger RNA and protein in the rat central nervous system, Proc. Natl. Acad. Sci. USA, 83 (1986) 817-821.
305
BRES 17655
Nerve growth factor increases calcium binding protein (Calbindin-D28K) in rat olfactory bulb A.M. Iacopino a, S. Christakos a, P.
Modi a and C.A. Altar b
aDepartment of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, Graduate School of Biomedical Sciences, Newark, NJ (USA) and bDepartment of Endocrine Research, Genentech Incorporated, San Francisco, CA (USA) (Accepted 10 December 1991) Key words: Calbindin-D2sK; Immunocytochemistry; Olfactory bulb; Nerve growth factor; Rat; Neurodegeneration
Calbindin-D28~ (CaBP2sK) is a soluble intracellular protein capable of sequestering micromolar concentrations of calcium. The in vivo regulation of CaBP28K by recombinant human nerve growth factor (rhNGF) was studied in adult, male rats. Via Alzet 2002 pumps, each rat received, for 14 days, a lateral ventricle infusion (i.c.v.; n = 5-6/group) of 12/A PBS/day containing 1.0/~g cytochrome C (control) or an equal amount of rhNGE Six other animals received a vehicle or rhNGF infusion into the central neostriatum. CaBP28K was elevated by 75% (P < 0.01) in the olfactory bulb following i.c.v, rhNGF in each of two experiments and was not altered in the temporal cortex, hippocampus, olfactory tubercle, cerebellum, or neostriatum. Direct striatal injections of rhNGF did not alter CaBPzsK in the neostriatum or other regions (including the olfactory bulb). The increases in olfactory bulb CaBPz8r protein levels were verified via Western blot analysis. CaBPEsK immunocytochemistry revealed that 33% of olfactory bulb neurons are immunoreactive for CaBP28K and that the number or proportion of immunoreactive neurons did not change with i.c.v, infusions of rhNGF, suggesting that exogenously delivered rhNGF augments the content of CaBP28K in olfactory bulb neurons that normally express the protein. Endogenous NGF may function as a neuroprotective factor by enhancing the ability of these cells to sequester cytoplasmic calcium and retard calcium-mediated neurodegeneration. INTRODUCTION The 28,000 M r vitamin D - d e p e n d e n t calcium binding protein was discovered in 1966 by Wasserman and Taylor 56. This protein has subsequently b e c o m e known as Calbindin-D2s K or CaBPzsK 55. CaBP28 K belongs to a family of calcium binding proteins which includes calmodulin, parvalbumin, troponin C, and S10054. Since its initial discovery in avian intestine, CaBP28 K has been identified in a variety of species 8'16'43'57 and in many other tissues including kidney 23'52, bone 9, pancreas 38'42, placenta 6, and brain 5A4'27. CaBP:8~ comprises up to 2% of the total soluble protein in some brain areas and is found in most major cell groups and fiber tracts 5'14'27. CaBP2s K immunoreactivity is detected throughout the soma, axon, dendrites, and nerve terminals, but not in glia. It is mainly a cytoplasmic protein, however, it is also associated with cytoskeletal elements 1l. In the brain, CaBP28 K can function as an intraneuronal calcium buffer that prevents accumulation of excessive levels of cytosolic free Ca 2+ 4,35,44, thereby protecting neurons against Ca2+-mediated degeneration 7"~3'~8'24-a6. In contrast to o t h e r tissues, where CaBP2s K gene expression is regulated by vitamin D,
CaBP28~ in the central nervous system is not responsive to vitamin D administration 45'5°. Until very recently, no h o r m o n e or chemical has d e m o n s t r a t e d the ability to regulate CaBP28K gene expression in the brain. Previous results from our l a b o r a t o r y indicate that corticosterone enhances CaBP28K gene expression in the rat hippocampus 25. It is possible that this relationship exists because type I corticosterone receptors and CaBP28K a r e both localized almost exclusively to the h i p p o c a m p a l areas C A x and dentate gyrus ~4'4°. The highest concentrations of nerve growth factor ( N G F ) 22, N G F m R N A 10, and N G F receptors 3"21"41 are located in those brain areas which also contain high amounts of CaBP28K 5'14"27. These brain areas include the cerebellum, olfactory bulb, hippocampus, neocortex, and c a u d a t e - p u t a m e n which can degenerate in A l z h e i m e r ' s disease, H u n t i n g t o n ' s Chorea, and advanced age. Decreased CaBP2s~: 24 and N G F 22'3° gene expression have been observed in these brain areas during aging and neurodegenerative diseases, suggesting that a direct or indirect relationship between N G F and CaBP28K may exist in the brain. A d r e n a l e c t o m y decreases N G F levels ~ and CaBP28K gene expression 25 in the rat hippocampus, further suggesting the possibility of an interaction between
Correspondence: A.M. Iacopino. Present address: Department of Anatomy, Baylor College of Dentistry, Dallas, TX 75246, USA.
306 t h e s e two proteins. I n d e e d , in P C 1 2 cells m a i n t a i n e d invitro, m u r i n e N G F induces the e x p r e s s i o n of genes for calcium binding p r o t e i n s w h o s e s e q u e n c e s are h o m o l o gous to t h o s e of the family of calcium binding p r o t e i n s which includes CaBP28K 34. In o r d e r to ascertain w h e t h e r N G F can effect CaBP28 K p r o t e i n levels in the brain, the p r e s e n t studies e x a m i n e d the effects of e x o g e n o u s adm i n i s t r a t i o n of r e c o m b i n a n t h u m a n N G F ( r h N G F ) on CaBP28K levels and CaBP28 K i m m u n o r e a c t i v i t y in the rat brain. MATERIALS AND METHODS
Animals Male Sprague-Dawley rats (200-220 g body weight) were subcutaneously implanted with osmotic minipumps (2002, 0.5 ~l/h, Alzet Corp., Palo Alto, CA) containing 216 /A of saline vehicle (PBS) with 18/~g cytochrome C or vehicle containing 18/ag of recombinant human NGF (rhNGF). Cytochrome C was used as a control protein because of its size and charge similarity to rhNGE The rhNGF was a generous gift of L.E. Burton (Genentech, Inc.) and has been characterized in prior reports 2"3. Twelve microliters of fluid (1 ~g cytochrome C or 1/~g rhNGF) were infused each day (n = 5-6 animals per group) into the left lateral ventricle via a 28 gauge stainless steel cannula (3280 P Plastics One, Roanoke, VA) that was stereotaxically implanted 0.8 mm posterior to bregma and 1.3 mm lateral to the midline. The cannula penetrated 4.0 mm below the surface of the skull to terminate in the lateral cerebral ventricle. A second series of animals (n = 5 animals per group) received identical vehicle or rhNGF infusions into the right or left caudate-putamen via 5.5 mm cannulas mounted 0.5 mm posterior to bregma and 3.0 mm from the midline suture. Animals were sacrificed 14 days later and brain areas were dissected and either snap frozen at -80°C or immersed in Bouin's solution (75% picric acid, 20% formalin, 5% glacial acetic acid) for immunocytochemical studies.
for 15 min and then incubated in a humid chamber for 60 min with dilutions of primary rabbit antisera generated against rat renal CaBPz8 K. The three dilutions used were 1:1000, 1:10,000 and 1:100,000. Some sections were incubated with CaBPesK antiserum exposed to CaBP28K (absorption controls). These sections were washed with PBS and primary antibody was detected using biotinylated donkey-antirabbit serum and streptavidin conjugated to horseradish peroxidase (Amersham Corp., Arlington Heights, IL). CaBP28 K was localized with peroxidase-antiperoxidase (PAP) and color formation was achieved with 3,3'-diaminobenzidine HC1 (DAB). The details of the PAP technique for CaBP28 K immunocytochemical localization have been described in a prior report 1°. Sections were counterstained with Cresyl violet, cover-slipped, and viewed under a Leitz Dialux 1I photomicroscope. Specificity of the DAB reaction product for CaBP28K was determined by the absence of staining in sections in which antiserum, preabsorbed with CaBP28 K, was substituted for the antiserum to CaBP28K (absorption control). The number of Cresyl violet-positive neurons and the number of CaBP28K-immunoreactive neurons were determined in the olfactory bulbs of rats that received vehicle or rhNGF (n = 5 animals per group). Stained neurons were counted in 3 randomly chosen microscopic fields in each of 5 sections located between 3 and 4 mm from the rostral tip of the olfactory bulb. The data were expressed as mean + S.E.M. of the ratio of CaBP28K-immunopositive cells per total cells observed (Cresyl violet-positive cells) in the microscopic field and tested for significance as in the RIA.
RESULTS CaBP28K levels d e t e r m i n e d via R I A in c e r e b e l l u m , c o r t e x , c a u d a t e - p u t a m e n , and h i p p o c a m p u s w e r e e q u a l to t h o s e r e p o r t e d p r e v i o u s l y 24'25. F o u r t e e n days of i.c.v. infusion with r h N G F e l e v a t e d CaBP28K by 75% in the o l f a c t o r y b u l b but not in the c e r e b e l l u m , cortex, caudate-putamen,
o r h i p p o c a m p u s (Fig. 1). R I A analysis
was r e p e a t e d on animals of a s e c o n d e x p e r i m e n t which i n c l u d e d g r o u p s that r e c e i v e d vehicle o r r h N G F
CaBP28K determination Tissue samples were homogenized (20% wt./vol.) in phosphatebuffered saline (4°C) and centrifuged at 38,000 x g for 30 min. The supernatant was stored at -20°C and total protein content was determined by the method of Lowry 31. CaBP28 K content was determined by radioimmunoassay (RIA) sl, using 12SI-labeled rat CaBP28K33. CaBP28 K was calculated as mean/ag CaBP28K/mg total protein + S.E.M. Group means were tested for significant differences by Student's t-test for two-group comparison or by analysis of variance (ANOVA) and Dunnett's t-test for multiple-group comparisons.
infu-
sions into the c a u d a t e - p u t a m e n in a d d i t i o n to the lateral
Effect of NGF on CaBP2sk Levels in Rat Brain 25 r
lmmunoblot (Western blot)
15
A modified Western blot procedure 2° was performed using supernatants from two randomly chosen vehicle or rhNGF-infused animals from each of the two RIA experiments. Autoradiographs were analyzed using scanning densitometry.
10
lmmunocytochemistry A specific antisera against CaBP28K51 (suitable for CaBP28 K immunocytochemistry in brain) was used to localize the protein in olfactory bulb sections from vehicle and rhNGF-infused animals. Within 5 rain of sacrifice, olfactory bulbs were immersed in Bouin's fixative and, within 4 days, each pair of olfactory bulbs was paraffin-embedded. Tissue sections (10/~m) were generated on a microtome and affixed to poly-L-lysine coated slides. Lipids were removed from the sections in xylene (3 changes, 30 min each) and sections were rehydrated in graded ethanols (95, 75, 70, 50% ethanol) and water. The sections were treated with 0.3% H20 2 in PBS
C| -
CO
Oil -I-
-
÷
--
CS "I-
--
HC ÷
--
AREA +
NGF
Fig. 1. Effect of 14 day i.c.v, infusion of PBS vehicle (-) or 1/~g/ day rhNGF (+) on CaBP28 K levels in rat cerebellum (Ce), olfactory bulb (OB), cortex (Co), caudate-putamen (CS), and hippocampus (HC). RIA values are reported as mean + S.E.M. (n = 5 animals per group) with significant differences (P < 0.01) marked by an asterisk.
307 Effects
of
NGF on CoBPaa K Levels in Rat Brain
CaBP2sr-positive neurons~total neurons in olfactory bulbs of norreal and rhNGF-treated animals
30
2s
The data represents CaBP28K-positive neurons/Cresyl violet stained neurons per microscopic field (data for each animal was obtained by pooling cell counts from 5 separate sections counting 3 separate fields per section generating 15 microscopic fields).
Q.
~ 20 2
e,l
~ (30 o
TABLE I
5
- +
C
CE
-
+
C
o0
-
+
R
L
OT
-
+
R
cs
L
-
+
Control
rhNGF-treated
1 2 3 4 5
1500/4818 1436/4244 1949/5772 1683/5239 1711/4712
1642/4581 1569/4594 1833/5421 1751/5002 1590/4436
RL
HC
Fig. 2. CaBP28K levels in rat brain areas (n = 6 animals per group) after 14 days of i.c.v, or intrastriatal infusions of vehicle (-) or rhNGF (+). Abbreviations are as in Fig. 1 with aT, olfactory tubercle. Bilateral brain areas were pooled and measured after intrastriatal infusions for CE and OB (c). Unilateral areas were measured separately in aT, CS, and HC after intrastriatal infusions (right, R; left, L). Dependent values, significance level, and other parameters are as described in Fig. 1.
ventricle. A g a i n , the only brain area to show a response to r h N G F administration was the olfactory bulb and, again, a 75% elevation in immunoreactive CaBP28 K was obtained (Fig. 2). Infusion of r h N G F into the caudatep u t a m e n had no effect on CaBP28 K levels in the olfactory bulb or o t h e r brain areas, including the caudatep u t a m e n itself. Western blot analysis was p e r f o r m e d to c o r r o b o r a t e
EFFECT OF NGF ON CoBP28k LEVELS (Western Blot Rot Olfactory Bulb)
28,000
Animal
M r-
the R I A results observed for the olfactory bulb. Fig. 3 shows the results for one set of randomly selected vehicle and r h N G F - t r e a t e d animals from one of the two experiments. CaBP28~: immunoreactivity on Western blot analysis was noticeably elevated by r h N G F administration. A l t h o u g h Western blot data are not strictly quantitative, densitometric analysis of autoradiographs revealed that r h N G F - i n f u s e d animals exhibited increases in protein levels ranging from 68-86%. These figures are in a g r e e m e n t with the R I A results. Immunocytochemical analyses were p e r f o r m e d to ascertain whether the increases in CaBP28 K observed in the R I A and Western blot studies were due to increased CaBP28K levels in neurons already expressing the protein, or whether the elevations in CaBP28 K were due to increased numbers of neurons expressing the protein. CaBP28K-positive neurons n u m b e r e d between 80 and 140 p e r microscopic field accounting for 30-40% of the total neurons observed. T h e labeled cells were observed in the glomerular, external plexiform, and internal granular layers of the olfactory bulb. T h e r e was no difference in the n u m b e r of CaBP28K-immunoreactive neurons in the olfactory bulbs of control and r h N G F - t r e a t e d animals. The total n u m b e r of neurons (Cresyl violet stained neurons) p e r microscopic field did not change following r h N G F administration (see Table I), thus there was no change in the p r o p o r t i o n of total neurons in the olfactory bulb that were counterstained with CaBP2s K antisera (Fig. 4), suggesting that the increase in CaBP2s K observed in the olfactory bulb represents an elevation of CaBP28K levels in neurons that normally express the protein.
C NGF Fig. 3. Western blot analysis of CaBP28K levels in rat olfactory bulb. Eight animals (2 vehicle and 2 rhNGF-infused animals from each of the RIA experiments) were compared according to rhNGF status. A representative animal pair is shown (NGF, rhNGF-treated; c, vehicle-treated). Elevation of CaBP28K in the olfactory bulb of rhNGF-treated animals was observed.
DISCUSSION The present results show that CaBP28K levels are relatively high in the rat olfactory bulb and that intraventricular infusions of r h N G F elevate CaBP28 K levels in the
308
Immunocytochemical Chora.cterzation of CaBP28 K Neurons in Rat Olfactory Bulb
Condition Animal# CoBP2/Totol (Mean SEM) Totals Control
NGF
1 2 3 4 5
.31 :~ .02 .34 + .02 .34 :~ .02 .32:1:.01 .36 + .01
.33 ± .02
I 2 3 4
.36 .34 .34 .35 .36
.35 i .01
5
:I: 4:t: • :~
.01 .03 .04 .03 .03
The data is expressedas a ratio of Calbindin-D2~immunopositive cells per total cells observedin the microscopicfield. The ratio for each animal was obtained by pooling cell counts from 5 separatesections counting 3 separate fields per section (15 total fields). Fig. 4. Immunocytochemical characterization of CaBP28K neurons in rat olfactory bulb. Representative photomicrograph showing CaBP28 ~ immunopositive neurons detected by immunocytochemistry in control (A) and NGF-treated animals (B). Results were quantitated by cell counting procedures (below). Results are expressed as means + S.E.M. of the number of CaBP28K immunopositive cells/total cells observed in each microscopic field. For 5 animals in each experimental group (control vs. rhNGF-treated), the relative number of CaBP28K immunopositive neurons did not differ.
bulb but not in 5 other brain areas tested. Administration of rhNGF increased CaBP2s K content, as measured by R I A and Western blot analyses, but not the number of bulb neurons that contained the protein. Thus, the CaBP28 K content per bulb neuron was increased. Immunocytochemical analysis is not quantitative and was utilized to address the question of cell number in rhNGFtreated animals. The R I A and Western blot assays were performed using whole tissue homogenates, thus it was impossible to determine which neuronal cell types within the olfactory bulb contained increased levels of CaBP28K following rhNGF treatment. Analysis of CaBP28 K m R N A in similarly-treated animals could help reveal whether increased protein levels resulted from augmented transcription or decreased degredation. In addition, in situ hybridization studies may reveal whether specific neuronal cell types within the olfactory bulb contain increased levels of CaBP28 K m R N A (i.e. mitral cells vs. all other neurons in the bulb). In any case, the augmentation of olfactory bulb CaBP28 K levels in response to rhNGF treatment is suggestive of a positive relationship between endogenous N G F and CaBP28 K gene expres-
sion. Previous results indicate that CaBP2sK plays a critical role in Ca 2+ homeostasis 4'35'44 and that CaBP28 K gene expression is reduced in aging and neurodegenerative diseases 24. Other studies have shown that excessive neuronal influx of Ca 2÷ is associated with neuronal death induced by excitatory amino acid stimulation 7"13. CaBP28 K may be a critical intracellular calcium buffer that prevents calcium-induced excitatory amino acid-dependent neurotoxicity 4'7'13A8"24-26'35'44. Thus, in addition to the ability of NGF to promote the survival of injured neurons 22'37, NGF may also function as a neuroprotective factor by augmenting calcium sequestration by CaBP28 K. It is well-known that the olfactory system degenerates in patients with Alzheimer's disease 12"39. Perhaps the prominent neurodegeneration of the olfactory bulb seen in Alzheimer's disease 39 represents Ca2÷-me diated neurotoxic sequelae resulting from decreased CaBP28 K levels secondary to a deficiency of human N G F synthesis, binding, or transport. There are two possible mechanisms which may explain the effect of rhNGF on CaBP2s K levels in the rat olfactory bulb. Firstly, augmentation of olfactory bulb CaBP2s K by ventricular delivery of rhNGF may be due to a transynaptic mechanism initiated at a nucleus where i.c.v. NGF is able to gain access and which projects to the olfactory bulb. The horizontal limb of the diagonal band (HDB) is one such area x5'46'47. Cholinergic neurons that innervate the olfactory bulb transport NGF in a retrograde, receptor-dependent manner a. These neurons originate in the HDB and innervate neurons in the periglomerular and glomerular cell layers of the olfactory bulb 32'28. Since the CaBP2sK-positive neurons in the bulb are also observed in these areas 17, the ability of the rhN G F to increase CaBP28 K may be mediated via cholinergic input from the HDB. Thus, transynaptic effects of rhNGF, via intrinsic connections between rhNGF-responsive cells and CaBP28K-positive cells are possible. Experiments with HDB lesions or cholinergic agonist/antagonist drug administration could help to identify such a mechanism. Secondly, NGF receptors are found in the periglomerular cell layer of the olfactory bulb 53. If these cells contain CaBPa8 K, then the rhNGF action could be mediated directly at the receptor level. Clearly, co-localization studies for CaBP28 K and NGF receptors in the olfactory bulb would help resolve these different mechanisms. Direct infusions of rhNGF into the HDB as well as into the olfactory bulb may resolve whether the 75% increase in olfactory bulb CaBP28K is due to local (receptor-mediated) or distal (transynaptic) influences of rhNGF. The biological activity of rhNGF would depend upon binding to high affinity (presumably biologically active) receptors rather than low affinity receptors 2"41'53. The high affinity binding sites in the rat brain have been
309 mapped using a protocol which distinguishes between these two types of receptors 3. It has been shown that each of the brain areas tested contain a high density of N G F receptors 3m and that while most brain regions share both high and low affinity receptors, the caudate/ putamen and neocortex are enriched with high affinity receptors 3. This, it is not likely that the selective effect of r h N G F on CaBP28 K levels in the olfactory bulb would be due to differences in the relative amount of high affinity N G F receptor present in the various brain regions under study. The olfactory bulb is unique among these brain areas as it receives innervation by neurons that are capable of regeneration 36. The sensory neurons of the nasal epithelium and their terminals in the olfactory bulb are in a continual cycle of mortality, development, and maturation. If the effects of r h N G F on CaBP28K levels are more pronounced in neuronal environments in which synaptic contacts are being established, then N G F may alter CaBP2s K in additional regions of developing brains or during reinnervation following neuronal injury. Additionally, CaBP28 K is found in many neuronal populations outside of the olfactory bulb 5'11'14't7'27 and may be regulated in these areas by other neurotrophic factors as well as by corticosteroids 1'25. Besides corticosterone, rhN G F is presently the only agent known to alter CaBP28K. Future research into exogenous manipulation of vitamin-D resistant brain CaBP28 K may lead to therapeutic regimens that can attenuate calcium-mediated neurodegeneration. Such studies may also help elucidate the cal-
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