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Acetyl-l -carnitine enhances the response of PC12 cells to nerve growth factor
Developmental Brain Research, 59 (1991) 221-230 © 1991 Elsevier Science Publishers B.V. 0165-3806/91/$03.50 ADONIS 0165380691512523
221
BRESD 51252
Acetyl-L-carnitine enhances the response of PC12 cells to nerve growth factor G. Taglialatela 1'3, L. Angelucci 2, M.T. Ramacci 3, K. Werrbach-Perez 1, G.R. Jackson 1 and J.R. Perez-Polo t 1Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.), 2Department of Pharmacology, University of Rome 'La Sapienza', Rome 00185 (Italy) and 31nstitutefor Research on Senescence--Sigma Tau, Pomezia 00040 (Italy) (Accepted 8 January 1991)
Key words: Nerve growth factor; Rat pheochromocytoma (PC12) cell; Neurite outgrowth; Acetyl-e-carnitine; Aging
We have demonstrated that treatment of rat pheochromocytoma (PC12) cells with acetyl-L-carnitine (ALCAR) stimulates the synthesis of nerve growth factor receptors (NGFR). ALCAR has also been reported to prevent some age-related impairments of the central nervous system (CNS). In particular, ALCAR reduces the loss of NGFR in the hippocampus and basal forebrain of aged rodents. On these bases, a study on the effect of NGF on the PC12 cells was carried out to ascertain whether ALCAR induction of NGFR resulted in an enhancement of NGF action. Treatment of PC12 cells for 6 days with ALCAR (10 mM) stimulated [~25I]NGF PC12 cell uptake, consistent with increased NGFR levels. Also, neurite outgrowth elicited in PC12 cells by NGF (100 ng/ml) was greatly augmented by ALCAR pretreatment. When PC12 cells were treated with 10 mM ALCAR and then exposed to NGF (1 ng/ml), an NGF concentration that is insufficient to elicit neurite outgrowth under these conditions, there was an ALCAR effect on neurite outgrowth. The concentration of NGF necessary for survival of serum-deprived PC12 cells was 100-fold lower for ALCAR-treated cells as compared to controls. The minimal effective dose of ALCAR here was between 0.1 and 0.5 mM. This is similar to the reported minimal concentration of ALCAR that stimulates the synthesis of NGFR in these cells. The data here presented indicate that one mechanism by which ALCAR rescues aged neurons may be by increasing their responsiveness to neuronotrophic factors in the CNS. INTRODUCTION The aged central nervous system (CNS) is characterized by a progressive onset of morphological and functional impairments. In aged rodents, the n u m b e r of h i p p o c a m p a l neurons in the A m m o n ' s horn and dentate gyrus is significantly r e d u c e d 1'27. Also, n u m e r o u s pycnotic cholinergic neurons b e c o m e manifest in the septum and basal forebrain nuclei of aged rats 8'9. A l o n g with these anatomical i m p a i r m e n t s , there are a n u m b e r of malfunctions of different neuronal systems that take place in the aged. Glucocorticoid receptors in the aged h i p p o c a m p u s are r e d u c e d and their physiological response is impaired 1'6'27'34'39. F u r t h e r m o r e , there is evidence that enzyme m a r k e r s for central cholinergic neurons are r e d u c e d during physiological and pathological aging 5"43. Some behavioral performances associated with the brain structures d a m a g e d in senescence, such as learning and spatial orientation, are impaired in aged subjects 8,11. O n e event that is c o m m o n to the physiology and
pathophysiology of the aging process in the CNS, is the reduction of nerve growth factor ( N G F ) receptors 3' 12,21,26,32. N G F is a p o l y p e p t i d e that is essential for the d e v e l o p m e n t and m a i n t e n a n c e of some neurons of the p e r i p h e r a l nervous system (PNS), where it plays a key role in the regulation of n e u r o n a l cell d e a t h 29'37. The N G F p r o d u c e d by target tissues binds to specific receptors ( N G F R ) at growth cones and is t r a n s p o r t e d retrogradely to the neuronal cell b o d y 22. T h e resulting continuous supply of target-derived N G F is essential for the preservation of the innervating n e u r o n 18. In the CNS, N G F also has trophic effects on the magnocellular cholinergic neurons of the basal forebrain and septum 19' zo. As in the PNS, N G F is released by target tissues, such as h i p p o c a m p u s and frontal cortex, where it binds to N G F R on cholinergic terminals and is r e t r o g r a d e l y t r a n s p o r t e d to the cell s o m a in the basal forebrain and septum25,41. The finding that N G F exerts n e u r o n o t r o p h i c activity in CNS has led to the hypothesis that the r e p o r t e d loss of N G F R in senescence with the resulting reduction of N G F
Correspondence: G. Taglialatela, Department of Human Biological Chemistry and Genetics, 440 G. Borden Bldg. (F-52), University of Texas Medical Branch, Galveston, TX 77550, U.S.A.
222 activity is r e s p o n s i b l e for n e u r o n a l celt d e a t h and shrinkage t h e r e . C o n s e q u e n t l y , the t h e r a p e u t i c use of N G F for the t r e a t m e n t of n e u r o l o g i c a l diseases associated with aging has b e e n p r o p o s e d 35'36. It is k n o w n carnitine,
that
treatment
of rats with acetyl-L-
a naturally occurring substance
i n v o l v e d in
m i t o c h o n d r i a l m e t a b o l i s m of fatty acids 1° p r e v e n t s certain C N S i m p a i r m e n t s in the a g e d 1'2. A L C A R t r e a t m e n t of s e n e s c e n t
rats p r e v e n t s
ioral p e r f o r m a n c e s that are r e l a t e d to the limbic system J l, partially p r e v e n t s the loss of N G F R
that
o c c u r s in the h i p p o c a m p u s and basal f o r e b r a i n of a g e d r o d e n t s 3. In o r d e r to study w h e t h e r this is a direct r e l a t i o n s h i p between
ALCAR
and
NGF iodination The iodination of NGF was attained using a modified lactoperoxidase technique, as described elsewhere 33. Specific activity of the ['25I]fl-NGF was 2000-3500 cprn/fmol with 85-90% of the counts being acid-precipitable. The iodinated NGF was then stored at 4 °C in phosphate buffered saline, pH 7.4, containing 2 mg/ml cytochrome C and used within 2 weeks.
the loss of g l u c o c o r t i c o i d
r e c e p t o r s in the h i p p o c a m p u s and i m p r o v e s the b e h a v 34 A L C A R
determined spectrophotometrically. Fhe purity of/;LNGF was assayed by isoelectric focusing on polyacrylamide disc gel and specific biological activity determined by the method developed by Greene ~.
NGF/NGFR
activity,
the
rat
p h e o c h r o m o c y t o m a (PC12) cell line was c h o s e n as an in
Cell culture Rat pheochromocytoma (PCI2) cells were kindly provided by Dr. Lloyd Greene, Columbia University, New York, NY. Cells were grown in RPMI 1640, supplemented with 5% heat-inactivated horse serum +5% heat-inactivated fetal calf serum, at 37 °C in a humified incubator with 5% CO2 atmosphere and fed on alternative days. At subconfluency, cells were dislodged by vigorous shaking and reseeded at a 1:1 ratio. Acetyl-L-carnitine was dissolved in RPMI and added to cells at the final concentrations indicated in the various experiments.
vitro m o d e l system for N G F - r e s p o n s i v e n e u r o n s 16. PC12
[1251]NGF uptake
cells are a r a t - d e r i v e d cell line that display N G F R similar
PC12 cells were plated out in 24-well plates at a density of 105 cells/ml. On the sixth day of A L C A R treatment (10 mM), [lzSI]NGF was added at a final concentration of 5 nM and the cells incubated at 37 °C for 45 min. After incubation, cells were washed 3 times with ice-cold PBS and solubilized with 0.5 N NaOH. Aliquots were then taken from each well for protein assay and the rest counted in a y-counter for radioactivity content. Protein content was measured by the bicinchoninic acid method 42. Results were expressed as fmol of labeled NGF per mg of protein.
to
those
described
for
sympathetic
and
sensory
n e u r o n s 44. P C 1 2 typically r e s p o n d to N G F by e l o n g a t i n g n e u r i t e s and d e v e l o p i n g into electrically e x c i t a b l e cells f e a t u r i n g s o m e c h a r a c t e r i s t i c of the p o s t - m i t o t i c cholinergic n e u r o n a l p h e n o t y p e 15. O t h e r k n o w n effects o f N G F on the P C 1 2 cells i n c l u d e i n d u c t i o n of o n c o g e n e s , such as c-f0s4"3°; p h o s p h o r y l a t i o n o f s o m e c y t o p l a s m i c p r o t e i n s 17' 38; i n d u c t i o n o f o r n i t h i n e d e c a r b o x y l a s e m R N A T ; p r o t e c t i o n against p e r o x i d a t i v e injury and stimulation of catalase activity and p r o t e i n 23"24. T r e a t m e n t of P C 1 2 cells with A L C A R mRNA
results in i n c r e a s e d N G F R
p r o t e i n and
( T a g l i a l a t e l a et al., s u b m i t t e d ) .
In the p r e s e n t study we d e m o n s t r a t e that, consistent with A L C A R
s t i m u l a t i o n of N G F R synthesis, the action
of N G F on P C 1 2 cells is e n h a n c e d by A L C A R t r e a t m e n t .
Neurite outgrowth experiment PCI2 cells were plated out into 35-ram Petri dishes at a density of 2 × 105 cells/ml. On the sixth day of A L C A R treatment (10 mM); the cells were added with NGF dissolved in RPMI 1640 at a final concentration of either 1 or 100 ng/ml (0.037 or 3.7 nM). On days 2, 5 and 7 after NGF addition, 100-120 cells from 5-12 randomly chosen microscope fields were counted and assayed for presence of neurites. All the counts were done independently by two investigators on coded samples. After all counts were carried out, codes were broken and the average of the two counts taken as final value estimation. The results were expressed as percent of cells bearing neurite processes.
Thus, ALCAR may prevent some degenerative processes in t h e a g e d brain by l o w e r i n g t h e r e s p o n s e t h r e s h o l d of s u s c e p t i b l e n e u r o n s to n e u r o n o t r o p h i c factors.
MATERIALS AND METHODS
Materials N,N'-methylene-bis-acrylamide, glycine and ammonium persulfate were purchased from Bio-Rad Laboratories; Cytochrome C, amberlite GC-400, lactoperoxidase and H20 2 were from Sigma; RMPI 1640, fetal calf serum and horse serum were purchased from Hazleton; Ampholines, pH range 3.5-10.0, were obtained from LKB Produkter AB, and ultrapure urea from Mallinckrodt Chemicals; carrier-free NaJ25I was from Amersham and [35S]methionine from ICN; acetyl-L-carnitine was supplied by the Sigma Tau Company, Italy. All other chemicals used were reagent grade.
NGF isolation The fl-NGF subunit was isolated from mouse submaxillary gland according to the method of Mobley et al. 3t After elution from an ion exchange column, the NGF was dialyzed overnight against 0.1% acetic acid. The dialysate was then concentrated by speed vacuum centrifugation to 1 mg/ml and final concentration of fl-NGF
Serum deprivation experiments PC12 were plated out into 24-well plates at a density of 5 x 104 cells/ml treated for 6 days with A L C A R 10 mM. After 6 days, the serum was removed from the culture medium and the cells were given NGF at concentrations 0.01-100 ng/ml (0.37 pM to 3~7 nM). After 48 h the medium was removed, replaced with fresh medium containing 2/~Ci/ml of [35S]methionine and cells were then incubated for 4 h at 37 °C as described elsewhere 23'24. After incubation, cells were washed in PBS, sotubilized with 0.5 N NaOH and the protein precipitated by addition of 50% trichtoroacetic acid (TCA). Prior to TCA precipitation, aliquots from each sample were taken for protein assay. Samples were then passed through:GF/F Whatman filters and washed with 5 vols. of 5% TCA. Filters were transferred into scintillation vials and, after addition of 5 ml of scintillation cocktail (Beckman), counted for radioactivity in a r-counter. In another experiment, PCI2 cells were treated for 6 days with ALCAR 0.1, 0.5, or 1 mM and supplemented, after serum deprivation, with NGF 1 ng/ml (0.037 nM). All the experiments were done in quadruplicate and the results (incorporation of [35S]methionine into TCA-precipitable proteins) expressed as cpm/ mg of protein.
Statistical analysis Statistical differences among groups were evaluated by analysis of
223 NGF
~-
5
120
4
100
NGF + Alcar"
..~
80
3
2 O
40 2O Control
Alcor
Fig. 1. [~25I]NGF uptake in PC12 cells treated for 6 days with 10 mM ALCAR. *P<0.05 (Two-tail Student's t-test); n = 8 per group.
variance (ANOVA) for unweighted means followed by Fischer's least significant difference (LSD) test. When appropriate, the two-tailed Student's t-test was applied instead. RESULTS
[125I]NGF uptake PC12 cells were treated for 6 days with A L C A R (10 mM) and then incubated for 45 min at 37 °C in the presence of 5 nM [125I]NGE This procedure led to an increased labelling of the cells that had been previously treated with A L C A R , as compared to control PC12 cells (Fig. 1). This finding is consistent with the reported increase in N G F R synthesis in PC12 cells following A L C A R treatment (Taglialatela et al., submitted). It is important to notice that we carried out our incubation at 37 °C for 45 min. Since at 37 °C the internalization of the N G F - N G F R complex in PC12 cells occurs within 20 to 30 min after NGF binding 44, the values reported here should include uptake of [125I]NGF into the cytoplasm, and not be limited to NGF bound to the plasma membrane. This assumes that A L C A R does not disrupt NGF internalization.
Neurite outgrowth experiments When we exposed to NGF (100 ng/ml) the PC12 cells treated for 6 days with A L C A R (10 mM), we observed a dramatic increase in the extent of neurite outgrowth elicited by NGF (Fig. 2 through Fig. 5). On day 2 after NGF exposure, the number of cells displaying neurites was 8-fold higher in the ALCAR-treated PC12 as compared to controls. Furthermore, after 5 days of NGF treatment, there were twice as many neurites in A L C A R treated PC12 than control cells. In both cases the difference in the extent of neurite outgrowth between the ALCAR-treated and control PC12 cells was statistically significant, as calculated by analysis of variance of the data (F = 29.21; df = 3; P<0.001). On day 7 of exposure to NGF, there was a slight, not statistically significant, difference in the neurite outgrowth of the A L C A R -
0 Day 2
Day 5 NGF t r e a t m e n t
time
Day • period
Fig. 2. Neurite outgrowth in PC12 cells treated for 2, 5, or 7 days with NGF (100 ng/ml). ALCAR treatment (10 mM) started 6 days prior to addition of the NGF. Results are expressed as percent of cells displaying neurites in 5-12 microscope fields per condition. *Statistical difference versus control cells; ANOVA: F = 29.21; df = 3; P<0.001. treated PC12 as compared to controls. Given that NGF-treated cells will clump with time, it was difficult at these latter times to draw definitive conclusions. In a second experiment, we used NGF at 1 ng/ml, a concentration that in our system does not stimulate neurite outgrowth. As shown in Fig. 6, there was neurite extension from the ALCAR-pre-treated PC12 cells, but not from the PC12 treated with NGF alone. A L C A R treatment alone did not elicit neurite outgrowth from PC12 cells under any conditions tested (Figs. 3-6, panel B).
NGF rescue of serum-deprived PC12 cells PC12 cells in a serum-free environment require NGF for survival TM. Increasing concentrations of NGF enhanced viability in a dose-response fashion (Fig. 7). The minimal concentration of NGF that increased survival of serum-deprived PC12 cells was 1 ng/ml. Furthermore, PC12 survival after serum deprivation was supported by 10 mM A L C A R and concentrations of NGF that were as low as 0.01 ng/ml. Thus, A L C A R treatment potentiated NGF trophic action on PC12 cells, as measured by [3~S]methionine incorporation. Since the vital dye exclusion test for viability in the presence of NGF may lead to errors due to PC12 aggregation, here we used the incorporation of [35S]methionine into TCA-precipitable proteins as a measure of viability. A detailed discussion of the reliability of this technique is given elsewhere (Jackson et al., in preparation). The addition of 1 ng/ml of NGF to serum-deprived, ALCAR-treated (0.1-1.0 mM) PC12 cells, showed that 0.5 mM of A L C A R is the minimal concentration that potentiates the rescue by NGF of PC12 after serum deprivation (Fig. 8). This A L C A R concentration is similar to the lowest dose of A L C A R reported to stimulate NGFR in PC12 ceils (Taglialatela et al., submitted).
224
Fig. 3. Phase-contrast micrograph of PC12 cells after 2 days of N G F treatment (100 ng/ml) in the presence or absence o~ A L C A R (10 mM) in the culture medium. A L C A R treatment started 6 days prior to addition of N G F to the cells. Reference bar = 100 ktm, A: control cells; B: A L C A R - t r e a t e d cells; C: NGF-treated cells; D: N G F - A L C A R - t r e a t e d cells.
225
Fig. 4. Phase-contrast micrograph of PC12 cells after 5 days of N G F treatment (100 ng/ml) in the presence or absence of A L C A R (10 mM) in the culture medium. A L C A R treatment started 6 days prior to addition of NGF to the cells. Reference bar = 100 ~m. A: control cells; B: A L C A R - t r e a t e d cells; C: NGF-treated cells; D: N G F - A L C A R - t r e a t e d cells.
226
I i
i
I
,
Fig. 5. Phase-contrast mierograph of PC12 cells after 7 days of NGF treatment (100 ng/ml) in presence or absence of ALCAR (10 raM) in the culture medium. ALCAR treatment started 6 days prior to addition of NGF to the cells. Reference bar = 100/~m. A: control cells; B: ALCAR-treated cells; C: NGF-treated cells; D: NGF-ALCAR-treated cells.
227
Fig. 6. Phase-contrast micrograph of PC12 cells after 5 days of NGF treatment (1 ng/ml) in presence or absence of A L C A R (10 mM) in the culture medium. ALCAR treatment started 6 days prior to addition of NGF to the cells. Reference bar = 75/~m. A: control cells; B: ALCAR-treated cells; C: NGF-treated ceils; D: NGF-ALCAR-treated cells.
228 [--~
Control
~
AIcQr
J
700 r / 6001
[ Control
~
NGF
(Ing/rn~]
]
I0
*
4001 -
~
F 300 4
1
,o O0
, 1 NGF" ( n g / m t )
1
1
Fig, 7. [3SS]methionine incorporation in serum deprived PC12 cells supplemented with NGF at various concentrations. ALCAR treatment: 10 mM for 6 days; n = 3-4 per condition; *significant difference versus respective control group (NGF 0 ng/ml); ANOVA: F = 71.36; df= 11; P < 0.001.
0
0
01 Alcar (raM)
05
~0
Fig. 8. [3SS]methionine incorporation in serum-deprived PCI2 cells supplemented with NGF t ng/ml. ALCAR treatment: 0.t, 0.5, or 1 mM for 6 days. n = 3-4 per condition; *statistical difference versus control group (NGF 0 ng/ml); *#statistical difference versus cells treated with NGF alone. ANOVA: F = 6.06; df = 4; P < 0.005.
DISCUSSION The results presented here are in agreement with the finding that A L C A R enhances N G F R expression in PC12 cells (Taglialatela et al. submitted). In that study, we reported that treatment of PC12 with A L C A R at varying concentrations for 6 days increased [125I]NGF binding, immunoprecipitable N G F R protein and m R N A for the NGFR. We therefore studied some of the known effects elicited by N G F in order to assess whether the increase in N G F R after A L C A R treatment resulted in an increased response of the PC12 cells to N G E T h e increase of [12SI]NGF uptake by ALCAR-treated PC12 can be explained as a direct consequence of the reported increase in N G F R at the membrane level. Supportive of this hypothesis is the finding that A L C A R treatment enhances both type I and type II N G F R in PC12 cells, as measured by [12SI]NGF equilibrium binding and by immunoprecipitation of crosslinked N G F N G F R complex (Taglialatela et al., submitted). Since only the type I (high-affinity) N G F R is responsible for internalization of N G F 45, increased levels of the type I N G F R on the cell surface would result in an increased rate of internalization and uptake of [~2SI]NGF in ALCAR-treated PC12. This assumes that A L C A R does not disrupt N G F internalization. Since A L C A R also enhanced the PC12 morphological response to N G F which has been demonstrated to rely on the internalization of N G F bound to high-affinity N G F binding sites in PC1244, any effects of A L C A R on the transition of NGF from a bound state to an internalized state would have to be a stimulatory nature. The best-characterized effect of N G F on PC12 cells is the neurite outgrowth and differentiation of PC12 into neuronal-like cells that display some features of a post-mitotic cholinergic phenotype 15. Treatment of PC12
with 10 mM A L C A R for 6 days led to a dramatic increase in the neurite outgrowth elicited by 100 ng/ml of N G E Although after 7 days of N G F treatment one could not differentiate by morphological criteria control cells from their ALCAR-treated counterparts, on the second day of NGF treatment the number of neurite-bearing PC12 cells was 8-fold lower in the control cells as compared to the ALCAR-treated PC12. These results would suggest that A L C A R treatment accelerates the response time of PC12 cells to N G E Furthermore, when PC12 were exposed to N G F concentrations that do not elicit extension of neurites, under our conditions, we could observe neurite outgrowth in the ALCAR-treated cells. This would suggest that A L C A R treatment, rather than shortening the response time of PC12 to NGF, enhances their responsiveness to N G E The mechanism of action for the A L C A R effect on NGF-stimulated neurite outgrowth may not be limited to an increased internalization of the N G F - N G F R complex. The N G F stimulation of neurite outgrowth in PC12 is a complex phenomenon that is preceeded by reduction of cellular divisions, ~mproved attachment to substrate and flattening of cells 16. Although A L C A R alone does not stimulate neurite outgrowth in PC12, we often observed that ALCAR-treated cells appeared to be more flattened and better attached to culture flasks (Figs. 3-6, panel B). Alternatively A L C A R , by means of its enhancement of fatty acid catabolism 1°, may increase the availability of the highenergy compounds required for morphological transformation of cells after N G F stimulation. These possible mechanisms of action are not mutually exclusive and may be independently involved in the A L C A R potentiation of NGF-stimulated neurite outgrowth. Since it has been demonstrated that N G F rescues PC12 cells which would normally die when cultured in the
229 absence of serum 14, we assayed the ability of A L C A R to enhance N G F protection of s e r u m - d e p r i v e d PC12 cells. The minimal concentration of N G F that is required to support PC12 cells after serum deprivation is 1 ng/ml. In the A L C A R - t r e a t e d cells, the same result could be achieved at concentrations of N G F as low as 0.01 ng/ml. The analysis of a b r o a d e r spectrum of A L C A R concentrations r e v e a l e d that the lower dose of A L C A R that is effective in potentiating the N G F rescue of serum d e p r i v e d PC12 is similar to that r e p o r t e d to increase the synthesis of N G F R in this cell line. This result suggests that the e n h a n c e m e n t of the N G F R expression by A L C A R may be primarily involved in the increased N G F protection of serum deprived PC12 in the presence of ALCAR. The aged CNS exhibits a n u m b e r of morphological and functional i m p a i r m e n t s 1'6'8'28'4° that are a c c o m p a n i e d by different behavioral disorders 27. The finding that during the physiological and pathological aging of the CNS there is a massive loss of N G F R and N G F activity, has led to the hypothesis that reduction of n e u r o n o t r o p h i c activities in CNS may be responsible for the onset of those i m p a i r m e n t s that are characteristic of brain senescence 3" 21,26,32,36. Consequently, a possible therapeutic role for N G F in the pathological disorders of the CNS associated with aging has been p r o p o s e d 35'36. H o w e v e r , the ex-
pected minimal t r a n s p o r t of N G F across the b l o o d - b r a i n b a r r i e r if a d m i n i s t e r e d systemically would not encourage the routine testing of N G F as a clinical agent. Treatment of rats with A L C A R prevents some of the CNS impairments that occur in senescence and some of the behavioral disorders r e p o r t e d for aged rodents ~' 2,11.34. A L C A R t r e a t m e n t partially prevents the loss of N G F R in the h i p p o c a m p u s and basal forebrain of aged rats and c o m p l e t e l y abolishes the reduction of N G F binding that follows e x p o s u r e to stress in h i p p o c a m p u s of adult rodents 3"46. O n these findings, a direct correlation between the effects of A L C A R on aged rats and its effects on N G F activity in CNS can be assumed. In conclusion, the data p r e s e n t e d here would indicate that A L C A R may p r e v e n t some d e g e n e r a t i v e events in the CNS of aged rodents by enhancing the responsiveness of neurons to e n d o g e n o u s n e u r o t r o p h i c factors. It follows that A L C A R should be considered as a synergistic or activating agent for e n d o g e n o u s trophic factors, this being of special interest for the p r o p o s e d use of exogenous N G F in pathologies associated with senescence.
Acknowledgement. The authors wish to thank Mrs. D. Masters for manuscript preparation. This work was supported in part by a grant from Sigma Tau Company, Italy, CNR, Italy and by NINDS Grant NS18708.
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15
16 17 18 19
atrophy and spatial memory impairment in aged rats by nerve growth factor, Nature, 32 (1987) 965-968. Flood, D.G. and Coleman, P.D., Neuron number and sizes in aging brain: comparison of human, monkey and rodent data, Neurobiol. Aging, 9 (1988) 453-463. Fritz, I.B., Carnitine and its role in fatty acid metabolism, Adv. Lipid Res., 1 (1963) 285-334. Ghirardi, O., Milano, S., Ramacci, M.T. and Angelucci, L., Effect of acetyl-L-carnitine chronic treatment on discrimination model in aged rats, Physiol. Behav., 44 (1988) 769-773. Gomez-Pinilla, F., Cotman, C.W. and Nieto-Sampedro, M., NGF receptor immunoreactivity in aged rat brain, Brain Res., 479 (1989) 255-262. Greene, L.A., A quantitative bioassay for nerve growth factor (NGF) activity employing a clonal pheochromocytoma cell line, Brain Res., 133 (1977) 350-353. Greene, L.A., Nerve growth factor prevents the death and stimulates the neuronal differentiation of clonal PC12 pheochromocytoma cells in serum-free media, J. Cell Biol., 78 (1978) 747-755. Greene, L.A. and Tischler, A.S., Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor, Proc. Natl. Acad. Sci. U.S.A., 73 (1975) 2424-2428. Greene, L.A. and Tischler, A.S., PC12 pheochromocytoma cultures in neurobiological research, Adv. Cell Neurobiol., 3 (1982) 373-414. Halegoua, S. and Patrick, J., Nerve growth factor mediates phosphorylation of specific proteins, Cell, 22 (1980) 571-581. Hamburger, V. and Oppenheimer, R.W., Naturally occurring neuronal death in vertebrates, Neurosci., Comment 1, (1982) 39-55. Hefti, F., Nerve growth factor promotes survival of septal cholinergic neurons after fimbrial transection, J. Neurosci., 6
230 (1986) 2155-2162. 20 Hefti, E, Hartikka, J.A., Montero, C.N. and Junard, E.O., Role of nerve growth factor in the central nervous system. In J . A Ferrendelli, R.C. Collins and E.M. Johnson (Eds.), Neurobiology of Amino Acids, Peptides and Trophic Factors, Kluwer, 1988, pp. 128-138. 21 Hefti, F. and Mash, D.C., Localization of nerve growth factor receptors in the normal human brain and Alzheimer's disease, Neurobiol. Aging, 10 (1989) 75-87. 22 Hendry, I.A., Stach, R.W. and Herrup, K., Characteristic of the retrograde axonal transport system for nerve growth factor in the sympathetic nervous system, Brain Res., 82 (1974) 117-128. 23 Jackson, G.R., Apffel, L., Werrbach-Perez, K. and Perez-Polo, J.R., Role of nerve growth factor in oxidant-antioxidant balance and neuronal injury. I. Stimulation of hydrogen peroxidase resistance, J. Neurosci. Res., 25 (1990) 360-368. 24 Jackson, G.R., Werrbach-Perez, K. and Perez-Polo, J.R., Role of nerve growth factor in oxidant-antioxidant balance and neuronal injury. II. A conditioning lesion paradigm, J. Neurosci. Res., 25 (1990) 369-374. 25 Johnson, Jr., E.M., Taniuchi, M., Clark, H.B., Springer, J.E., Koh, S., Tayrien, M.W. and Loy, R., Demonstration of retrograde transport of nerve growth factor receptor in the peripheral and central nervous system, J. Neurosci., 7 (1987) 923-929. 26 Koh, S. and Loy, R., Age-related loss of nerve growth factor sensitivity in rat basal forebrain neurons, Brain Res. 440 (1988) 396-401. 27 Landfield, P.W., Hippocampal neurobiological mechanisms of age-related memory dysfunction, Neurobiol. Aging, 9 (1988) 571-579. 28 Landfield, P.W., Aging-related increase in voltage-sensitive, inactivating calcium currents in rat hippocampus. Implications for mechanisms of brain aging and Alzheimer's disease, Ann. Natl. Acad. Sci. U.S.A., 568 (1989) 95-105. 29 Levi-Montalcini, R., The nerve growth factor 35 years later, Science, 273 (1987) 1154-1162. 30 Mildbrandt, J., Nerve growth factor rapidly induces c-fos mRNA in PC12 rat pheochromocytoma cells, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 4780-4793. 31 Mobley, W.C., Schenker, A. and Shooter, E.M., Characterization and isolation of proteolytically modified nerve growth factor, Biochemistry, 15 (1981) 5543-5551. 32 Mufson, E.J., Boyhwell, M. and Kordower, J.H., Loss of nerve growth factor receptor-containing neurons in Alzheimer's disease: a quantitative analysis across subregions of the basal forebrain, Exp. Neurol., 105 (1989) 221-232. 33 Olender, E.J. and Stach, R.W., Sequestration of lZSl-labeiled Beta nerve growth factor by sympathetic neurons, J. Biol.
Chem., 255 (1980) 9338-9343. 34 Patacchioli, ER., Amenta, E, Ramacci, M.T., Taglialatela, G., Maccari, S. and Angelucci, L., Acetyl-L-carnitine reduces the age dependent loss of adrenocorticoid receptors in the rat hippoeampus: an autoradiographic study, J. Neurosci. Res., 23 (1989) 462-466. 35 Phelps, C.H., Gage, EM., Growdon, J.H., Hefti, E, Harbaugh, R., Johnston, M.V., Khacheturian, Z.S., Mobley, W.C., Price, D.L., Reskind, M., Simpkins, J., Thai, L.J. and Woodcock, J., Potential use of nerve growth factor to treate Alzheimer disease, Neurobiol. Aging, 10 (1989) 205-207. 36 Perez-Polo, J.R., Werrbach-Perez, K., Ramacci, M.T., Taglialatela, G., Morgan, B. and Angelucci, L., Role of nerve growth factor in neurological disease. In A. Agnoli, J. Cahn, N. Lassen and R. Mayeux (Eds.), Senile Demendtia, H International Symposium, John Libbey Eurotext, Paris, 1988, pp. 15-25. 37 Perez-Polo, J.R., Foreman, P.J., Jackson, G.R., Shan, D., Taglialatela, G., Thorpe, L.W. and Werrbach-Perez, K., Nerve growth factor and neuronal cell death, Mol. Neurobiol., in press. 38 Romano, C., Nichols, R.A. and Greengard, P., Synapsin I in PCI2 cells. II. Evidence for regulation by NGF of phosphorylation at a novel site, J. Neurosci., 7 (1987) 1300-1306. 39 Sapolsky, R.M., Krey, L.C. and McEwen, B.S., Prolonged glucocorticoid exposure reduces hippocampal neuron number: implications for aging, J. Neurosci., 5 (1985) 1222-1227. 40 Sapolsky, R.M., Krey, L.C. and McEwen, B.S., The neurobiology of stress and aging: the glucocorticoid cascade hypothesis, Endocr. Rev., 7 (1986) 284-301. 41 Seiler, M. and Schwab, M.E., Specific retrograde transport of nerve growth factor from neocortex to nucleus basalis in the rat, Brain Res., 300 (1984) 33-39. 42 Smith, P.K., Krohn, R.I., Hermanson, G.T., MaUia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klen, D.C., Measurement of protein using bicinchoninic acid, Ann. Biochem., 150 (1985) 76-85. 43 Springer, J.E., Tayrien, M.W. and Loy, R., Regional analysis of age-related changes in the cholinergic system of the hippocampal formation and basal forebrain of the rat, Brain Res., 407 (1987) 180-184. 44 Stach, R.W. and Perez-Polo, J.R., Binding of nerve growth factor to its receptors, J. Neurosci. Res., 17 (1987) 1-10. 45 Stach, R.W. and Wagner, B.J., Sequestration requirements for the degradation of lZSl-labeled beta nerve growth factor bound to embryonic sensory neurons, J. Neurosci. Res., 7 (1982) 403-411. 46 Taglialatela, G., Angelucci, L., Ramacci, M.T., Foreman, P.J. and Perez-Polo, J.R., lzsI-fl-nerve growth factor binding is reduced in rat brain after stress exposure, J. Neurosci. Res., 25 (1990) 331-335.
221
BRESD 51252
Acetyl-L-carnitine enhances the response of PC12 cells to nerve growth factor G. Taglialatela 1'3, L. Angelucci 2, M.T. Ramacci 3, K. Werrbach-Perez 1, G.R. Jackson 1 and J.R. Perez-Polo t 1Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.), 2Department of Pharmacology, University of Rome 'La Sapienza', Rome 00185 (Italy) and 31nstitutefor Research on Senescence--Sigma Tau, Pomezia 00040 (Italy) (Accepted 8 January 1991)
Key words: Nerve growth factor; Rat pheochromocytoma (PC12) cell; Neurite outgrowth; Acetyl-e-carnitine; Aging
We have demonstrated that treatment of rat pheochromocytoma (PC12) cells with acetyl-L-carnitine (ALCAR) stimulates the synthesis of nerve growth factor receptors (NGFR). ALCAR has also been reported to prevent some age-related impairments of the central nervous system (CNS). In particular, ALCAR reduces the loss of NGFR in the hippocampus and basal forebrain of aged rodents. On these bases, a study on the effect of NGF on the PC12 cells was carried out to ascertain whether ALCAR induction of NGFR resulted in an enhancement of NGF action. Treatment of PC12 cells for 6 days with ALCAR (10 mM) stimulated [~25I]NGF PC12 cell uptake, consistent with increased NGFR levels. Also, neurite outgrowth elicited in PC12 cells by NGF (100 ng/ml) was greatly augmented by ALCAR pretreatment. When PC12 cells were treated with 10 mM ALCAR and then exposed to NGF (1 ng/ml), an NGF concentration that is insufficient to elicit neurite outgrowth under these conditions, there was an ALCAR effect on neurite outgrowth. The concentration of NGF necessary for survival of serum-deprived PC12 cells was 100-fold lower for ALCAR-treated cells as compared to controls. The minimal effective dose of ALCAR here was between 0.1 and 0.5 mM. This is similar to the reported minimal concentration of ALCAR that stimulates the synthesis of NGFR in these cells. The data here presented indicate that one mechanism by which ALCAR rescues aged neurons may be by increasing their responsiveness to neuronotrophic factors in the CNS. INTRODUCTION The aged central nervous system (CNS) is characterized by a progressive onset of morphological and functional impairments. In aged rodents, the n u m b e r of h i p p o c a m p a l neurons in the A m m o n ' s horn and dentate gyrus is significantly r e d u c e d 1'27. Also, n u m e r o u s pycnotic cholinergic neurons b e c o m e manifest in the septum and basal forebrain nuclei of aged rats 8'9. A l o n g with these anatomical i m p a i r m e n t s , there are a n u m b e r of malfunctions of different neuronal systems that take place in the aged. Glucocorticoid receptors in the aged h i p p o c a m p u s are r e d u c e d and their physiological response is impaired 1'6'27'34'39. F u r t h e r m o r e , there is evidence that enzyme m a r k e r s for central cholinergic neurons are r e d u c e d during physiological and pathological aging 5"43. Some behavioral performances associated with the brain structures d a m a g e d in senescence, such as learning and spatial orientation, are impaired in aged subjects 8,11. O n e event that is c o m m o n to the physiology and
pathophysiology of the aging process in the CNS, is the reduction of nerve growth factor ( N G F ) receptors 3' 12,21,26,32. N G F is a p o l y p e p t i d e that is essential for the d e v e l o p m e n t and m a i n t e n a n c e of some neurons of the p e r i p h e r a l nervous system (PNS), where it plays a key role in the regulation of n e u r o n a l cell d e a t h 29'37. The N G F p r o d u c e d by target tissues binds to specific receptors ( N G F R ) at growth cones and is t r a n s p o r t e d retrogradely to the neuronal cell b o d y 22. T h e resulting continuous supply of target-derived N G F is essential for the preservation of the innervating n e u r o n 18. In the CNS, N G F also has trophic effects on the magnocellular cholinergic neurons of the basal forebrain and septum 19' zo. As in the PNS, N G F is released by target tissues, such as h i p p o c a m p u s and frontal cortex, where it binds to N G F R on cholinergic terminals and is r e t r o g r a d e l y t r a n s p o r t e d to the cell s o m a in the basal forebrain and septum25,41. The finding that N G F exerts n e u r o n o t r o p h i c activity in CNS has led to the hypothesis that the r e p o r t e d loss of N G F R in senescence with the resulting reduction of N G F
Correspondence: G. Taglialatela, Department of Human Biological Chemistry and Genetics, 440 G. Borden Bldg. (F-52), University of Texas Medical Branch, Galveston, TX 77550, U.S.A.
222 activity is r e s p o n s i b l e for n e u r o n a l celt d e a t h and shrinkage t h e r e . C o n s e q u e n t l y , the t h e r a p e u t i c use of N G F for the t r e a t m e n t of n e u r o l o g i c a l diseases associated with aging has b e e n p r o p o s e d 35'36. It is k n o w n carnitine,
that
treatment
of rats with acetyl-L-
a naturally occurring substance
i n v o l v e d in
m i t o c h o n d r i a l m e t a b o l i s m of fatty acids 1° p r e v e n t s certain C N S i m p a i r m e n t s in the a g e d 1'2. A L C A R t r e a t m e n t of s e n e s c e n t
rats p r e v e n t s
ioral p e r f o r m a n c e s that are r e l a t e d to the limbic system J l, partially p r e v e n t s the loss of N G F R
that
o c c u r s in the h i p p o c a m p u s and basal f o r e b r a i n of a g e d r o d e n t s 3. In o r d e r to study w h e t h e r this is a direct r e l a t i o n s h i p between
ALCAR
and
NGF iodination The iodination of NGF was attained using a modified lactoperoxidase technique, as described elsewhere 33. Specific activity of the ['25I]fl-NGF was 2000-3500 cprn/fmol with 85-90% of the counts being acid-precipitable. The iodinated NGF was then stored at 4 °C in phosphate buffered saline, pH 7.4, containing 2 mg/ml cytochrome C and used within 2 weeks.
the loss of g l u c o c o r t i c o i d
r e c e p t o r s in the h i p p o c a m p u s and i m p r o v e s the b e h a v 34 A L C A R
determined spectrophotometrically. Fhe purity of/;LNGF was assayed by isoelectric focusing on polyacrylamide disc gel and specific biological activity determined by the method developed by Greene ~.
NGF/NGFR
activity,
the
rat
p h e o c h r o m o c y t o m a (PC12) cell line was c h o s e n as an in
Cell culture Rat pheochromocytoma (PCI2) cells were kindly provided by Dr. Lloyd Greene, Columbia University, New York, NY. Cells were grown in RPMI 1640, supplemented with 5% heat-inactivated horse serum +5% heat-inactivated fetal calf serum, at 37 °C in a humified incubator with 5% CO2 atmosphere and fed on alternative days. At subconfluency, cells were dislodged by vigorous shaking and reseeded at a 1:1 ratio. Acetyl-L-carnitine was dissolved in RPMI and added to cells at the final concentrations indicated in the various experiments.
vitro m o d e l system for N G F - r e s p o n s i v e n e u r o n s 16. PC12
[1251]NGF uptake
cells are a r a t - d e r i v e d cell line that display N G F R similar
PC12 cells were plated out in 24-well plates at a density of 105 cells/ml. On the sixth day of A L C A R treatment (10 mM), [lzSI]NGF was added at a final concentration of 5 nM and the cells incubated at 37 °C for 45 min. After incubation, cells were washed 3 times with ice-cold PBS and solubilized with 0.5 N NaOH. Aliquots were then taken from each well for protein assay and the rest counted in a y-counter for radioactivity content. Protein content was measured by the bicinchoninic acid method 42. Results were expressed as fmol of labeled NGF per mg of protein.
to
those
described
for
sympathetic
and
sensory
n e u r o n s 44. P C 1 2 typically r e s p o n d to N G F by e l o n g a t i n g n e u r i t e s and d e v e l o p i n g into electrically e x c i t a b l e cells f e a t u r i n g s o m e c h a r a c t e r i s t i c of the p o s t - m i t o t i c cholinergic n e u r o n a l p h e n o t y p e 15. O t h e r k n o w n effects o f N G F on the P C 1 2 cells i n c l u d e i n d u c t i o n of o n c o g e n e s , such as c-f0s4"3°; p h o s p h o r y l a t i o n o f s o m e c y t o p l a s m i c p r o t e i n s 17' 38; i n d u c t i o n o f o r n i t h i n e d e c a r b o x y l a s e m R N A T ; p r o t e c t i o n against p e r o x i d a t i v e injury and stimulation of catalase activity and p r o t e i n 23"24. T r e a t m e n t of P C 1 2 cells with A L C A R mRNA
results in i n c r e a s e d N G F R
p r o t e i n and
( T a g l i a l a t e l a et al., s u b m i t t e d ) .
In the p r e s e n t study we d e m o n s t r a t e that, consistent with A L C A R
s t i m u l a t i o n of N G F R synthesis, the action
of N G F on P C 1 2 cells is e n h a n c e d by A L C A R t r e a t m e n t .
Neurite outgrowth experiment PCI2 cells were plated out into 35-ram Petri dishes at a density of 2 × 105 cells/ml. On the sixth day of A L C A R treatment (10 mM); the cells were added with NGF dissolved in RPMI 1640 at a final concentration of either 1 or 100 ng/ml (0.037 or 3.7 nM). On days 2, 5 and 7 after NGF addition, 100-120 cells from 5-12 randomly chosen microscope fields were counted and assayed for presence of neurites. All the counts were done independently by two investigators on coded samples. After all counts were carried out, codes were broken and the average of the two counts taken as final value estimation. The results were expressed as percent of cells bearing neurite processes.
Thus, ALCAR may prevent some degenerative processes in t h e a g e d brain by l o w e r i n g t h e r e s p o n s e t h r e s h o l d of s u s c e p t i b l e n e u r o n s to n e u r o n o t r o p h i c factors.
MATERIALS AND METHODS
Materials N,N'-methylene-bis-acrylamide, glycine and ammonium persulfate were purchased from Bio-Rad Laboratories; Cytochrome C, amberlite GC-400, lactoperoxidase and H20 2 were from Sigma; RMPI 1640, fetal calf serum and horse serum were purchased from Hazleton; Ampholines, pH range 3.5-10.0, were obtained from LKB Produkter AB, and ultrapure urea from Mallinckrodt Chemicals; carrier-free NaJ25I was from Amersham and [35S]methionine from ICN; acetyl-L-carnitine was supplied by the Sigma Tau Company, Italy. All other chemicals used were reagent grade.
NGF isolation The fl-NGF subunit was isolated from mouse submaxillary gland according to the method of Mobley et al. 3t After elution from an ion exchange column, the NGF was dialyzed overnight against 0.1% acetic acid. The dialysate was then concentrated by speed vacuum centrifugation to 1 mg/ml and final concentration of fl-NGF
Serum deprivation experiments PC12 were plated out into 24-well plates at a density of 5 x 104 cells/ml treated for 6 days with A L C A R 10 mM. After 6 days, the serum was removed from the culture medium and the cells were given NGF at concentrations 0.01-100 ng/ml (0.37 pM to 3~7 nM). After 48 h the medium was removed, replaced with fresh medium containing 2/~Ci/ml of [35S]methionine and cells were then incubated for 4 h at 37 °C as described elsewhere 23'24. After incubation, cells were washed in PBS, sotubilized with 0.5 N NaOH and the protein precipitated by addition of 50% trichtoroacetic acid (TCA). Prior to TCA precipitation, aliquots from each sample were taken for protein assay. Samples were then passed through:GF/F Whatman filters and washed with 5 vols. of 5% TCA. Filters were transferred into scintillation vials and, after addition of 5 ml of scintillation cocktail (Beckman), counted for radioactivity in a r-counter. In another experiment, PCI2 cells were treated for 6 days with ALCAR 0.1, 0.5, or 1 mM and supplemented, after serum deprivation, with NGF 1 ng/ml (0.037 nM). All the experiments were done in quadruplicate and the results (incorporation of [35S]methionine into TCA-precipitable proteins) expressed as cpm/ mg of protein.
Statistical analysis Statistical differences among groups were evaluated by analysis of
223 NGF
~-
5
120
4
100
NGF + Alcar"
..~
80
3
2 O
40 2O Control
Alcor
Fig. 1. [~25I]NGF uptake in PC12 cells treated for 6 days with 10 mM ALCAR. *P<0.05 (Two-tail Student's t-test); n = 8 per group.
variance (ANOVA) for unweighted means followed by Fischer's least significant difference (LSD) test. When appropriate, the two-tailed Student's t-test was applied instead. RESULTS
[125I]NGF uptake PC12 cells were treated for 6 days with A L C A R (10 mM) and then incubated for 45 min at 37 °C in the presence of 5 nM [125I]NGE This procedure led to an increased labelling of the cells that had been previously treated with A L C A R , as compared to control PC12 cells (Fig. 1). This finding is consistent with the reported increase in N G F R synthesis in PC12 cells following A L C A R treatment (Taglialatela et al., submitted). It is important to notice that we carried out our incubation at 37 °C for 45 min. Since at 37 °C the internalization of the N G F - N G F R complex in PC12 cells occurs within 20 to 30 min after NGF binding 44, the values reported here should include uptake of [125I]NGF into the cytoplasm, and not be limited to NGF bound to the plasma membrane. This assumes that A L C A R does not disrupt NGF internalization.
Neurite outgrowth experiments When we exposed to NGF (100 ng/ml) the PC12 cells treated for 6 days with A L C A R (10 mM), we observed a dramatic increase in the extent of neurite outgrowth elicited by NGF (Fig. 2 through Fig. 5). On day 2 after NGF exposure, the number of cells displaying neurites was 8-fold higher in the ALCAR-treated PC12 as compared to controls. Furthermore, after 5 days of NGF treatment, there were twice as many neurites in A L C A R treated PC12 than control cells. In both cases the difference in the extent of neurite outgrowth between the ALCAR-treated and control PC12 cells was statistically significant, as calculated by analysis of variance of the data (F = 29.21; df = 3; P<0.001). On day 7 of exposure to NGF, there was a slight, not statistically significant, difference in the neurite outgrowth of the A L C A R -
0 Day 2
Day 5 NGF t r e a t m e n t
time
Day • period
Fig. 2. Neurite outgrowth in PC12 cells treated for 2, 5, or 7 days with NGF (100 ng/ml). ALCAR treatment (10 mM) started 6 days prior to addition of the NGF. Results are expressed as percent of cells displaying neurites in 5-12 microscope fields per condition. *Statistical difference versus control cells; ANOVA: F = 29.21; df = 3; P<0.001. treated PC12 as compared to controls. Given that NGF-treated cells will clump with time, it was difficult at these latter times to draw definitive conclusions. In a second experiment, we used NGF at 1 ng/ml, a concentration that in our system does not stimulate neurite outgrowth. As shown in Fig. 6, there was neurite extension from the ALCAR-pre-treated PC12 cells, but not from the PC12 treated with NGF alone. A L C A R treatment alone did not elicit neurite outgrowth from PC12 cells under any conditions tested (Figs. 3-6, panel B).
NGF rescue of serum-deprived PC12 cells PC12 cells in a serum-free environment require NGF for survival TM. Increasing concentrations of NGF enhanced viability in a dose-response fashion (Fig. 7). The minimal concentration of NGF that increased survival of serum-deprived PC12 cells was 1 ng/ml. Furthermore, PC12 survival after serum deprivation was supported by 10 mM A L C A R and concentrations of NGF that were as low as 0.01 ng/ml. Thus, A L C A R treatment potentiated NGF trophic action on PC12 cells, as measured by [3~S]methionine incorporation. Since the vital dye exclusion test for viability in the presence of NGF may lead to errors due to PC12 aggregation, here we used the incorporation of [35S]methionine into TCA-precipitable proteins as a measure of viability. A detailed discussion of the reliability of this technique is given elsewhere (Jackson et al., in preparation). The addition of 1 ng/ml of NGF to serum-deprived, ALCAR-treated (0.1-1.0 mM) PC12 cells, showed that 0.5 mM of A L C A R is the minimal concentration that potentiates the rescue by NGF of PC12 after serum deprivation (Fig. 8). This A L C A R concentration is similar to the lowest dose of A L C A R reported to stimulate NGFR in PC12 ceils (Taglialatela et al., submitted).
224
Fig. 3. Phase-contrast micrograph of PC12 cells after 2 days of N G F treatment (100 ng/ml) in the presence or absence o~ A L C A R (10 mM) in the culture medium. A L C A R treatment started 6 days prior to addition of N G F to the cells. Reference bar = 100 ktm, A: control cells; B: A L C A R - t r e a t e d cells; C: NGF-treated cells; D: N G F - A L C A R - t r e a t e d cells.
225
Fig. 4. Phase-contrast micrograph of PC12 cells after 5 days of N G F treatment (100 ng/ml) in the presence or absence of A L C A R (10 mM) in the culture medium. A L C A R treatment started 6 days prior to addition of NGF to the cells. Reference bar = 100 ~m. A: control cells; B: A L C A R - t r e a t e d cells; C: NGF-treated cells; D: N G F - A L C A R - t r e a t e d cells.
226
I i
i
I
,
Fig. 5. Phase-contrast mierograph of PC12 cells after 7 days of NGF treatment (100 ng/ml) in presence or absence of ALCAR (10 raM) in the culture medium. ALCAR treatment started 6 days prior to addition of NGF to the cells. Reference bar = 100/~m. A: control cells; B: ALCAR-treated cells; C: NGF-treated cells; D: NGF-ALCAR-treated cells.
227
Fig. 6. Phase-contrast micrograph of PC12 cells after 5 days of NGF treatment (1 ng/ml) in presence or absence of A L C A R (10 mM) in the culture medium. ALCAR treatment started 6 days prior to addition of NGF to the cells. Reference bar = 75/~m. A: control cells; B: ALCAR-treated cells; C: NGF-treated ceils; D: NGF-ALCAR-treated cells.
228 [--~
Control
~
AIcQr
J
700 r / 6001
[ Control
~
NGF
(Ing/rn~]
]
I0
*
4001 -
~
F 300 4
1
,o O0
, 1 NGF" ( n g / m t )
1
1
Fig, 7. [3SS]methionine incorporation in serum deprived PC12 cells supplemented with NGF at various concentrations. ALCAR treatment: 10 mM for 6 days; n = 3-4 per condition; *significant difference versus respective control group (NGF 0 ng/ml); ANOVA: F = 71.36; df= 11; P < 0.001.
0
0
01 Alcar (raM)
05
~0
Fig. 8. [3SS]methionine incorporation in serum-deprived PCI2 cells supplemented with NGF t ng/ml. ALCAR treatment: 0.t, 0.5, or 1 mM for 6 days. n = 3-4 per condition; *statistical difference versus control group (NGF 0 ng/ml); *#statistical difference versus cells treated with NGF alone. ANOVA: F = 6.06; df = 4; P < 0.005.
DISCUSSION The results presented here are in agreement with the finding that A L C A R enhances N G F R expression in PC12 cells (Taglialatela et al. submitted). In that study, we reported that treatment of PC12 with A L C A R at varying concentrations for 6 days increased [125I]NGF binding, immunoprecipitable N G F R protein and m R N A for the NGFR. We therefore studied some of the known effects elicited by N G F in order to assess whether the increase in N G F R after A L C A R treatment resulted in an increased response of the PC12 cells to N G E T h e increase of [12SI]NGF uptake by ALCAR-treated PC12 can be explained as a direct consequence of the reported increase in N G F R at the membrane level. Supportive of this hypothesis is the finding that A L C A R treatment enhances both type I and type II N G F R in PC12 cells, as measured by [12SI]NGF equilibrium binding and by immunoprecipitation of crosslinked N G F N G F R complex (Taglialatela et al., submitted). Since only the type I (high-affinity) N G F R is responsible for internalization of N G F 45, increased levels of the type I N G F R on the cell surface would result in an increased rate of internalization and uptake of [~2SI]NGF in ALCAR-treated PC12. This assumes that A L C A R does not disrupt N G F internalization. Since A L C A R also enhanced the PC12 morphological response to N G F which has been demonstrated to rely on the internalization of N G F bound to high-affinity N G F binding sites in PC1244, any effects of A L C A R on the transition of NGF from a bound state to an internalized state would have to be a stimulatory nature. The best-characterized effect of N G F on PC12 cells is the neurite outgrowth and differentiation of PC12 into neuronal-like cells that display some features of a post-mitotic cholinergic phenotype 15. Treatment of PC12
with 10 mM A L C A R for 6 days led to a dramatic increase in the neurite outgrowth elicited by 100 ng/ml of N G E Although after 7 days of N G F treatment one could not differentiate by morphological criteria control cells from their ALCAR-treated counterparts, on the second day of NGF treatment the number of neurite-bearing PC12 cells was 8-fold lower in the control cells as compared to the ALCAR-treated PC12. These results would suggest that A L C A R treatment accelerates the response time of PC12 cells to N G E Furthermore, when PC12 were exposed to N G F concentrations that do not elicit extension of neurites, under our conditions, we could observe neurite outgrowth in the ALCAR-treated cells. This would suggest that A L C A R treatment, rather than shortening the response time of PC12 to NGF, enhances their responsiveness to N G E The mechanism of action for the A L C A R effect on NGF-stimulated neurite outgrowth may not be limited to an increased internalization of the N G F - N G F R complex. The N G F stimulation of neurite outgrowth in PC12 is a complex phenomenon that is preceeded by reduction of cellular divisions, ~mproved attachment to substrate and flattening of cells 16. Although A L C A R alone does not stimulate neurite outgrowth in PC12, we often observed that ALCAR-treated cells appeared to be more flattened and better attached to culture flasks (Figs. 3-6, panel B). Alternatively A L C A R , by means of its enhancement of fatty acid catabolism 1°, may increase the availability of the highenergy compounds required for morphological transformation of cells after N G F stimulation. These possible mechanisms of action are not mutually exclusive and may be independently involved in the A L C A R potentiation of NGF-stimulated neurite outgrowth. Since it has been demonstrated that N G F rescues PC12 cells which would normally die when cultured in the
229 absence of serum 14, we assayed the ability of A L C A R to enhance N G F protection of s e r u m - d e p r i v e d PC12 cells. The minimal concentration of N G F that is required to support PC12 cells after serum deprivation is 1 ng/ml. In the A L C A R - t r e a t e d cells, the same result could be achieved at concentrations of N G F as low as 0.01 ng/ml. The analysis of a b r o a d e r spectrum of A L C A R concentrations r e v e a l e d that the lower dose of A L C A R that is effective in potentiating the N G F rescue of serum d e p r i v e d PC12 is similar to that r e p o r t e d to increase the synthesis of N G F R in this cell line. This result suggests that the e n h a n c e m e n t of the N G F R expression by A L C A R may be primarily involved in the increased N G F protection of serum deprived PC12 in the presence of ALCAR. The aged CNS exhibits a n u m b e r of morphological and functional i m p a i r m e n t s 1'6'8'28'4° that are a c c o m p a n i e d by different behavioral disorders 27. The finding that during the physiological and pathological aging of the CNS there is a massive loss of N G F R and N G F activity, has led to the hypothesis that reduction of n e u r o n o t r o p h i c activities in CNS may be responsible for the onset of those i m p a i r m e n t s that are characteristic of brain senescence 3" 21,26,32,36. Consequently, a possible therapeutic role for N G F in the pathological disorders of the CNS associated with aging has been p r o p o s e d 35'36. H o w e v e r , the ex-
pected minimal t r a n s p o r t of N G F across the b l o o d - b r a i n b a r r i e r if a d m i n i s t e r e d systemically would not encourage the routine testing of N G F as a clinical agent. Treatment of rats with A L C A R prevents some of the CNS impairments that occur in senescence and some of the behavioral disorders r e p o r t e d for aged rodents ~' 2,11.34. A L C A R t r e a t m e n t partially prevents the loss of N G F R in the h i p p o c a m p u s and basal forebrain of aged rats and c o m p l e t e l y abolishes the reduction of N G F binding that follows e x p o s u r e to stress in h i p p o c a m p u s of adult rodents 3"46. O n these findings, a direct correlation between the effects of A L C A R on aged rats and its effects on N G F activity in CNS can be assumed. In conclusion, the data p r e s e n t e d here would indicate that A L C A R may p r e v e n t some d e g e n e r a t i v e events in the CNS of aged rodents by enhancing the responsiveness of neurons to e n d o g e n o u s n e u r o t r o p h i c factors. It follows that A L C A R should be considered as a synergistic or activating agent for e n d o g e n o u s trophic factors, this being of special interest for the p r o p o s e d use of exogenous N G F in pathologies associated with senescence.
Acknowledgement. The authors wish to thank Mrs. D. Masters for manuscript preparation. This work was supported in part by a grant from Sigma Tau Company, Italy, CNR, Italy and by NINDS Grant NS18708.
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