Excitatory Amino Acid Receptors in Human Spinal Cord Evaluation in Amyotrophic Lateral Sclerosis Patients? H. ALLAOUA, I. CHAUDIEU, P. BOKSA, T. L. PERBY! C. KRIEGEqb AND R. QUIRION Dotghs Hop’zulRwclwch Gm&r and Dtpartmnzt ofPsychiany McGU UnipnritY 6875 Latalk BM. Vdun, Q&btz H4H 1R3, C i z d br;lcuAty ofA4edicinc
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2176 H d h SciincW Mau British Columbia V6T 1W5,G a d
Amyotrophic lateral sclerosis (ALS) is a progressive id neuromuscular disease that attacks nerve cells and pathways in the brain and spinal cord. Its etiological basis is still unknown. Recent data’-3 suggest that ALS may result h m motoneuron damage induced by endogenous or exogenous excitotoxins, and particularly by excitatory amino acids.‘ A similar neuroexcitotoxic mechanism has been suggested as contributing to neurodegenerative processes in disorders such as Huntington’s and Alzheimer‘s w . 5 . 6
On the basis of an excitotoxic hypothesis of ALS, we investigated, using quantitative receptor autoradiognphy, whether there may be selective losses in any of the three subtypes of excitatory amino acid receptors in the spinal cord of ALS patients as compared to controls. Frozen postmortem human spinal cords were obtained fiom five control patients (age range 59-76 years) and five ALS patients (age range 43-91 years) with postmortem intervals < 24 hours. All h z e n samples were sectioned and tissue slices (20 pm) were cut using a uyostat at - 17OC, thaw mounted onto gelatinkhrome alum coated slides, air dried under vacuum at 4OC ovemq$t, and then stored at -7OOC. The PCP binding site of the NMDA receptor was labelled with 40 nM [3H]1-1-(2-thienyl)-cyclohexyl pipendine ([jH]KP). The AMPA binding site of the quisqualate ionotropic receptor was assessed using 50 nM alpha-[JH]amino-3-hydroxy-5-methyl-Q-isole propionic acid ([JHIAMPA) and the kainate binding site of the kainate receptor was labelled with 20 nM [jHIkainate using conditions previously desc~ibed.~-~ Autoradiograms were generated by apposing labelled sections to [JH]Hyperfilm (Amersham) This work was supported by Fonds de la Rccherche en SantC du Qutbec and the Medical bearch Council of Canada. 260
ALLAOUA ct al.: E X C m R Y AMINO ACID RECEPrORS
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LamInr II
261
Vent. Horn
FIGURE 1. Sections of spinal cord from normal control and ALS patients were incubated with 40 nM [3H]XP to label NMDA binding sites. Rcsults are exprrssed as [3H]TCP bound in ALS patients (n = 5) compared to normal control patients (n = 5) and are the mean f SEM of two separate experiments. :significantlydifirent from the control values,p< 0.005 (Student's t tat).
tbr 2-4 weeks, and the optical density of the autoradiogmns was analyzed by microcomputer image analyses (MCID) using a tritium standard b r film calibration. Under these experimental conditions, the specific binding of [3H]'ICP, [JHIAMPA and [3H]kainate represented 40%, 9096 and 50% of the total binding, respectively. In normal control spinal c o d , N-methyl-waspartate (NMDA), quisqualate (QUIS) and kainate receptor binding sites are almost exclusively concentrated in the gray matter. NMDA receptors are localized both in the ventral and dorsal horns with higher densities observed in lamina 11. QUIS and kainate receptors are especiallyabundant in the substantia gelatinosa of the dorsal horn of the spinal cord. In ALS patients, the discrete localization of these three classes of receptors was unchanged as compared to control tissues. However the apparent density of [JHI'ICP sites associated with NMDA receptors was decreased in both dorsal and ventral horn (67%and 55%of control values, respectively). The decrease in [3H]KPbinding sites was not related to the age of the subject. [3H]AMPA binding was not affected in ALS. The preferential alteration in the NMDA receptor suggests that, should an excitotoxic mechanism be operative in ALS, the NMDA sub-type of EAA receptor is a likely candidate as a mediator of this mechanism. Results are shown in FIGURE1. In summary, using [3H]TCP to label NMDA receptors, we have reported significant decreases in this binding site not only in the ventral horn which contains motoneurons, but also in the dorsal horn of the spinal cord of ALS patients. This may have implications fbr the clinical profile of ALS patients.
ACKNOWLEDGMENT We thank the National Neurological Tissues Bank.
262
ANNALS NEW YORK ACADEMY OF SCIENCES
REFERENCES 1. 2. 3. 4. 5. 6.
7. 8. 9.
PERRY,T. L., S. HANSEN & K. JONES. 1987. Neurology 37: 1845-1848. PLAITAKIS,A. 1990. Ann. Neurol. 28: 3-8. SPENCER, P. S., P. B. NUNM& J. HUGON.1987. Science 237: 517-522. YOUNG, A. B. 1990. Ann. Neurol. 28: 9-11. BEAL,M. F., N. W. KOWALL, D. W. ELLISON, M. F. MAZWREK, K. J. SWARTZ & J. B. MARTIN. 1986. Nature 321: 168-171. GREENAMYRE, J . T., J. B. PE”EY, C. T. D’AMATO &A. B. YOUNG. 1987. J. Neumhem. 48: 543-551. CONTRERAS, P.C., R QumoN & T. L.O’DONOHUB. 1986. Neurosci. Lett. 67: 101-106. NIEISEN, E. O., J. H. J. CHA,J. B. PENNEY & A . B. YOUNG. 1988. Eur. J. Pharmacol. 157: 197-203. REPRESA, A., E. TREMBLAY & Y. BEN ARI. 1987. Neuroscience 20: 739-745.
V ,-
2176 H d h SciincW Mau British Columbia V6T 1W5,G a d
Amyotrophic lateral sclerosis (ALS) is a progressive id neuromuscular disease that attacks nerve cells and pathways in the brain and spinal cord. Its etiological basis is still unknown. Recent data’-3 suggest that ALS may result h m motoneuron damage induced by endogenous or exogenous excitotoxins, and particularly by excitatory amino acids.‘ A similar neuroexcitotoxic mechanism has been suggested as contributing to neurodegenerative processes in disorders such as Huntington’s and Alzheimer‘s w . 5 . 6
On the basis of an excitotoxic hypothesis of ALS, we investigated, using quantitative receptor autoradiognphy, whether there may be selective losses in any of the three subtypes of excitatory amino acid receptors in the spinal cord of ALS patients as compared to controls. Frozen postmortem human spinal cords were obtained fiom five control patients (age range 59-76 years) and five ALS patients (age range 43-91 years) with postmortem intervals < 24 hours. All h z e n samples were sectioned and tissue slices (20 pm) were cut using a uyostat at - 17OC, thaw mounted onto gelatinkhrome alum coated slides, air dried under vacuum at 4OC ovemq$t, and then stored at -7OOC. The PCP binding site of the NMDA receptor was labelled with 40 nM [3H]1-1-(2-thienyl)-cyclohexyl pipendine ([jH]KP). The AMPA binding site of the quisqualate ionotropic receptor was assessed using 50 nM alpha-[JH]amino-3-hydroxy-5-methyl-Q-isole propionic acid ([JHIAMPA) and the kainate binding site of the kainate receptor was labelled with 20 nM [jHIkainate using conditions previously desc~ibed.~-~ Autoradiograms were generated by apposing labelled sections to [JH]Hyperfilm (Amersham) This work was supported by Fonds de la Rccherche en SantC du Qutbec and the Medical bearch Council of Canada. 260
ALLAOUA ct al.: E X C m R Y AMINO ACID RECEPrORS
-
E
LamInr II
261
Vent. Horn
FIGURE 1. Sections of spinal cord from normal control and ALS patients were incubated with 40 nM [3H]XP to label NMDA binding sites. Rcsults are exprrssed as [3H]TCP bound in ALS patients (n = 5) compared to normal control patients (n = 5) and are the mean f SEM of two separate experiments. :significantlydifirent from the control values,p< 0.005 (Student's t tat).
tbr 2-4 weeks, and the optical density of the autoradiogmns was analyzed by microcomputer image analyses (MCID) using a tritium standard b r film calibration. Under these experimental conditions, the specific binding of [3H]'ICP, [JHIAMPA and [3H]kainate represented 40%, 9096 and 50% of the total binding, respectively. In normal control spinal c o d , N-methyl-waspartate (NMDA), quisqualate (QUIS) and kainate receptor binding sites are almost exclusively concentrated in the gray matter. NMDA receptors are localized both in the ventral and dorsal horns with higher densities observed in lamina 11. QUIS and kainate receptors are especiallyabundant in the substantia gelatinosa of the dorsal horn of the spinal cord. In ALS patients, the discrete localization of these three classes of receptors was unchanged as compared to control tissues. However the apparent density of [JHI'ICP sites associated with NMDA receptors was decreased in both dorsal and ventral horn (67%and 55%of control values, respectively). The decrease in [3H]KPbinding sites was not related to the age of the subject. [3H]AMPA binding was not affected in ALS. The preferential alteration in the NMDA receptor suggests that, should an excitotoxic mechanism be operative in ALS, the NMDA sub-type of EAA receptor is a likely candidate as a mediator of this mechanism. Results are shown in FIGURE1. In summary, using [3H]TCP to label NMDA receptors, we have reported significant decreases in this binding site not only in the ventral horn which contains motoneurons, but also in the dorsal horn of the spinal cord of ALS patients. This may have implications fbr the clinical profile of ALS patients.
ACKNOWLEDGMENT We thank the National Neurological Tissues Bank.
262
ANNALS NEW YORK ACADEMY OF SCIENCES
REFERENCES 1. 2. 3. 4. 5. 6.
7. 8. 9.
PERRY,T. L., S. HANSEN & K. JONES. 1987. Neurology 37: 1845-1848. PLAITAKIS,A. 1990. Ann. Neurol. 28: 3-8. SPENCER, P. S., P. B. NUNM& J. HUGON.1987. Science 237: 517-522. YOUNG, A. B. 1990. Ann. Neurol. 28: 9-11. BEAL,M. F., N. W. KOWALL, D. W. ELLISON, M. F. MAZWREK, K. J. SWARTZ & J. B. MARTIN. 1986. Nature 321: 168-171. GREENAMYRE, J . T., J. B. PE”EY, C. T. D’AMATO &A. B. YOUNG. 1987. J. Neumhem. 48: 543-551. CONTRERAS, P.C., R QumoN & T. L.O’DONOHUB. 1986. Neurosci. Lett. 67: 101-106. NIEISEN, E. O., J. H. J. CHA,J. B. PENNEY & A . B. YOUNG. 1988. Eur. J. Pharmacol. 157: 197-203. REPRESA, A., E. TREMBLAY & Y. BEN ARI. 1987. Neuroscience 20: 739-745.
