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Reductions in acidic amino acids andN-acetylaspartylglutamate in amyotrophic lateral sclerosis CNS
Brain Research, 556 (1991) 151-156 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939124759P
151
BRES 24759
Reductions in acidic amino acids and N-acetylaspartylglutamate in amyotrophic lateral sclerosis CNS Guochuan Tsai, Barbara Stauch-Slusher, Lulu Sim, John C. Hedreen, Jeffrey D. Rothstein, Ralph Kuncl and Joseph T. Coyle Departments of Psychiatry, Neuroscience, Pathology and Neurology, The Johns Hopkins University School of Medicine Baltimore, MD 21205 (U.S.A.)
(Accepted 23 April 1991) Key words: Amyotrophic lateral sclerosis; Aspartate; Glutamate; N-Acetylaspartate; N-Acetylaspartylglutamate; N-Acetylated-a-linked-amino dipeptidase (NAALADase); Excitoxin
Acidic excitatory amino acids have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). We now report that, in addition to selective regional reductions in endogenous aspartate and glutamate, N-acetylaspartate (NAA), and N-acetylaspartylgiutamate (NAAG) are also decreased in the CNS, whereas the activity of N-acetylated-a-linked-amino dipeptidase (NAALADase) is increased. In cervical cord, the concentrations of aspartate and glutamate were decreased significantlyin the ventral horn; NAb, was decreased in the ventral horn, dorsal horn and ventral column, whereas NAAG was decreased in all regions of the cord examined, except the posterior column. NAALADase activity was increased in the ventral column. In motor cortex of ALS patients, aspartate and glutamate were decreased and NAALADase activity was increased in both gray and white matter; whereas NAAG was decreased in gray matter alone. None of these parameters was affected in the cerebral cortex of the Huntington's patients. Of the markers examined, the alterations in the levels of NAAG most closely parallel the cellular neuropathology in ALS.
Amyotrophic lateral sclerosis (ALS) is a progressive disorder of upper and lower motor neurons which is characterized by the combination of muscle weakness, wasting, hyperreflexia and spasticity. Although the pathogenesis of ALS remains unknown, there is evidence that acidic excitatory amino acids may be involved. The metabolism of glutamate (Glu) in CNS and in the periphery has been reported to be abnormal in ALS patients 21'22'26. The levels of Glu and aspartate (Asp) are significantly reduced in ALS spinal cord a t autopsy 22'26. Asp and Glu are considered to be the major excitatory neurotransmitters in brain, and persistent activation of their receptors can cause selective neuronal degeneration through the so-called 'excitotoxic' mechanism 5. N-acetylaspartylglutamate ( N A A G ) is an acidic dipeptide found in high concentrations in the CNS with the greatest levels in the spinal cord and brainstem 1°. Immunocytochemical studies have revealed that N A A G is concentrated in a number of putative glutamatergic neuronal systems, including the Betz pyramidal cells of the motor cortex; but, in addition, it is also co-localized to the motor neurons of the brainstem and ventral horn of the spinal cord 16'19. An enzyme which cleaves N A A G
into N-acetylaspartate (NAA) and Glu, designated Nacetylated-a-linked-amino dipeptidase (NAALADase), has been characterized and purified to homogeneity 24"32. Whereas N A A may serve as a precursor to and catabolite of N A A G , N A A is also highly concentrated in neurons throughout the CNS, and N A A is rapidly de-acetylated to Asp after intracerebral injection 15. N A A G is released by a calcium-dependent process with electrical stimulation or potassium depolarization from excitatory nerve terminals 3a,34. Neurophysiologic studies indicate that high concentrations of N A A G can activate the N M D A receptor aS, which has been implicated in excitotoxic responses 25. Furthermore, N A A G has been suggested to act as a mixed agonist-antagonist at the N M D A receptor based upon ligand binding studies 23, and on intracellular recordings in the Xenopus expression system 27. While the functional roles of N A A G and N A A and their relationship to neurotransmitter pools of Glu and Asp remain poorly understood, N A A G itself, or its products, Glu and Asp, could participate in excitotoxic responses 1. Recently, we demonstrated that the concentrations of Asp, Glu, N A A G and N A A are significantly elevated by
Correspondence: J.T. Coyle, Department of Psychiatry, Meyer 4-163, The Johns Hopkins School of Medicine, 600 N. Wolfe St., Baltimore, MD 21205, U.S.A.
152 2- to 3-fold in the CSF of living patients with ALS 26, whereas their levels were significantly reduced in the spinal cord ventral horus in a cohort of patients who died with ALS. Since N A A G and NAA are co-localized to motoneurons, it is unclear if reductions in their ventral horn levels represent merely an epiphenomenon of ventral horn neuronal cell loss. Accordingly, in the present study, we carried out a more extensive postmortem neurochemical analysis to determine whether these neurochemical abnormalities occur elsewhere in the CNS in ALS, whether they are selective for acidic amino acids and N A A G in ALS, and whether they are specific for ALS or occur in another neurodegenerative disorder, Huntington's disease. Cervical spinal cords were obtained at autopsy from 8 pathologically confirmed ALS patients. The control group includes 8 patients without neurological disease and 1 patient with Parkinson's disease. Sections of cortical tissue from medial frontal gyrus and motor cortex were also obtained at autopsy from a different population of 9 ALS, 5 Huntington's disease and 10 control brains. Both spinal cord and cortical tissues came from the Johns Hopkins Brain Bank. The mean delay between death and freezing were 9.9 h (range, 2-22 h) after death. The tissues were stored at -80 °C for up to 5 years. Mean age was similar in ALS, Huntington's disease and control patients (mean age: ALS, 63.0 years; Huntington's, 61.0; controls, 65.7 years); whereas postmortem delay was somewhat shorter in ALS (ALS, 7.4 h; Huntington's, 11.0 h; controls, 11.8 h). Tissue slabs of 2-3 mm thickness were punched by using a 19 gauge needle, according to the method of Palkowitz et al. TM. In spinal cord, bilateral punches from ventral horn, dorsal horn, ventral column, dorsal column and lateral column were assayed. In motor cortex and medial prefrontal gyrus, double samples of deep gray matter and the underlying white matter were assayed (Fig. 1). The punches were sonicated in 90% methanol in water (v/v), centrifuged at 10,000 g, and aliquots of supernate were assayed. All biochemical analyses were performed in a blinded fashion. For NAALADase assay, the tissues were sonicated in 0.1% Triton in 50 mM Tris-HCl buffer, pH 7.4. Protein was determined by the method of Lowry et al. a2 with bovine serum albumen as standard. Amino acids were analyzed by o-phathaldehyde precolumn derivatization coupled with reverse phase C18 column HPLC separation and fluorescent detection 8, Absolute concentrations of the amino acids were determined using computer analysis of peak height with internal and external standards. NAA and NAAG were assayed by anion-exchange HPLC separation and UV detection at the wavelength of
214 nm m. Their concentrations were also determined by peak height calculation. Activity of NAALADase was measured by the hydrolysis of N-acetyl-L-aspartyl-L-[3H]glutamate as described by Robinson et al. 24. The levels of Asp and Glu in cervical cord in the control subjects were similar to previously reported values (Table I) 22'26. In patients with ALS, Asp and Glu were significantly decreased in ventral horn (-31 and -22%, respectively; Table I) but not in the other gray and white matter areas assayed. Taurine, GABA, glycine and glutamine were unchanged in all sampled regions. NAAG and NAA were substantially decreased not only in the ventral horn (-40 and -60%), but in the dorsal horn (-44 and -45%), and the ventral column (-49 and -37%). In the lateral column, the concentration of NAAG was decreased by 46%, whereas NAAG was increased by 49% in the posterior column (Table I). NAALADase activity tended to be higher in ALS patients, but was significantly elevated only in the ventral column (Table I). In precentral motor cortex of ALS patients, Asp and
A. Cervical Cord
B. Cortex
Fig. 1. Schematic representation of the sites sampled by the micropunch which are represented as shaded circles in the cervical cord and cortex.
153 TABLE I Excitatory transmitter markers in cervical cord o r A L S
Values are nmol/mg protein (mean ± S.E.M.) The values of NAA and NAAG in ventral horn have been published, see ref. 26.
NAAG Control ALS NAA Control ALS NAALADase (pmol/mg protein/h) Control ALS Aspartate Control ALS Glutamate Control ALS Glutamine Control ALS Taurine Control ALS
Ventral horn
Dorsal horn
Ventral column
Lateral column
Dorsal column
11.1 _+2.1 4.4 ± 0.9***
11.6±2.7 6.4±0.7*
9.0±0.5 5.7±0.8**
10.9±1.0 5.9±1.0"*
8.0±0.8 11.9±1.2"
92.5 ± 9.6 55.3 ± 9.7**
22.2±2.6 12.3±1.7"*
14.0±1.0 7.1±0.7"**
15.0±2.2 11.8±1.9
15.7±1.4 19.3±1.7
0.6 ± 0.2 1.0 ± 0.3
1.0±0.2 1.1±0.2
0.3±0.1 0.7±0.2*
0.3±0.1 0.5±0.1
0.6±0.2 0.6±0.2
9.3 ± 0.4 6.5 ± 0.7*
6.9±0.6 8.4±1.3
4.2±0.3 4.1±0.4
5.6±0.2 4.7±0.4
6.0±0.5 6.7±0.6
49.3 ± 4.1 38.2 ± 2.2*
~.3±4.9 41.3±4.5
20.8±1.6 21.6±2.2
21.8±1.7 21.6±1.7
28.5±2.0 26.9±1.6
42.7 ± 4.0 38.2 ± 2.1
~.5±2.1 ~.3±1.5
39.2±2.1 33.6±1.8
32.3±1.7 34.4±1.1
42.2±2.2 35.3±1.6
12.7 ± 0.9 12.0 ± 1.3
10.5±1.1 14.3±1.3
6.4±0.4 6.4±1.0
4.7±0.3 5.5±0.5
7.6±0.8 7.4±0.6
16.9 ± 1.8 13.6 ± 0.9
14.4±0.8 16.4±0.8
9.0±1.0 11.6±1.1
8.9±0.9 10.8±3.0
10.6±1.0 10.3±0.7
2.7 ± 0.3 2.8 ± 0.2
2.8±0.1 2.5±0.2
2.9±0.1 2.7±0.4
1.9±0.2 2.3±0.2
2.5±0.2 2.2±0.2
GABA
Control ALS Glycine Control ALS
*p < 0.05, **P < 0.01, ***P < 0.005 versus control values by Student's two-tailed t-test.
Glu were decreased in both gray (-23 and - 2 1 % respectively) and white m a t t e r ( - 4 2 and - 4 0 % ) . The decrease of N A A G was limited to gray m a t t e r ( - 3 4 % ) , and N A A was unchanged in m o t o r cortex. N A A L A D a s e activity increased significantly both in gray ( + 157%) and white ( + 9 5 % ) matter. M o t o r cortex from H u n t i n g t o n ' s brains did not show any significant alterations in N A A , N A A G , N A A L A D a s e and the o t h e r amino acids examined (Table II). In medial frontal cortex, A L S values were not significantly different from control values for N A A , N A A G , N A A L A D a s e and the amino acids (Table II). Since there was a difference in the p o s t m o r t e m delay b e t w e e n A L S patients (mean = 7.4 h) as c o m p a r e d to controls ( m e a n = 11.8 h), we e x a m i n e d the correlation coefficients b e t w e e n time to autopsy and sample content of the substance for all controls in which a significant difference between A L S samples was observed. O n l y one d a t a set, ventral horn glutamate, exhibited a significant correlation between p o s t m o r t e m delay and levels (r 2 = 0.771; P < 0.05), which might have occurred by chance with 18 d a t a sets.
O u r study extends the previous findings of alterations in brain levels of A s p and G l u in A L S 2°'22'26 to include N A A , N A A G and N A A L A D a s e . Notably, the reductions in N A A and N A A G in A L S tissues are m o r e p r o n o u n c e d than those for G i n and A s p and m o r e closely parallel the cellular p a t h o l o g y o f the disorder. While m o r p h o m e t r i c studies reveal a t r o p h y and d e g e n e r a t i o n of the large m o t o r neurons and the large m y e l i n a t e d fibers of the ventral spinal roots in A L S 9'28, in a d v a n c e d cases, medium-sized a n d large n e u r o n s in the i n t e r m e d i a t e zone are also severely affected 17. D e g e n e r a t i o n of the lateral and ventral columns in A L S results from the loss of the myelinated axons in the corticospinal tract; however, the posterior white column is conspicuously spared 7. Notably, N A A G and N A A are r e d u c e d in the dorsal and ventral horns and the ventral and lateral columns, but not the dorsal column of the cervical cord, whereas significant alterations in A s p and G i n are restricted to the ventral horn. Neuronal loss from the pre-central gyrns is p r o m i n e n t in A L S 6. T h e decrease o f N A A G , A s p and G l u in the m o t o r cortex, but not in the frontal cortex, further
154 TABLE II Excitatory transmitter markers in cortex
Values are nmol/mg of protein (mean ± S.E.M.). Motor cortex
NAAG Control ALS Huntington's NAA Control ALS Huntington's NAALADase (pmoFmgprotei~h) Control ALS Huntington's Aspartate Control ALS Huntington's Glutamate Control ALS Huntington's Glutamine Control ALS Huntington's ~urine Control ALS Huntington's GABA Control ALS Huntington's Glycine Control ALS Huntington's
Frontal cortex
Gray matter
White matter
14.3±1.0 9.4±1.1'* 12.7±0.8
16.3±2.6 16.3±2.7 16.7±4.3
4.7±0.7 3.8±0.5
5.9±1.0 4.6±1.0
93.9±4.9 ~.5±9.4 78.0±4.5
73.5±4.5 ~.6±7.6 69.1±9.2
29.4±4.0 24.1±2.9
33.9±3.4 28.4±4.0
3.9±0.6 7.5±1.8" 5.9±0.9
2.8±0.5 4.5±1.2
4.3±1.2 6.8±0.9
15.5±1.2 11.9±1.0" 13.5 ± 1.0
13.4±1.6 7.8±0.6*** 12.3 ± 1.2
13.4±1.2 11.5±0.7
11.7±1.2 9.4±0.7
70.4 ± 2.8 55.7±2.9** 65.3 ± 3.6
~ . 4 ± 3.8 35.9±3.8** 53.7 ± 2.6
74.2±4.6 63.6±2.9
72.7±3.8 73.2±3.0
~ . 7 ± 1.7 46.5±1.8 42.5±1.4
49.3 ± 2.1 47.1±2.1 45.1±1.9
50.2±3.4 ~.8±3.6
51.5±4.3 45.3±1.3
9.5±0.8 11.0±0.5 10.9±0.5
11.4±0.8 14.0±1.0 14.4±1.4
11.6±0.8 10.3±1.2
13.6±0.7 15.3±0.9
53.7±2.7 48.6±2.0 45.4±3.4
9.4±0.8 9.0±0.5 7.6±0.5
20.3±1.4 17.57±0.9
22.2±2.3 25.7±2.1
10.3±1.6 9.4±0.8 8.8±1.5
10.3±1.7 9.5±1.2 12.4±0.7
9.4±1.2 7.5±1.1
10.0±1.0 11.5±1.3
3.3±0.3 8.5±1.9"** 4.0±0.8
Gray matter
White matter
*P < 0.05, **P < 0.01, ***P < 0.005 versus control values by Student's two-tailed t-test.
d e m o n s t r a t e s congruence between the n e u r o p a t h o l o g y and neurochemical abnormalities of the acidic amino acids and peptides in A L S . F u r t h e r m o r e , these alterations a p p e a r to be specific for the amino acids that we studied. No significant alterations in glutamine, taurine or glycine were o b s e r v e d in any areas of the cervical cord or cerebral cortex in the A L S samples. T h e levels of the neurotransmitter, GABA, which is concentrated in interneurons of affected regions, were also unaffected. F u r t h e r m o r e , neither the acidic amino acids nor N A A G levels were altered in the cerebral cortex in Huntington's disease, indicating that the reductions in A L S are not simply a secondary consequence of a primary neurodegenerative process. The increased C S F levels and the decreased spinal
cord levels of A s p , Glu, N A A and N A A G suggest that factors that m o d u l a t e the levels of these 4 substances are closely interrelated. It is interesting to note that N A A L A D a s e activity is increased in b o t h ventral column and m o t o r cortex. F u r t h e r m o r e , m o l a r concentrations of N A A G and N A A in C S F are g r e a t e r than the m o l a r concentrations of A s p and Glu 26, which raises the possibility that C S F A s p and G l u are metabolites of N A A and N A A G . Such an inference is consistent with evidence that the catabolic enzyme, N A A L A D a s e , is located on the extracellular surface of m e m b r a n e s and that N A A G and N A A themselves are not subject to active transport, whereas the G l u l i b e r a t e d from N A A G is very efficiently t a k e n up 3. H o w e v e r , the relationship b e t w e e n intracellular N A A G and potential neurotran-
155 smitter pools of A s p and Glu remains unclear, since N A A G is localized to a n u m b e r of putative glutamatergic and aspartergic n e u r o n a l systems 1. The co-localization of N A A G to m o t o n e u r o n s , while consistent with the reductions in its levels in the ventral horn in A L S , does not seem to c o r r e s p o n d with any evidence that glutamate or glutamate-like substances serve a n e u r o t r a n s m i t t e r function in the v e r t e b r a t e m o t o r neurons. H o w e v e r , it is i n d e e d curious that G l u appears to be the neurotransmitter o f the m o t o r neurons in invertebrates, such as insects 4.
evidence that exogenous g l u t a m a t e analogs m a y p r o d u c e neurotoxicity with A L S - l i k e s y m p t o m s and neuropathology 29-31. F u r t h e r m o r e , alterations in the levels of N A A and N A A G in o t h e r neurologlc disorders are not without precedent. Notably, elevation of N A A with concomitant aciduria and the deficiency of aspartoacylase has been r e p o r t e d to be associated with neuronal degeneration in C a n a v a n disease 5'11'14. F u r t h e r m o r e , N A A 13 and N A A G 2 have b e e n shown to b e significantly r e d u c e d in the brain and in the spinal cord of h e r e d i t a r y myodystrophic mice. Nevertheless, because of our current ignorance about the relationship a m o n g N A A and N A A G , A s p and Glu, and N A A L A D a s e , the possibility that these alterations are an e p i p h e n o m e n o n o f some, m o r e fundamental, n e u r o p a t h o l o g i c process cannot be excluded.
Since A s p , Glu and N A A G are all capable of activating N M D A receptors, which can lead to selective n e u r o n a l d e g e n e r a t i o n , a plausible scenario that would account for b o t h the elevated extracellular levels and d e p l e t e d tissue levels o f N A A G and the acidic amino acids would be a defect in the storage process for N A A G . Such a defect would result in elevated extracellular concentrations of N A A G , G l u and A s p that ultimately causes excitotoxic d a m a g e in the affected neuronal systems. This hypothesis is consistent with accumulating
This research was supported by the McKnight Foundation, the Muscular Dystrophy Association, the Jay Slotkin Fund for Neuromuscular Research, and a USPHS Grant NS-13584. We thank Alice Trawinski for her editorial assistance.
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151
BRES 24759
Reductions in acidic amino acids and N-acetylaspartylglutamate in amyotrophic lateral sclerosis CNS Guochuan Tsai, Barbara Stauch-Slusher, Lulu Sim, John C. Hedreen, Jeffrey D. Rothstein, Ralph Kuncl and Joseph T. Coyle Departments of Psychiatry, Neuroscience, Pathology and Neurology, The Johns Hopkins University School of Medicine Baltimore, MD 21205 (U.S.A.)
(Accepted 23 April 1991) Key words: Amyotrophic lateral sclerosis; Aspartate; Glutamate; N-Acetylaspartate; N-Acetylaspartylglutamate; N-Acetylated-a-linked-amino dipeptidase (NAALADase); Excitoxin
Acidic excitatory amino acids have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). We now report that, in addition to selective regional reductions in endogenous aspartate and glutamate, N-acetylaspartate (NAA), and N-acetylaspartylgiutamate (NAAG) are also decreased in the CNS, whereas the activity of N-acetylated-a-linked-amino dipeptidase (NAALADase) is increased. In cervical cord, the concentrations of aspartate and glutamate were decreased significantlyin the ventral horn; NAb, was decreased in the ventral horn, dorsal horn and ventral column, whereas NAAG was decreased in all regions of the cord examined, except the posterior column. NAALADase activity was increased in the ventral column. In motor cortex of ALS patients, aspartate and glutamate were decreased and NAALADase activity was increased in both gray and white matter; whereas NAAG was decreased in gray matter alone. None of these parameters was affected in the cerebral cortex of the Huntington's patients. Of the markers examined, the alterations in the levels of NAAG most closely parallel the cellular neuropathology in ALS.
Amyotrophic lateral sclerosis (ALS) is a progressive disorder of upper and lower motor neurons which is characterized by the combination of muscle weakness, wasting, hyperreflexia and spasticity. Although the pathogenesis of ALS remains unknown, there is evidence that acidic excitatory amino acids may be involved. The metabolism of glutamate (Glu) in CNS and in the periphery has been reported to be abnormal in ALS patients 21'22'26. The levels of Glu and aspartate (Asp) are significantly reduced in ALS spinal cord a t autopsy 22'26. Asp and Glu are considered to be the major excitatory neurotransmitters in brain, and persistent activation of their receptors can cause selective neuronal degeneration through the so-called 'excitotoxic' mechanism 5. N-acetylaspartylglutamate ( N A A G ) is an acidic dipeptide found in high concentrations in the CNS with the greatest levels in the spinal cord and brainstem 1°. Immunocytochemical studies have revealed that N A A G is concentrated in a number of putative glutamatergic neuronal systems, including the Betz pyramidal cells of the motor cortex; but, in addition, it is also co-localized to the motor neurons of the brainstem and ventral horn of the spinal cord 16'19. An enzyme which cleaves N A A G
into N-acetylaspartate (NAA) and Glu, designated Nacetylated-a-linked-amino dipeptidase (NAALADase), has been characterized and purified to homogeneity 24"32. Whereas N A A may serve as a precursor to and catabolite of N A A G , N A A is also highly concentrated in neurons throughout the CNS, and N A A is rapidly de-acetylated to Asp after intracerebral injection 15. N A A G is released by a calcium-dependent process with electrical stimulation or potassium depolarization from excitatory nerve terminals 3a,34. Neurophysiologic studies indicate that high concentrations of N A A G can activate the N M D A receptor aS, which has been implicated in excitotoxic responses 25. Furthermore, N A A G has been suggested to act as a mixed agonist-antagonist at the N M D A receptor based upon ligand binding studies 23, and on intracellular recordings in the Xenopus expression system 27. While the functional roles of N A A G and N A A and their relationship to neurotransmitter pools of Glu and Asp remain poorly understood, N A A G itself, or its products, Glu and Asp, could participate in excitotoxic responses 1. Recently, we demonstrated that the concentrations of Asp, Glu, N A A G and N A A are significantly elevated by
Correspondence: J.T. Coyle, Department of Psychiatry, Meyer 4-163, The Johns Hopkins School of Medicine, 600 N. Wolfe St., Baltimore, MD 21205, U.S.A.
152 2- to 3-fold in the CSF of living patients with ALS 26, whereas their levels were significantly reduced in the spinal cord ventral horus in a cohort of patients who died with ALS. Since N A A G and NAA are co-localized to motoneurons, it is unclear if reductions in their ventral horn levels represent merely an epiphenomenon of ventral horn neuronal cell loss. Accordingly, in the present study, we carried out a more extensive postmortem neurochemical analysis to determine whether these neurochemical abnormalities occur elsewhere in the CNS in ALS, whether they are selective for acidic amino acids and N A A G in ALS, and whether they are specific for ALS or occur in another neurodegenerative disorder, Huntington's disease. Cervical spinal cords were obtained at autopsy from 8 pathologically confirmed ALS patients. The control group includes 8 patients without neurological disease and 1 patient with Parkinson's disease. Sections of cortical tissue from medial frontal gyrus and motor cortex were also obtained at autopsy from a different population of 9 ALS, 5 Huntington's disease and 10 control brains. Both spinal cord and cortical tissues came from the Johns Hopkins Brain Bank. The mean delay between death and freezing were 9.9 h (range, 2-22 h) after death. The tissues were stored at -80 °C for up to 5 years. Mean age was similar in ALS, Huntington's disease and control patients (mean age: ALS, 63.0 years; Huntington's, 61.0; controls, 65.7 years); whereas postmortem delay was somewhat shorter in ALS (ALS, 7.4 h; Huntington's, 11.0 h; controls, 11.8 h). Tissue slabs of 2-3 mm thickness were punched by using a 19 gauge needle, according to the method of Palkowitz et al. TM. In spinal cord, bilateral punches from ventral horn, dorsal horn, ventral column, dorsal column and lateral column were assayed. In motor cortex and medial prefrontal gyrus, double samples of deep gray matter and the underlying white matter were assayed (Fig. 1). The punches were sonicated in 90% methanol in water (v/v), centrifuged at 10,000 g, and aliquots of supernate were assayed. All biochemical analyses were performed in a blinded fashion. For NAALADase assay, the tissues were sonicated in 0.1% Triton in 50 mM Tris-HCl buffer, pH 7.4. Protein was determined by the method of Lowry et al. a2 with bovine serum albumen as standard. Amino acids were analyzed by o-phathaldehyde precolumn derivatization coupled with reverse phase C18 column HPLC separation and fluorescent detection 8, Absolute concentrations of the amino acids were determined using computer analysis of peak height with internal and external standards. NAA and NAAG were assayed by anion-exchange HPLC separation and UV detection at the wavelength of
214 nm m. Their concentrations were also determined by peak height calculation. Activity of NAALADase was measured by the hydrolysis of N-acetyl-L-aspartyl-L-[3H]glutamate as described by Robinson et al. 24. The levels of Asp and Glu in cervical cord in the control subjects were similar to previously reported values (Table I) 22'26. In patients with ALS, Asp and Glu were significantly decreased in ventral horn (-31 and -22%, respectively; Table I) but not in the other gray and white matter areas assayed. Taurine, GABA, glycine and glutamine were unchanged in all sampled regions. NAAG and NAA were substantially decreased not only in the ventral horn (-40 and -60%), but in the dorsal horn (-44 and -45%), and the ventral column (-49 and -37%). In the lateral column, the concentration of NAAG was decreased by 46%, whereas NAAG was increased by 49% in the posterior column (Table I). NAALADase activity tended to be higher in ALS patients, but was significantly elevated only in the ventral column (Table I). In precentral motor cortex of ALS patients, Asp and
A. Cervical Cord
B. Cortex
Fig. 1. Schematic representation of the sites sampled by the micropunch which are represented as shaded circles in the cervical cord and cortex.
153 TABLE I Excitatory transmitter markers in cervical cord o r A L S
Values are nmol/mg protein (mean ± S.E.M.) The values of NAA and NAAG in ventral horn have been published, see ref. 26.
NAAG Control ALS NAA Control ALS NAALADase (pmol/mg protein/h) Control ALS Aspartate Control ALS Glutamate Control ALS Glutamine Control ALS Taurine Control ALS
Ventral horn
Dorsal horn
Ventral column
Lateral column
Dorsal column
11.1 _+2.1 4.4 ± 0.9***
11.6±2.7 6.4±0.7*
9.0±0.5 5.7±0.8**
10.9±1.0 5.9±1.0"*
8.0±0.8 11.9±1.2"
92.5 ± 9.6 55.3 ± 9.7**
22.2±2.6 12.3±1.7"*
14.0±1.0 7.1±0.7"**
15.0±2.2 11.8±1.9
15.7±1.4 19.3±1.7
0.6 ± 0.2 1.0 ± 0.3
1.0±0.2 1.1±0.2
0.3±0.1 0.7±0.2*
0.3±0.1 0.5±0.1
0.6±0.2 0.6±0.2
9.3 ± 0.4 6.5 ± 0.7*
6.9±0.6 8.4±1.3
4.2±0.3 4.1±0.4
5.6±0.2 4.7±0.4
6.0±0.5 6.7±0.6
49.3 ± 4.1 38.2 ± 2.2*
~.3±4.9 41.3±4.5
20.8±1.6 21.6±2.2
21.8±1.7 21.6±1.7
28.5±2.0 26.9±1.6
42.7 ± 4.0 38.2 ± 2.1
~.5±2.1 ~.3±1.5
39.2±2.1 33.6±1.8
32.3±1.7 34.4±1.1
42.2±2.2 35.3±1.6
12.7 ± 0.9 12.0 ± 1.3
10.5±1.1 14.3±1.3
6.4±0.4 6.4±1.0
4.7±0.3 5.5±0.5
7.6±0.8 7.4±0.6
16.9 ± 1.8 13.6 ± 0.9
14.4±0.8 16.4±0.8
9.0±1.0 11.6±1.1
8.9±0.9 10.8±3.0
10.6±1.0 10.3±0.7
2.7 ± 0.3 2.8 ± 0.2
2.8±0.1 2.5±0.2
2.9±0.1 2.7±0.4
1.9±0.2 2.3±0.2
2.5±0.2 2.2±0.2
GABA
Control ALS Glycine Control ALS
*p < 0.05, **P < 0.01, ***P < 0.005 versus control values by Student's two-tailed t-test.
Glu were decreased in both gray (-23 and - 2 1 % respectively) and white m a t t e r ( - 4 2 and - 4 0 % ) . The decrease of N A A G was limited to gray m a t t e r ( - 3 4 % ) , and N A A was unchanged in m o t o r cortex. N A A L A D a s e activity increased significantly both in gray ( + 157%) and white ( + 9 5 % ) matter. M o t o r cortex from H u n t i n g t o n ' s brains did not show any significant alterations in N A A , N A A G , N A A L A D a s e and the o t h e r amino acids examined (Table II). In medial frontal cortex, A L S values were not significantly different from control values for N A A , N A A G , N A A L A D a s e and the amino acids (Table II). Since there was a difference in the p o s t m o r t e m delay b e t w e e n A L S patients (mean = 7.4 h) as c o m p a r e d to controls ( m e a n = 11.8 h), we e x a m i n e d the correlation coefficients b e t w e e n time to autopsy and sample content of the substance for all controls in which a significant difference between A L S samples was observed. O n l y one d a t a set, ventral horn glutamate, exhibited a significant correlation between p o s t m o r t e m delay and levels (r 2 = 0.771; P < 0.05), which might have occurred by chance with 18 d a t a sets.
O u r study extends the previous findings of alterations in brain levels of A s p and G l u in A L S 2°'22'26 to include N A A , N A A G and N A A L A D a s e . Notably, the reductions in N A A and N A A G in A L S tissues are m o r e p r o n o u n c e d than those for G i n and A s p and m o r e closely parallel the cellular p a t h o l o g y o f the disorder. While m o r p h o m e t r i c studies reveal a t r o p h y and d e g e n e r a t i o n of the large m o t o r neurons and the large m y e l i n a t e d fibers of the ventral spinal roots in A L S 9'28, in a d v a n c e d cases, medium-sized a n d large n e u r o n s in the i n t e r m e d i a t e zone are also severely affected 17. D e g e n e r a t i o n of the lateral and ventral columns in A L S results from the loss of the myelinated axons in the corticospinal tract; however, the posterior white column is conspicuously spared 7. Notably, N A A G and N A A are r e d u c e d in the dorsal and ventral horns and the ventral and lateral columns, but not the dorsal column of the cervical cord, whereas significant alterations in A s p and G i n are restricted to the ventral horn. Neuronal loss from the pre-central gyrns is p r o m i n e n t in A L S 6. T h e decrease o f N A A G , A s p and G l u in the m o t o r cortex, but not in the frontal cortex, further
154 TABLE II Excitatory transmitter markers in cortex
Values are nmol/mg of protein (mean ± S.E.M.). Motor cortex
NAAG Control ALS Huntington's NAA Control ALS Huntington's NAALADase (pmoFmgprotei~h) Control ALS Huntington's Aspartate Control ALS Huntington's Glutamate Control ALS Huntington's Glutamine Control ALS Huntington's ~urine Control ALS Huntington's GABA Control ALS Huntington's Glycine Control ALS Huntington's
Frontal cortex
Gray matter
White matter
14.3±1.0 9.4±1.1'* 12.7±0.8
16.3±2.6 16.3±2.7 16.7±4.3
4.7±0.7 3.8±0.5
5.9±1.0 4.6±1.0
93.9±4.9 ~.5±9.4 78.0±4.5
73.5±4.5 ~.6±7.6 69.1±9.2
29.4±4.0 24.1±2.9
33.9±3.4 28.4±4.0
3.9±0.6 7.5±1.8" 5.9±0.9
2.8±0.5 4.5±1.2
4.3±1.2 6.8±0.9
15.5±1.2 11.9±1.0" 13.5 ± 1.0
13.4±1.6 7.8±0.6*** 12.3 ± 1.2
13.4±1.2 11.5±0.7
11.7±1.2 9.4±0.7
70.4 ± 2.8 55.7±2.9** 65.3 ± 3.6
~ . 4 ± 3.8 35.9±3.8** 53.7 ± 2.6
74.2±4.6 63.6±2.9
72.7±3.8 73.2±3.0
~ . 7 ± 1.7 46.5±1.8 42.5±1.4
49.3 ± 2.1 47.1±2.1 45.1±1.9
50.2±3.4 ~.8±3.6
51.5±4.3 45.3±1.3
9.5±0.8 11.0±0.5 10.9±0.5
11.4±0.8 14.0±1.0 14.4±1.4
11.6±0.8 10.3±1.2
13.6±0.7 15.3±0.9
53.7±2.7 48.6±2.0 45.4±3.4
9.4±0.8 9.0±0.5 7.6±0.5
20.3±1.4 17.57±0.9
22.2±2.3 25.7±2.1
10.3±1.6 9.4±0.8 8.8±1.5
10.3±1.7 9.5±1.2 12.4±0.7
9.4±1.2 7.5±1.1
10.0±1.0 11.5±1.3
3.3±0.3 8.5±1.9"** 4.0±0.8
Gray matter
White matter
*P < 0.05, **P < 0.01, ***P < 0.005 versus control values by Student's two-tailed t-test.
d e m o n s t r a t e s congruence between the n e u r o p a t h o l o g y and neurochemical abnormalities of the acidic amino acids and peptides in A L S . F u r t h e r m o r e , these alterations a p p e a r to be specific for the amino acids that we studied. No significant alterations in glutamine, taurine or glycine were o b s e r v e d in any areas of the cervical cord or cerebral cortex in the A L S samples. T h e levels of the neurotransmitter, GABA, which is concentrated in interneurons of affected regions, were also unaffected. F u r t h e r m o r e , neither the acidic amino acids nor N A A G levels were altered in the cerebral cortex in Huntington's disease, indicating that the reductions in A L S are not simply a secondary consequence of a primary neurodegenerative process. The increased C S F levels and the decreased spinal
cord levels of A s p , Glu, N A A and N A A G suggest that factors that m o d u l a t e the levels of these 4 substances are closely interrelated. It is interesting to note that N A A L A D a s e activity is increased in b o t h ventral column and m o t o r cortex. F u r t h e r m o r e , m o l a r concentrations of N A A G and N A A in C S F are g r e a t e r than the m o l a r concentrations of A s p and Glu 26, which raises the possibility that C S F A s p and G l u are metabolites of N A A and N A A G . Such an inference is consistent with evidence that the catabolic enzyme, N A A L A D a s e , is located on the extracellular surface of m e m b r a n e s and that N A A G and N A A themselves are not subject to active transport, whereas the G l u l i b e r a t e d from N A A G is very efficiently t a k e n up 3. H o w e v e r , the relationship b e t w e e n intracellular N A A G and potential neurotran-
155 smitter pools of A s p and Glu remains unclear, since N A A G is localized to a n u m b e r of putative glutamatergic and aspartergic n e u r o n a l systems 1. The co-localization of N A A G to m o t o n e u r o n s , while consistent with the reductions in its levels in the ventral horn in A L S , does not seem to c o r r e s p o n d with any evidence that glutamate or glutamate-like substances serve a n e u r o t r a n s m i t t e r function in the v e r t e b r a t e m o t o r neurons. H o w e v e r , it is i n d e e d curious that G l u appears to be the neurotransmitter o f the m o t o r neurons in invertebrates, such as insects 4.
evidence that exogenous g l u t a m a t e analogs m a y p r o d u c e neurotoxicity with A L S - l i k e s y m p t o m s and neuropathology 29-31. F u r t h e r m o r e , alterations in the levels of N A A and N A A G in o t h e r neurologlc disorders are not without precedent. Notably, elevation of N A A with concomitant aciduria and the deficiency of aspartoacylase has been r e p o r t e d to be associated with neuronal degeneration in C a n a v a n disease 5'11'14. F u r t h e r m o r e , N A A 13 and N A A G 2 have b e e n shown to b e significantly r e d u c e d in the brain and in the spinal cord of h e r e d i t a r y myodystrophic mice. Nevertheless, because of our current ignorance about the relationship a m o n g N A A and N A A G , A s p and Glu, and N A A L A D a s e , the possibility that these alterations are an e p i p h e n o m e n o n o f some, m o r e fundamental, n e u r o p a t h o l o g i c process cannot be excluded.
Since A s p , Glu and N A A G are all capable of activating N M D A receptors, which can lead to selective n e u r o n a l d e g e n e r a t i o n , a plausible scenario that would account for b o t h the elevated extracellular levels and d e p l e t e d tissue levels o f N A A G and the acidic amino acids would be a defect in the storage process for N A A G . Such a defect would result in elevated extracellular concentrations of N A A G , G l u and A s p that ultimately causes excitotoxic d a m a g e in the affected neuronal systems. This hypothesis is consistent with accumulating
This research was supported by the McKnight Foundation, the Muscular Dystrophy Association, the Jay Slotkin Fund for Neuromuscular Research, and a USPHS Grant NS-13584. We thank Alice Trawinski for her editorial assistance.
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