Abstract
Some cases of familial amyotrophic lateral sclerosis (FALS) are associated with copper/zinc superoxide dismutase (Cu/Zn-SOD) mutations, which are implicated in the death of motor neurons. Because Cu/Zn-SOD is present in high amounts in nigrostriatal dopaminergic neurons, we considered the possibility that FALS may be associated with subclinical nigrostriatal dopaminergic dysfunction. We used [(18) F]fluorodopa (FDOPA) and PET to study 14 FALS patients (50 +/- 11 years [mean +/- SD]): seven with (FALS-1) and seven without (FALS-0) Cu/Zn-SOD mutations. Fourteen age-matched normal volunteers (48 +/- 18 years) served as controls. Striato-occipital ratios (SORs) for the caudate and the putamen were calculated. Five of the 14 FALS patients had reduced striatal FDOPA uptake in the caudate nucleus, putamen, or both. Mean caudate SOR did not differ among FALS-1, FALS-0, and control subjects. Mean putamen SOR was significantly abnormal in FALS-0 but not in FALS-1 patients. These findings indicate that subclinical nigrostriatal dopaminergic dysfunction is present in some FALS patients and that FDOPA/PET abnormalities are more likely to be associated with FALS-0 status. This suggests that SOD mutations are less cytotoxic to dopaminergic than to motor neurons.
NEUROLOGY 1996;47: 1546-1551
ALS is a fatal paralytic neurodegenerative disorder of unknown cause characterized by a progressive loss of upper and lower motor neurons. [1] Among the formulated physiopathogenic hypotheses [2,3] is that mutations of the copper/zinc superoxide dismutase (Cu/Zn-SOD) gene, found in some patients affected with familial ALS (FALS), [4,5] may reduce erythrocyte Cu/Zn-SOD activity [2] and decrease the half-life of the mutated Cu/Zn-SOD enzyme. [6] Chronic inhibition of Cu/Zn-SOD can produce apoptotic death of spinal neurons. [7] Moreover, at least three of the Cu/Zn-SOD mutations can cause motor neuron disease in transgenic mice. [8-10] The mechanism responsible for degeneration of motor neurons in FALS-associated Cu/Zn-SOD mutations is not known. [2-5]
Pardo et al. [11] studied the cellular distribution of Cu/Zn-SOD in the nervous system. The motor neurons of the spinal cord were among the cells most heavily immunostained for the enzyme protein. [11] Dopaminergic neurons of the substantia nigra were also conspicuously immunostained for Cu/Zn-SOD. Because FALS-associated Cu/Zn-SOD mutations are likely to be neurotoxic, we considered the possibility that subsets of neurons containing high amounts of Cu/Zn-SOD may be more prone to degeneration in the presence of these mutations. Accordingly, FALS patients carrying Cu/Zn-SOD mutations may lose not only motor neurons of the spinal cord but also dopaminergic neurons of the substantia nigra. Of relevance is the postmortem demonstration of reductions in the number of dopaminergic neurons in substantia nigra in sporadic ALS, [12] as well as striatal dopaminergic deficits in living patients studied with PET. [13] Degeneration of the nigrostriatal dopaminergic pathway is a key feature of the pathology of Parkinson's disease (PD). [14] Although similar Cu/Zn-SOD mutations have not been identified in PD, [15] alterations in Cu/Zn-SOD may be toxic to dopaminergic neurons [16-18] and may potentially be involved in the pathogenesis of parkinsonism as well as ALS.
Previously, we and others have used [(18) F]fluorodopa (FDOPA) and PET to demonstrate dysfunction in the nigrostriatal dopaminergic pathway. [19-21] In the present study, we used quantitative FDOPA/PET to study FALS patients with and without identified Cu/Zn-SOD mutations to test the hypothesis that nigrostriatal neurons may undergo subclinical degeneration in this disease.
Methods.
ALS patients.
We studied 14 patients (mean age, 50.4 +/- 11.4 years; range, 32 to 67) who fulfilled the criteria of definite ALS. [22] Thirteen of the 14 patients were from separate pedigrees with at least two other family members and two consecutive generations affected with ALS and were thus designated as FALS; at the time of the study, patient 2 had no positive family history for ALS, but was carrying a Cu/Zn-SOD mutation and thus was also considered as FALS. Patients were diagnosed in the Eleanor and Lou Gehrig ALS Research Center at Columbia-Presbyterian Medical Center, New York, NY or the Cecil B. Day Laboratory for Neuromuscular Research at the Massachusetts General Hospital, Boston, MA. Patients did not receive anticonvulsants, levodopa, or deprenyl (selegiline) for at least 6 weeks prior to the study. However, four ALS patients were taking vitamin E (300 to 3,000 U per day), and three were also taking vitamin C (1.5 to 3 grams per day), beta-carotene (25,000 U per day), or both. On the day of the PET scanning procedure, all of the patients had neurological examinations, and blood screening for Cu/Zn-SOD mutations [4] and enzyme activity. [23] Brain MRI was normal in all of the patients.
Control subjects.
Fourteen age-matched neurological normal volunteer subjects (mean age, 47.9 +/- 18.9 years; range, 23 to 77) were used in this study.
Positron emission tomography.
Patients and volunteers fasted overnight before PET scanning. PET studies were performed with the Superpett 3000 tomograph (Scanditronix, Essex, MA). These studies were authorized by the Institutional Review Board of North Shore University Hospital/Cornell University Medical College. Written consent for all subjects was obtained after a detailed explanation of the scanning procedure. The performance characteristics of this instrument have been described elsewhere. [24] This four-ring, BaF2 time-of-flight, whole-body tomograph acquires 14 PET slices with Z-axis translation of one half-ring distance every 30 seconds. Each slice is 8 mm thick and reconstructed with an in-plane resolution of 7.5 mm (full width at half maximum) in high resolution mode.
The PET procedure and FDOPA synthesis were performed as previously reported. [21] In brief, subjects were positioned with a Laitinen stereoadaptor and three-dimensional laser alignment. The gantry angle of the tomograph was adjusted to be parallel to the orbitomeatal line (OM). A cylindrical tube filled with68 Ge was placed in the field of view to provide real-time calibration for each slice. All studies were performed with eyes open in a dimly lit room and with minimal auditory stimulation. FDOPA was 95% radiochemically pure (specific activity, [approximately]400 mCi/micro mol). To inhibit peripheral decarboxylation, all subjects received 200 mg of carbidopa 60 minutes before the IV administration of 6 to 10 mCi of FDOPA in 20 to 40 ml of saline. The transit time from injection point to the brain was measured by the coincidence counter and the scanner. Emission scanning began simultaneously with the start of the FDOPA injection, and continuous scan data acquired in list mode between 0 and 100 minutes postinjection. PET images were reconstructed with a correction for tissue attenuation of 511 keV gamma-radiation measured with an external68 Ge sector source. PET reconstructions were also corrected for random coincidences, electronic dead time, and scatter effects.
Region of interest (ROI) analysis was performed on 256 x 256 PET reconstructions by a SUN microcomputer (490 SPARC server) and Scan/VP software. [25] ROIs for caudate nucleus, putamen, and occipital cortex were identified by visual inspection with reference to a standard neuroanatomic atlas. [26] Elliptical ROIs were placed interactively on composite (40 to 100 minutes) PET brain slices to out-line the caudate and the putamen (mean 50 pixels per caudate; mean 90 pixels per putamen; pixel size, 4 mm2). Irregular occipital ROIs (16 to 20 cm2) were defined on the first 10-minute scan (0 to 10 minutes) to avoid sampling activity in the transverse sinuses and torcula.
All PET scans were acquired and analyzed with the investigater blinded to SOD genetic status and erythrocyte SOR activity. For each subject, we calculated the right and left ratio of striatal to occipital activity (SOR) by dividing caudate and putamen count rates by the occipital count rate measured on the last 10-minute scan (90 to 100 minutes postinjection). In all patients and controls, we averaged right and left values to calculate mean hemispheric SOR measures for both the caudate and the putamen. (In previous studies, we have found SOR to be the optimal FDOPA/PET parameter for discriminating normal controls and patients with mild dopaminergic defects [21,27]).
Statistics.
All values represent mean +/- SD. Differences between FALS-0 and FALS-1 were tested by two-tailed unpaired Student's t-test. Differences among FALS-0, FALS-1, and control groups were tested by one-way ANOVA. When ANOVA showed significant differences, pairwise comparisons were tested by Student-Newman-Keuls post-hoc test. Pearson's product correlation coefficient, and subsequent linear regression, was performed with a least-squares curve fit model. In all analyses, the null hypothesis was rejected at the 0.05 level. All statistical analyses were performed with the program SigmaStat for Windows (version 1.0, Jandel Corporation, San Rafael, CA).
Results.
FALS patients.
Seven of the 14 FALS patients had Cu/Zn-SOD mutations (FALS-1) (Table 1). Erythrocyte SOD activity in all 14 FALS patients is also presented in Table 1. Age at the time of study, age at onset, and duration of symptoms were not significantly different between FALS-1 and FALS-0 patients (t-test, p > 0.05). At the time of PET, all FALS patients had signs of lower and upper motor neuron involvement, except for patient 11, who had no clinical evidence of upper motor neuron disease. Bulbar involvement was noticed in four patients (patients 1, 2, 12, and 13), but was severe only in patients 1 and 12. Legs were predominantly affected in nine patients and arms in five (Table 1). Minimal limb bradykinesia was observed in two patients (patients 7 and 12); tremor, rigidity, autonomic dysfunction, eye movement abnormalities, and dementia were absent in all patients.
Table 1. Clinical data and superoxide dismutase (SOD) activity
FDOPA/PET.
Representative abnormal FDOPA/PET from a FALS-0 patient (patient 7) and normal FDOPA/PET from a FALS-1 patient (patient 8) are shown in Figure 1. Mean caudate SOR in FALS-0 (1.86 +/- 0.37) and FALS-1 (2.04 +/- 0.33) patients did not differ significantly from control values (2.08 +/- 0.15; p = 0.22). However, in three FALS-0 (patients 7, 12, and 13) and one FALS-1 (patient 3), caudate SOR was reduced >2 SD below the control mean (Figure 2). By contrast, mean putamen SOR was significantly reduced in the FALS-0 group, but not in the FALS-1 group (p = 0.04). Three FALS-0 (patients 6, 7, and 13) had abnormal (>2 SD) reductions in putamen SOR below the control mean (Figure 2). Most patients exhibited some degree of clinical asymmetry, and among those who had an abnormal FDOPA/PET, only two (patients 6 and 7) had abnormal (>2 SD) striatal SOR asymmetry indices above the control mean (Table 2). In patient 6, striatal SOR reduction was greatest ipsilateral to the most affected body side, while in patient 7, striatal SOR reduction was greatest contralateral to the most affected body side. Among the FALS patients with abnormal FDOPA/PET, two (patients 3 and 13) had an abnormal caudate/putamen ratio (reduced >2 SD below the control mean), indicating a greater SOR reduction in caudate nucleus than in putamen (Table 2).
Figure 1. FDOPA PET images from a normal volunteer (Normal), a familial ALS patient (patient 7) without identified Cu/Zn-SOD mutations (FALS-0), and a familial ALS patient (patient 8) with an identified Cu/Zn-SOD mutation (FALS-1). The color quantifies (18) F activity at 2 hours after FDOPA injection relative to an internal calibration source. A decrease in both caudate and putamen FDOPA uptake is evident in patient 7, who does not carry a Cu/Zn-SOD mutation. In contrast, striatal FDOPA uptake was normal in patient 8, who carries a Cu/Zn-SOD exon-1 mutation and a reduced erythrocyte SOD activity.
Figure 2. Individual and mean +/- SD FDOPA PET striatal to occipital ratios (SORs) for caudate (A) and putamen (B) in normal volunteers (Control: n = 14), familial ALS without Cu/Zn-SOD mutation (FALS-0; n = 7), and familial ALS with Cu/Zn-SOD mutation (FALS-1; n = 7). Caudate SOR values did not differ among the three groups. Putamen SOR values were significantly reduced in the FALS-0 patients, but not in FALS-1 patients compared with control subjects. Asterisk represents a value different from controls (ANOVA, Student Newman-Keuls post-hoc test, p < 0.05).
Table 2. Striato-occipital ratio (SOR) [(18) F]fluorodopa (FDOPA)/PET asymmetry index and caudate/putamen ratio
We found no significant correlation between erythrocyte SOD activity and caudate or putamen SOR in FALS-0 or FALS-1 (r2 < 0.45, p > 0.06). Likewise, we found no correlation between age at onset or duration of symptoms and caudate or putamen SOR values in either group of patients (r2 < 0.20, p > 0.16).
Discussion.
The present study shows that five of the 14 FALS (35%) patients had significantly reduced striatal FDOPA uptake in the caudate nucleus, putamen, or both (Figure 2). Consistent with our finding is the postmortem demonstration of significant neuronal loss in the substantia nigra of FALS patients. [28-32] Clinical features of parkinsonism have been reported in only two of these five patients with nigral degeneration at autopsy. [29,30] Likewise, in our study, bradykinesia, as the sole sign of parkinsonism, was present in two of the five patients with abnormal FDOPA/PET (patients 7 and 12). Thus, in some of our patients, the reduction in striatal dopaminergic function may not have been sufficient to produce clinical parkinsonism. Indeed, in most of the FALS patients with abnormal FDOPA/PET, we observed the SOR reductions to be smaller in magnitude than those associated with early stage PD. [21] However, two of the patients (patients 3 and 13) without identifiable parkinsonism had SOR values in the range of early-stage PD. [21] Therefore, the major loss of upper and lower motor neurons possibly may mask extrapyramidal signs in some of these patients.
The combination of clinical evidence of degeneration of the anterolateral columns of the spinal cord and the FDOPA/PET finding of nigrostriatal dopaminergic pathway dysfunction in some of our patients indicates the potential for widespread degenerative changes in FALS. This suggests that a subgroup of FALS patients may in fact be affected with a form of multisystem degeneration, [33,34] rather than "pure ALS." Conversely, nine of our 14 FALS patients had normal striatal FDOPA uptake on PET (Figure 2), suggesting that in many other FALS patients the degenerative process excludes dopaminergic neurons. However, diffuse degeneration including the substantia nigra has been reported in ALS patients living beyond respiratory failure. [35] Therefore, nigrostriatal dopaminergic pathway alteration may have been detected in more of our patients had they progressed further by the time of PET.
In the present study, we found no significant linear correlation between SOR and duration of symptoms in FALS. Conversely, Takahashi et al. [13] found a negative correlation between the FDOPA uptake rate constant and duration of symptoms in sporadic ALS. This discrepancy may be related to differences in the ALS population studied (i.e., familial versus sporadic). We also noted that the reported correlation may have been driven by a distinct subset of ALS patients with an unusually long duration of symptoms. Thus, our data suggest that it is unlikely that a major relationship exists between duration of symptoms and severity of nigrostriatal dysfunction in ALS patients.
Among the patients with FALS, half carried Cu/Zn-SOD mutations, and half did not. Clinically, FALS-1 and FALS-0 were indistinguishable. The two groups were also indistinguishable in age of onset and duration of disease at the time of study (Table 1). Despite these clinical similarities, four of the seven FALS-0 patients (57%) had significantly reduced striatal SOR, while only one out of seven FALS-1 patients (14%) had an abnormal FDOPA/PET. This indicates that SOR values for putamen may be a reliable predictor of Cu/Zn-SOD mutation status. A FALS patient without the Cu/Zn-SOD mutation may be more likely to harbor subclinical nigrostriatal damage compared with another FALS patient bearing such a mutation.
We found that while putamen SOR was significantly reduced in FALS-0 but not in FALS-1 patients, caudate reductions did not reach statistical significance. Nevertheless, from looking at the individual caudate/putamen ratios (Table 2), it does not appear that the putamen is more vulnerable than the caudate. This suggests that in FALS patients with abnormal FDOPA/PET, nigral dopaminergic projections to the putamen may not be more affected than those projecting to the caudate nucleus. This pattern of FDOPA/PET changes is distinct from that seen in PD, wherein the putamen is predominately affected. [19] The apparent neurochemical difference between ALS and PD does not exclude the possibility of common pathogenic mechanisms in these two conditions. [2,36-38] For instance, shared genetic substrates for both ALS and PD have previously been suggested. [39] Because transgenic mice with increased Cu/Zn-SOD are resistant to N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, [16] abnormal Cu/Zn-SOD activity may be associated with increased sensitivity of dopaminergic neurons to certain neurotoxins. Additionally, reductions in Cu/Zn-SOD activity in cell cultures are associated with apoptotic cell death in several types of primary neurons and neuronal cell lines. [7,17,18] Against this notion, however, is our finding that nigrostriatal dopaminergic function is more frequently abnormal in FALS-0 patients, in spite of their normal erythrocyte SOD activity. We can further speculate that in ALS, and possibly in PD, the loss of dopaminergic neurons is likely not to be a direct consequence of altered SOD activity, but perhaps is related to another factor whose intrinsic neurotoxicity is modulated by SOD.
There are several arguments in favor of the gain of an adverse property by the mutant enzymes, [2,37] including the observations that (1) transgenic mice overexpressing the mutated Cu/Zn-SOD enzyme develop paralysis and motor neuron death; [8] (2) familial ALS is dominantly inherited; (3) mutant but not wild-type Cu/Zn-SOD cDNA induces apoptotic death in a neuronal cell line in vitro; [40] and (4) there are no true nulls yet defined in Cu/Zn-SOD mutations. However, the specific characteristics of the vulnerable cells that confer sensitivity to Cu/Zn-SOD mutations are not known. We found that FALS-1 patients, despite having severe upper and lower motor neuron loss, have relatively normal nigrostriatal dopaminergic function in life. This suggests that some property specific to motor neurons, but not to dopaminergic neurons, is required for neuronal death in the presence of a mutation in Cu/Zn-SOD.
Acknowledgment
We are grateful to Dr. L.P. Rowland for his comments and advice on the study.
- Copyright 1996 by Advanstar Communications Inc.
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