Address correspondence and reprint requests to Dr John W. Haycock, LSUHSC-BIOCHEM, 1100 Florida Ave., New Orleans, LA 70119, USA. E-mail: email@example.com
Because age-related changes in brain dopaminergic innervation are assumed to influence human disorders involving dopamine (DA), we measured the levels of several presynpatic DAergic markers [DA, homovanillic acid, tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase (AADC), vesicular monoamine transporter 2 (VMAT2), and dopamine transporter (DAT)] in post-mortem human striatum (caudate and putamen) from 56 neurologically normal subjects aged 1 day to 103 years. Striatal DA levels exhibited pronounced (2- to 3-fold) post-natal increases through adolescence and then decreases during aging. Similarly, TH and AADC increased almost 100% during the first 2 post-natal years; however, the levels of TH and, to a lesser extent, AADC then declined to adult levels by approximately 30 years of age. Although VMAT2 and DAT levels closely paralleled those of TH, resulting in relatively constant TH to transporter ratios during development and aging, a modest but significant decline (13%) in DAT levels was observed in only caudate during aging. This biphasic post-natal pattern of the presynaptic markers suggests that striatal DAergic innervation/neuropil appears to continue to develop well past birth but appears to become overelaborated and undergo regressive remodeling during adolescence. However, during adulthood, a striking discrepancy was observed between the loss of DA and the relative preservation of proteins involved in its biosynthesis and compartmentation. This suggests that declines in DA-related function during adulthood and senescence may be explained by losses in DA per se as opposed to DAergic neuropil.
The dopamine (DA) neurotransmitter system is fundamentally involved in several human brain disorders occurring in both development and adulthood. These include idiopathic Parkinson's disease (Ehringer and Hornykiewicz 1960) and other Parkinsonisms occurring at an early age (< 21 years), including aromatic l-amino acid decarboxylase (AADC) deficiency (Hyland et al. 1992) and l-DOPA-responsive dystonia (Rajput et al. 1994). A hypothetical role for brain DA has also been suggested in other conditions such as schizophrenia (Seeman and Lee 1975), drug abuse (DiChiara 1995), and the mild motor impairment which accompanies normal senescence (cf. Kish et al. 1992). Because the clinical expression of all of the above human conditions is highly age-dependent, information on the status of the brain DA system and its biosynthetic capacity during normal development and aging is required for use as baseline data for comparison with the pathological disorders.
A general consensus has emerged that in normal human brain the number of nigrostriatal DA neurons declines during adulthood, as indicated by age-related losses in pigmented cell bodies in substantia nigra (Hirai 1968; McGeer et al. 1977; Mann and Yates 1979; Thiessen et al. 1990; Fearnley and Lees 1991) and in ligand-binding to the dopamine (Zelnik et al. 1986; Allard and Marcusson 1989) and vesicular monoamine transporters (Scherman et al. 1989) (DAT and VMAT2, respectively) in striatum (caudate and putamen). However, the magnitude of such decreases can be modest (see Discussion), and recent studies have questioned whether midbrain DAergic cell bodies do, in fact, decline during aging (Kubis et al. 2000). Post-mortem studies of striatal DA neurotransmitter content and, more particularly, biosynthetic capacity [tyrosine hydroxylase (TH) and AADC activity levels] provide conflicting data such that neither the extents nor the aging patterns of the DAergic changes are certain either in post-natal development or during adulthood (cf. Kish et al. 1992).
In general, these investigations have put forward three competing scenarios regarding age-related changes in striatal DA biosynthetic capacity: (i) the capacity for DA biosynthesis is maintained both during post-natal development and adulthood (Wolf et al. 1991); (ii) a precipitous decline takes place during the first two decades of life with only a moderate decrement in adulthood (McGeer & McGeer 1976a, b; McGeer et al. 1977); and (iii) a marked decline occurs in aging primarily restricted to middle age and senescence (Côté and Kremzner 1983; Bannon et al. 1992). Several factors may account for such discrepancies in the literature. The total number of subjects in many of the previous studies has often been small, and data from human infants and adolescents have been particularly sparse. Secondly, many investigations have relied upon enzyme activity measurements of TH and AADC in post-mortem brain, and the within-group variability of individual values is marked, possibly reflecting the susceptibility of the enzymes to pre- and post-mortem variables such as agonal status and freezing and thawing of the tissue for biochemical analysis (McGeer and McGeer 1975, 1976b; Lloyd and Hornykiewicz 1972; Black and Green 1975). This is in contrast to the much lower variability in post-mortem enzyme protein determinations for TH and AADC (cf. Kish et al. 1995; Zhong et al. 1995). Lastly, most individual studies have measured a limited number of DA markers, making comparisons between DA and presynaptic DAergic markers, no less among presynaptic DAergic markers themselves, difficult.
Previously, we reported a substantial age-related decline in dopamine (Kish et al. 1992), but not AADC levels (Kish et al. 1995) in post-mortem striatum of normal adult subjects. Thus, to address the above issues relating to the presence or absence of age-related changes of all of the major dopamine nerve terminal markers, during the entire period from post-natal development to senescence, we have measured DA, homovanillic acid (HVA), TH, AADC, VMAT2, and DAT levels in a relatively large cohort of subjects ranging in age from 1 day to 103 years old.
Materials and methods
Caudate and putamen samples were taken from the intermediate portion of slices 4 and 7, respectively, as described (Kish et al. 1988), from the autopsied brains of 56 neurologically and neuropathologically normal subjects (31 male, 25 female) who ranged in age from 1 day to 103 years. The mean post-mortem interval (between death and freezing of one half-brain at − 80°C) of the 56 subjects was 13 ± 1 h (mean ± SE). A one-way anova did not disclose a main effect of post-mortem time among the eight age groups ( p > 0.05).
The 56 subjects were separated into eight groups (n = 7 each) with mean ages of 1 month (mean ± SEM, 0.08 ± 0.04 years; range, 1 day−3.5 month), 8 months (0.65 ± 0.07; 4–10 months), 2 years (1.8 ± 0.2; 1.0–2.8 years), 9 years (8.5 ± 1.6; 3.5–14), 30 years (30 ± 3; 17–41), 52 years (52 ± 2; 42–58), 69 years (69 ± 2; 60–73), and 87 years (87 ± 4; 75–103). Levels of DA, HVA, TH and AADC were determined for all 56 subjects. Analysis of the remaining complement of markers required that several pairs of similar-age samples be combined, resulting in 47 samples separated into the same eight age groups as follows: 0.14 ± 0.05 years, 4 days−3.7 months, n = 5; 0.73 ± 0.07, 6.5 months−1 years, n = 6; 2.2 ± 0.3, 1.3–3.5 years, n = 6; 11 ± 2; 5.2–17, n = 6; 35 ± 3, 28–42, n = 6; 55 ± 2, 46–60, n = 6; 70 ± 1, 66–73, n = 6; 85 ± 3, 75–97, n = 6.
Although freezer storage time (interval between freezing of the brain at autopsy and sample preparation) for the 28 adults was about twice that for the 28 young subjects (7.7 vs. 3.3 years, respectively), neither one-way anovas of freezer storage time nor regression analyses of freezer storage time versus DA or TH levels within the four groups of the young subjects or of the adults revealed an effect of freezer storage time (p > 0.05).
Primary antibody production
Polyclonal rabbit antisera were raised by Bethyl Laboratories (Montgomery, TX, USA) and Macromolecular Resources (Fort Collins, CO, USA). For polyclonal anti-protein antibodies, animals were immunized initially with 200 μg protein/animal and boosted with 100 μg after 2, 4, and 6 weeks and 50 μg at 4 week intervals thereafter. For polyclonal antipeptide antibodies, peptides were coupled to keyhole limpet hemocyanin via the N-terminal Cys, and animals were immunized initially with 500 μg conjugate protein/animal and boosted with 250 μg and 125 μg as above. Sera were collected every other week for 6–12 months. The development and progression of immune responses were monitored by strip-blot immunolabeling assays of solubilized human caudate samples. Monoclonal mouse antibody against AADC peptide (see below) was generated by Sigma Israel (Rehovot, Israel). BALB/c mice were immunized and boosted with 200 μg conjugate at 3-week intervals. Fusion between spleen cells of immunized mice and NS-1 mouse myeloma cells was performed 3 days after the third booster injection, using polyethylene glycol (MW 1500). Mixed cell cultures were subsequently grown in Dulbecco's modified Eagle medium containing 100 μm hypoxanthine, 16 μm thymidine, 0.4 μm aminopterin and supplemented with 10% heat-inactivated horse serum. After 10–14 days of culture, supernatants were screened for AADC antibodies by ELISA and strip-blot immunolabeling assays of solubilized caudate. Positive wells were isolated and cells were cloned by limiting dilution. Supernatants were rescreened for AADC immunoreactivity by strip-blot immunolabeling assays of striatum, and an IgG1-producing clone (available under the product name DDC-109; Sigma Chemical Company) was selected for generating ascites fluid.
Working dilutions for each of the antibodies were chosen as approximately ‘half saturating,’ established empirically by antibody saturation curves in strip-blot immunolabeling assays of pooled human striatal tissue standards. The specificity of all antibodies was tested and confirmed by blot immunolabeling assays of human caudate samples from normal subjects and from subjects with advanced Parkinson's disease. Immunoreactivity with the latter samples was reduced by ≥ 90% (data not shown).
Affinity-purified polyclonal rabbit antibodies, raised against purified, sodium dodecyl sulfate (SDS)-denatured rat PC12 cell TH, were as described (Haycock 1989) and used at 0.5 μg IgG/mL. These antibodies recognize all four isoforms of human TH. Affinity-purified, human TH isoform-specific, polyclonal antipeptide antibodies were as described (Haycock 1991, 1993a) and used at 0.5–1.5 μg IgG/mL.
Aromatic l-amino acid decarboxylase
Polyclonal rabbit and monoclonal mouse antibodies were raised against a peptide from exon 1 of human AADC (C-GIEGRQVYPDVEPGYL-NH2) (Ichinose et al. 1989). The polyclonal antibodies were affinity-purified by column chromatography on a peptide-SulfoLink (Pierce, Rockford, IL. USA) resin. After extensive rinsing (Smith et al. 1978), bound antibody was eluted with HEPES-buffered MgCl2/ethylene glycol (Tsang and Wilkins 1991), dialyzed, concentrated, and used at 1.2 μg IgG/mL. The data reported below were obtained using these polyclonal antibodies, but results were confirmed with two additional antibodies: The monoclonal antipeptide antibody described above was used at a 1 : 4000 dilution of ascites fluid. A second polyclonal rabbit antibody was raised against full-length recombinant bovine AADC expressed in Escherichia coli (a gift from Dr John Reinhardt). AADC was isolated as inclusion bodies and purified by preparative SDS–polyacrylamide gel electrophoresis (PAGE) (Bio-Rad Laboratories, Hercules, CA, USA). Antibodies were affinity-purified as above by column chromatography on an AADC-AminoLink column after preadsorption of the sera on an AminoLink (Pierce) resin to which solubilized inclusion body protein from E. coli expressing a different protein (tryptophan hydroxylase) were bound. These antibodies were used at 1.0 μg/mL.
Polyclonal rabbit antibodies were raised against a peptide from the 2nd extracellular loop (C-HLHQSHGIDDLGPPRW-OH) and against a peptide from the C-terminal tail (C-EKDRELVDRGEVRQFTLRHWL-OH) (Giros et al. 1992). Antibodies were affinity-purified by column chromatography on a peptide-SulfoLink column as above and used at 1.4 and 1.7 μg IgG/mL, respectively. Samples were analyzed independently twice with each of the antibodies and mean values were used for statistical analyses shown in Table 1; however, the results with each of these antibodies are shown separately in Fig. 3.
Table 1. Influence of age on presynaptic dopaminergic markers in autopsied human caudate
Polyclonal rabbit antibodies were raised against a peptide from the C-terminal tail (C-TQNNIQSYPIGEDEESESD-OH) (cf. Erickson et al. 1996). Antibodies were affinity-purified by column chromatography on a peptide-SulfoLink column as above and used at 1.3 μg IgG/mL.
Polyclonal rabbit antibodies that specifically recognize the gamma subunits of neuron-specific enolase were obtained from BioGenex Laboratories (San Ramon, CA, USA) in concentrated format and used at a dilution of 1 : 1000 (Ordway et al. 1994).
Tissue samples were solubilized by sonication in 1% SDS, 5 mm Tris, 2 mm EDTA (final pH 8.0–8.3) and heating. Protein concentrations, as measured using the bicinchoninic acid method (Smith et al. 1985) with an albumin/IgG standard (Sigma 540–10), were adjusted to 1 mg/mL in sample buffer. Initially, equal volume aliquots of the samples and a range of pooled tissue standard aliquots were subjected to SDS–PAGE in slab gels (1.5 mm thick, 9% T) and transferred electrophoretically (3 V/cm, 16 h, room temperature (22–24°C)) to nitrocellulose sheets (0.2 μm, Bio-Rad). Ponceau S staining was recorded xerographically after background destaining with 0.2% (v/v) HCl (cf. Figure 1, top). In subsequent runs, aliquot volumes were adjusted as necessary to produce equal Ponceau S staining across the sample lanes. Transfers were then destained/quenched in blot buffer (Dulbecco's phosphate-buffered saline (Gibco, Rockville, MS, USA), 10 mm Tris-HCl (pH 7.6), 0.05% (w/v) Tween20, 0.01% sodium azide) containing 1% (w/v) polyvinylpyrrolidone (Haycock 1993b). Transfers were then incubated (1 h, room temperature) sequentially with primary antibody, secondary antibody (0.8–1.0 μg/mL), and 125I-protein A (400–600 kcpm/mL; Amersham) in blot buffer containing polyvinylpyrrolidone. Transfers were rinsed five times (2 × 2 min, 3 × 5 min) with blot buffer after incubation with each of the reagents. In early experiments, separate transfers were analyzed for TH and AADC immunoreactivity. Subsequently, relatively long gels (5–5.5′) were run and transfers were cut into three segments for probing with different antibodies (upper, DAT or VMAT2; middle, TH; lower, AADC or NSE) in order to conserve sample.
The secondary antibodies used were affinity-purified rabbit anti-mouse IgG1 (1 μg IgG/mL; DAKO) and affinity-purified swine anti-rabbit Ig (0.8 μg IgG/mL; DAKO). Immunoreactivity was visualized autoradiographically using X-ray film (XAR or BMS film; Kodak) and quantitated by gamma counting of excised bands and blanks. The levels of immunoreactivity were translated into protein levels by interpolation to a pooled tissue standard curve that was run on the same gel, and values were expressed as μg of tissue standard protein per μg sample protein. The levels of each antigen were determined independently at least three times and median values were taken for analysis. Except as noted, TH levels (from the type 1 plus type 2 band) were subsequently converted to ng TH/μg protein based upon calibration of the tissue standard against a sample of purified recombinant human TH containing equal amounts of types 1 and 2 TH (Haycock 1993a). An independent analysis of striatal AADC protein levels in the 28 adults has been reported previously (Kish et al. 1995).
Levels of DA and its HVA metabolite were measured using a HPLC procedure (Wilson et al. 1994). Striatal DA and HVA levels have been previously reported for 23 of the present 28 adult subjects (Kish et al. 1992).
Except as noted above, a minimum of three independent determinations was made for each of the protein markers, and median values were used for statistical analyses: First, the eight age groups were analyzed by one-way anova, followed by separate one-way anovas of the four youngest and the four oldest age groups. Secondly, Pearson product-moment correlation coefficients were also used to evaluate the effects of age in early post-natal development (the two youngest groups), early post-natal development through adolescence (the four youngest groups), and adulthood (the four oldest groups). Analyses of male and female subjects separately showed regression coefficients generally similar to those of the combined sujects (data not shown). An alpha of p < 0.05 was used as the criterion for statistical significance.
Representative blot immunolabeling by antibodies to the DAergic markers and NSE in caudate and putamen samples run on a 3.5′ gel is shown in Fig. 1. TH types 1 and 2 are present as a single band below the pre-stained 70 kDa marker, while TH types 3 and 4 are barely visible just above in the caudate sample. AADC is present as a much less intense single band between the 47 and 55 kDa markers, and NSE migrates as a single band (Ordway et al. 1994), just below AADC. VMAT2 and DAT migrate as a broad set of poorly resolved bands just above the pre-stained marker (Fig. 1, asterisk), which were used to quantitate these markers. (The heterogeneity of the transporter immunoreactivity, including the lower bands seen in the VMAT2 lanes, presumably reflects heterogeneity in glycosylation.) Specificity of the antibodies to the DAergic markers was confirmed by substantial (≥ 85%) decreases of immunoreactivities in striatum from Parkinson's disease subjects (not shown). Quantitation of striatal levels of DA and the presynaptic DAergic markers in the eight age groups is summarized in Tables 1 and 2. There was a signifcant main effect of age across all age groups on all markers (not presented), with the exception of AADC levels in caudate (F = 1.44).
Table 2. Influence of age on presynaptic dopaminergic markers in autopsied human putamen
Striatal DA levels exhibited a pronounced increase (2- to 3-fold) during development and then, as previously reported in a subset of these samples (Kish et al. 1992), a pronounced decrease (∼3-fold) from maximal levels during aging (Tables 1 and 2 and Fig. 2). Mean striatal HVA levels exhibited a similar developmental increase and decrease during aging, paralleling those of DA levels, and a statistically significant correlation between DA and HVA levels was found in each region in the two age subgroups (caudate; young, r = 0.74, old, r = 0.52; putamen: young, r = 0.67; old, r = 0.50). As a measure of DA turnover, however, the ratio of HVA to DA levels decreased during development and increased during aging (Tables 1 and 2).
TH and AADC levels in autopsied human striata
Striatal TH and AADC levels also exhibited a biphasic development/aging profile; however, these biosynthetic markers increased significantly during early development (1 month and 8 month age groups), reaching maximal levels in the two-year-old group, whereafter relatively stable levels were maintained throughout adulthood (Table 1 and Fig. 2). Unlike with DA levels, correlation coefficients between age and TH or AADC levels were not statistically significant for subjects in either the four youngest or the four oldest age groups (Table 1).
VMAT2 and DAT levels in autopsied human striata
While the mean transporter levels exhibited a biphasic profile similar to TH and AADC, with maximal values in the 8 month−2-year-old groups and relatively stable levels thereafter, neither anova nor regression analyses revealed any statistically significant effects (with the exception of a modest but statistically significant decline (13%) in DAT levels in the caudate during aging) (Table 1 and Fig. 3). Whereas DAT levels were quantitated with both the DAT-EL2 and DAT-CT antibodies (shown separately in Fig. 3), the values obtained with these antibodies were highly correlated (caudate, r = 0.81; putamen, r = 0.81), and mean values were subjected to the statistical analyses shown in Table 1.
TH isoform levels in autopsied human striata
In initial experiments, the upper band (types 3 and 4) and lower band (types 1 and 2) immunoreactivities with the affinity-purfied, polyclonal rabbit anti-TH (which recognizes all types equally; Haycock 1993a) were quantitated separately and the levels of types 3 plus 4 were expressed as a percentage of total TH. Whereas a main effect of age across all age groups in caudate and putamen was observed, there were no significant main effects or correlation coefficients in the subgroups (data not shown). Portions of selected samples were pooled and subsequently analyzed using the type-specific antibodies (Fig. 4). In agreement with previous studies in human adrenal (Haycock 1991) and human brain (Lewis et al. 1993), all four forms of TH are present in human striatum. The data in Fig. 4 presents the first quantitative analysis of the relative abundances of the four isoforms in brain. Types 3 and 4 comprise 5–6% of total TH, and types 1 and 2 each comprise approximately equal amounts of the preponderance of total TH. As judged by the expression of TH protein isoforms shown in Fig. 4, alternative splicing of TH RNA does not appear to vary post-natally. Moreover, the existence of TH protein isoforms in addition to types 1–4, as suggested by Dumas et al. (1996), could not be demonstrated either in human striatum (data not shown) or in human adrenals and pheochromocytomas (Haycock 2002).
NSE levels in autopsied human striata
NSE is considered to be a ubiquitous neuronal ‘housekeeping’ enzyme and, hence, not expressed specifically in DAergic elements. As such, it has been gainfully employed in previous studies of age-matched post-mortem human brain samples (Ordway et al. 1994) as an alternative/complement to total protein for normalizing specific antigen levels. As shown in Fig. 5, unlike the DAergic markers, NSE levels increased over the entire post-natal period studied (caudate: F = 9.70, r = 0.73; putamen: F = 4.47, r = 0.61), increasing on average 2.7% (caudate) and 0.1% (putamen) per decade from 8 month to 87 years of age.
Our investigation is the first to examine simultaneously the striatal levels of the DAergic markers DA, HVA, TH, AADC, VMAT2 and DAT in autopsied human brain of a large number of subjects spanning, not only the age range from birth to adolescence (1 day to 14 years), but the entire range of human post-natal development and aging.
The major findings from our study are that (i) striatal levels of presynaptic DAergic markers increase during up to the first 9 y of post-natal life, with transporters preceding biosynthetic enzymes preceding DA and HVA, (ii) most presynaptic DAergic markers exhibit a developmental peak which exceeds their adult levels, and (iii) although DA and HVA decline with age, the presynaptic DAergic protein markers show either no or only a modest age-related decline in adults. In addition, we found that the neuronal ‘housekeeping’ protein NSE, perhaps a marker for neuronal/neuropil density, showed a modest increase in the very young neonates and a gradual increase throughout life thereafter, consistent with a gradual decrease in volume and increase in neuropil density in the human corpus striatum from 20 to 90 years of age (Haug and Eggers 1991). And, despite the biphasic developmental increase/peak/plateau observed for total TH protein, the relative abundances of the four human isoforms remained unchanged throughout post-natal life.
Presynaptic DAergic markers in striata of young subjects
We found that of the presynaptic DAergic markers examined, striatal DA levels increased the most markedly after birth, reaching a plateau by 2–9 years of age. During this time span DA levels increased to 2- to 3-times those in the 1-month age group. And, while HVA levels exhibited a similar but smaller and not statistically significant increase over this period, a significant correlation between DA and HVA levels was found (caudate, r = 0.74; putamen, r = 0.52). By contrast, the developmental increases in TH and AADC were much smaller and appeared to peak within 8 months to 2 years of birth and no significant differences in either DAT or VMAT2 were observed, although the highest mean values of DAT and VMAT2 were observed in the 8-month-old age group.
Our observation in the human that striatal levels of DA, TH, and AADC increase from birth through infancy is consistent with comparable results from a large body of animal studies (cf. Irwin et al. 1994; Rosenberg and Lewis 1994, 1995; Erickson et al. 1998) which have shown a developmental increase in DA and TH that peaks in development before decreasing to adult levels, in the case of TH, or continuing to decline, in the case of DA. In humans, Kalaria et al. (1993) reported a less exaggerated increase in DA levels but significantly higher HVA levels in 19- versus 4-month-old-subjects. In fact, both DA and HVA levels exhibited an inverted-U shape in their cohort of 28 subjects ranging from 1 month to 55 years old. In sharp contrast to our findings, however, are the reports of a precipitous age-related decline in human striatal TH (McGeer et al. 1967:; McGeer and McGeer 1976b) and AADC (McGeer and McGeer 1976a,b) activity during the first 20 years of age. This discrepancy could be explained by problems associated with measuring TH and AADC activity in autopsied human brain or by the inclusion in these investigations of only four subjects aged less than 10 years and none under the age of five.
While the full complement of nigral DAergic perikarya appears to be established during the fetal period (cf. Sailaja and Gopinath 1994), our results indicate that full development of the nigrostriatal DAergic system is not completed at birth. The order of ‘maturation’ appears to be sequestration mechanisms (DAT, VMAT2), biosynthetic mechanisms (TH, AADC), and then transmitter/metabolite (DA, HVA). From the perspective that DA is potentially cytotoxic unless compartmentalized, such an ordering could be considered developmentally sage. Nonetheless, the biochemical basis by which DA levels lag behind expression of the biosynthetic enzymes remains open. A partial explanation may be that tetrahydrobiopterin, which serves, not only as a co-factor for TH activity, but as a reductant of Fe(III)-TH to Fe(II)-TH (the active form of TH) (cf. Fitzpatrick 1999), limits the production of DA. As reported by Furukawa and Kish (1998), a post-natal increase (2-fold) in total biopterin levels was observed in post-mortem human putamen (n = 27) over the same age range (1 month to 9 years of age) as in the present study. As such, measurement of the post-natal expression of GTP cyclohydrolase 1 (a key enzyme in tetrahydrobiopterin biosynthesis) in humans might provide further insight. In mice, for example, there is a dramatic post-natal increase in cerebral pteridine levels and GTP cyclohydrolase 1 activity (Yoshida et al. 2000).
Although the mean levels of DAT and VMAT2 protein in developing striatum were suggestive of an early increase and subsequent decrease to adult levels, no statistically significant differences emerged. As would be expected for proteins exported from the cell bodies, the molecular masses of the transporters that were quantitated reflected post-translationally ‘mature’ forms. However, total homogenates were analyzed and, as such, the present data cannot address the subcellular provenance of the levels reported. For example, Tennyson et al. (1972) reported a 67% increase in the number of synaptic junctions and vesicle-filled processes in DA neurons between post-natal day 9 and 45 in rabbit striatum. Thus, while VMAT2 is generally regarded as an index of DAergic neuropil in the adult, additional studies will be required to determine whether this also holds for the developing brain.
Presynaptic DAergic markers in striata of adults
Our data demonstrate, as previously reported (Kish et al. 1992), a substantial loss of DA in both subdivisions of the striatum in the aging adult. This is paralleled by an approximately 50% decrease in HVA levels which, although statistically significant by anova only in the putamen, were significantly correlated within subjects with the decreases in DA levels in both striatal regions (see also Kalaria et al. 1993). This is in general agreement with many other post-mortem investigations showing an age-related decline in striatal DA levels during adulthood (cf. Kish et al. 1992). The failure of some studies (Bird and Iversen 1974; Robinson et al. 1977; Mackay et al. 1978; Adolfsson et al. 1979) to detect significant decreases in DA levels is most likely due to the smaller number of cases at the young and very old ends of the adult age scale employed in these investigations.
Unlike DA and HVA levels, TH and AADC protein levels were essentially stable during adulthood and senescence after declining modestly from pre-adolescence peak values. These data support the observations of Lewis and co-workers (Rosenberg and Lewis 1994, 1995; Erickson et al. 1998) that there is a developmental ‘overshoot’ in the elaboration of TH-positive neuropil in monkey brain. In contrast to the results of one study reporting a marked decline in striatal TH and AADC activities during adulthood (Coté and Kremzner 1983), several previous studies in the human did not detect statistically significant age-associated decline of striatal TH activity or TH protein (Robinson et al. 1977; Mackay et al. 1978; Gaspar et al. 1980; Girault et al. 1989; Wolf et al. 1991) with no or only a moderate reduction after the age of 20 years. Similarly, in vivo positron emission tomography studies using [18F]fluoroDOPA, an index of AADC activity, have shown either no change or only a modest decline during adulthood (cf. Kish et al. 1995). Our data suggest that in the aging human striatum, the capacity for DA biosynthesis as reflected by TH and AADC protein levels is at most only slightly diminished. However, the present data cannot address whether the in situ activity of TH, which is rate-limiting for DA biosynthesis, was unaffected by age. For example, oxidizing compounds or radicals, thought to increase with aging (Floyd 1999), can inactivate TH (cf. Fitzpatrick 1999). Moreover, as described by Furukawa and Kish (1998), total biopterin levels in human post-mortem putamen decreased by more than 50% with age in a cohort of 30 subjects ranging in age from 17 to 92 years old. These observations are consistent with reports of decreased cerebral pteridine levels and GTP cyclohydrolase 1 activity in aging mice (Yoshida et al. 2000) and with decreased GTP cyclohydrolase 1-immunoreactivity in the substantia nigra of aged monkeys and humans (Chen et al. 2000).
One of the more surprising observations in the present study was the relative stability of both DAT and VMAT2 protein levels in the adult striatum. Despite relatively low variances, only DAT levels in the caudate showed a significant, albeit modest (∼10%), decline with age in the adult groups. By contrast, several PET and SPECT radioligand binding studies have reported that DAT decreases with age (Volkow et al. 1994, 1996; Tissingh et al. 1997; Volkow et al. 1998; Mozley et al. 1999; Pirker et al. 2000). However, the majority of the observed declines often occurred during young adulthood, as observed in the present studies of transporter protein levels (cf. Tables 1 and 2). In fact, when data from imaging studies were fit to a biphasic model (Mozley et al. 1999), the rate of DAT decline with age above the ‘break point’ (approximately 40 years old) was not only several fold lower in striatum but actually reversed (i.e. DAT increased with age) in caudate. Whether or not the other differences between the DAT imaging and present results may reflect functional differences in DAT is unclear. As discussed below, values from imaging studies are susceptible to volume changes (Haug and Eggers 1991), which are more likely to affect striatal neurochemical measurements in living versus autopsied brain. Conversely, whole homogenates were analyzed in the present studies, allowing for the detection of DAT molecules with potentially occult radioligand binding sites in vivo. Irrespective of methodological differences, the maintenance of protein levels does not necessarily equate to maintenance of protein function. However, both the present and previous studies do agree that both VMAT2 and DAT levels decrease post-natally. For example, in the present study VMAT2 levels decreased 30–31% and DAT levels decreased 42–43% from the peak levels observed in the 8-month-old groups. The fundamental disagreement between the present and previous studies lies in the temporal course of the decrease. The present data, based upon a broad range of post-natal ages, indicates that transporter levels are relatively stable during adulthood.
In the present study, VMAT2 protein levels in both caudate and putamen remained constant with age in the adult groups. Of particular importance is that the levels of VMAT2 (unlike TH, AADC, or DAT) appear to be less susceptible to transcriptional regulation in adult animals (Vander Borght et al. 1995; Wilson and Kish 1996; Vilpoux et al. 2000; but see Rehavi et al. 1998) and, as such, are generally considered to be representative of the density of DAergic neuropil in striatum (Frey et al. 2001), wherein noradrenergic and serotoninergic innervation is very low. Previous studies in human have used the binding of dihydrotetrabenazine (DHTB), a selective vesicular monoamine transporter ligand, to estimate VMAT2 levels. Scherman et al. (1989) reported that [3H]-DHTB binding in total homogenates of post-mortem caudate samples decreased approximately 40% from age 65–95 years (n = 49); however, analysis of VMAT2 protein levels and age in the two oldest age groups from the present study failed to reveal any correlation, either in caudate or putamen (data not presented). In another study, Frey et al. (1996) used [11C]-DHTB and PET imaging to determine VMAT2 binding in vivo in the putamen of 15 subjects of ages 22–70 years and found VMAT2 levels to decrease approximately 40% over this period. However, no decrease was observed in the subjects over 60 years of age (n = 5), and elimination of an outlier value from one of the young subjects essentially flattened the regression line.
A general issue raised by these studies concerns differences related to measurement of DAT and VMAT2 by radioligand binding versus blot immunolabeling techniques. While striatal levels of DAT and VMAT2 assessed by either procedure can be considered ‘valid’ markers of DAergic/monoaminergic neuropil in a general sense and are, as expected, severely decreased in patients with Parkinson's disease, the extent of reduction of at least one of the transporters (DAT) differs when assessed by binding versus protein measurement (Wilson et al. 1996b). Moreover, as evidenced by several radioligand binding studies of drug-induced changes in DAT in human brain, substantially different results can be produced by different radioligands (Little et al. 1998). In fact, in studies of brains of human cocaine users, data from three independent laboratories indicate that changes or lack of changes in DAT levels as measured by radioligand binding techniques (Little et al. 1993; Staley et al. 1994; Wilson et al. 1996a) are not associated with changes in DAT protein levels as determined by the same investigator (Staley et al. 1995; Wilson et al. 1996a; Oakman et al. 2000). In the present study, essentially identical DAT levels were measured with either of two antibodies against two different epitopes in DAT. Given the variation in results from studies using different radioligands, but not from the present study using different antibodies, blot immunolabeling measurement of monoaminergic transporter proteins would appear to provide more reliable data. As such, it is important that the disparity between ligand binding and protein levels be acknowledged even though it is difficult to ascertain which of the values is of more relevant physiological import.
Implications for nigrostriatal pathophysiology in aging and neurological disorders
Our observations are consistent with a developmental scenario in which the biochemical development of the nigrostriatal DA neurons continues after birth and may involve some degree of overelaboration of DAergic neuropil. DA itself appears to be the lagging indicator of what could potentially be considered a measure of the ‘mature’ density of innervation. However, perhaps the most unexpected finding from these studies is the contrast between the dramatic decrease in DA levels with age compared with the relative stabilities of the presynaptic DAergic protein markers. While several studies have indicated that the number of neuromelanin-containing neurons in substantia nigra decreases with age (see Introduction), leading to the long-standing assumption that striatal DAergic neuropil similarly decreases with age, there is not consensus on this point. For example, Kubis et al. (2000), who used TH immunohistochemical techniques to identify and count DAergic neurons, suggest that the discrepancies could be due to the failure of most investigators to sample a sufficiently representative number of sections throughout the entirety of the cell body region or to use a marker, such as TH, that is more specifically DAergic. To the extent that the levels of VMAT2 are representative of DAergic neuropil density in the striatum, our data suggest that it is DA as opposed to DAergic neuropil which decreases in aging/senescence. This interpretation accommodates the emergence of ‘non-pathological’ movement-related anomalies that arise in aging without positing an underlying neurodegenerative basis. As qualified above, in the absence of deficits in protein levels of either the biosynthetic or transport/sequestration machinery (TH and AADC, DAT and VMAT2, respectively), several candidate mechanisms for the decrease in DA levels seem likely. One is a relative deficit in tetrahydrobiopterin and, consequently, a decrease in tyrosine hydroxylation in vivo. This is supported by the age-dependent decrease in total biopterin, as reported in putamen by Furukawa and Kish (1998). While the decreases in total biopterin and DA are not completely commensurate, it is difficult to estimate how much of an effect a decrease in the former would have on the latter. Although not addressed in the present study, age-dependent increases in DA and/or TH oxidation may also contribute to the observed decreases in DA levels late in life (Romero-Ramos et al. 1997). However, with either of the above mechanisms, a much closer correlation between DA and HVA levels than was observed would be predicted. Thus, it is also possible that age-related losses of D2 DA receptors could result in enhanced release of DA, thereby contributing to decreased DA levels. In summary, whether or not valid, the authors' interpretations of the present results – that it is DA and not DAergic neuropil which diminishes in adulthood/senescence – may lead to alternative therapeutics for non-degenerative movement and cognitive disorders that arise during aging/senescence.
This research was supported by USPHS grants MH00967, MH55208 (JWH), DA07182 and NS26034 (SJK).