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Keywords:

  • axon;
  • blood–brain barrier;
  • dopamine;
  • myelin;
  • phenylketonuria;
  • tyrosine

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mice
  5. Histologic staining of cerebral hemisphere brain sections
  6. Western blots
  7. Gas chromatography mass spectrometry (GC-MS) quantification of Phe, tyrosine, and dopamine in blood and brain tissues
  8. Statistics
  9. Results
  10. Blood and brain Phe levels drop to near normal levels following placement of PKU mice on a low Phe diet for 4 weeks
  11. Tyrosine levels do not rebound to normal levels following placement of PKU mice on the low Phe diet for 4 weeks
  12. Dopamine levels increase to near normal in frontal cortex and striatum following placement of PKU mice on diet for 4 weeks
  13. Placement of PKU mice on diet for 4 weeks induces myelination in the frontal cortex and striatum
  14. Myelin and axonal proteins increase in PKU mouse frontal cortex and striatum during the low Phe diet time course
  15. Discussion
  16. Acknowledgements
  17. References

Phenylketonuria is caused by specific mutations in the phenylalanine hydroxylase gene and is characterized by elevated blood phenylalanine levels, hypomyelination in forebrain structures, reduced dopamine levels, and cognitive difficulties. To determine whether brain tyrosine levels and/or myelination play a role in the up-regulation of dopamine, phenylketonuric mice were placed on a low phenylalanine diet for 4 weeks and as blood phenylalanine levels dropped to normal, the relationships between phenylalanine, tyrosine, dopamine, myelin proteins, and axonal proteins in frontal cortex and striatum were determined using gas chromatography mass spectrometry, histology, and western blotting techniques. Blood phenylalanine rapidly decreased from an eight-fold elevation to near control levels, and blood tyrosine gradually rose from about 50% to near normal values. In frontal cortex and striatum, phenylalanine levels dropped to 2- and 1.5-fold elevations above control, respectively, and tyrosine levels increased but remained less than 70% of control in both structures. In frontal cortex, increases in dopamine and myelin basic protein occurred in a similar biphasic pattern, reaching near normal levels by week 4. In striatum, dopamine and MBP dramatically increased to near normal levels in the first week. Myelination was confirmed histologically and by western blot quantification of phosphorylated neurofilaments. In summary, our results showed: (i) an increase in dopamine despite low brain tyrosine levels and (ii) similar recovery patterns for myelination and dopamine. Since myelin/axonal interactions trigger signaling pathways that result in axonal maturation, we speculate that this interaction also may trigger signals that up-regulate neurotransmitter synthesis.

Abbreviations used
GC-MS

gas chromatography mass spectrometry

lCPSleu

local rate of leucine incorporation into cerebral protein

MBP

myelin basic protein

PAH

phenylalanine hydroxylase

PKU

phenylketonuria

Phe

phenylalanine

TH

tyrosine hydroxylase

In the CNS, oligodendrocytes extend numerous processes, and from the distal tip of each process, a membrane sheet is assembled and wrapped around a segment of axon as an internode of myelin. Myelin is a highly metabolically active membrane that, under normal conditions, remains connected to and is supported by the oligodendrocyte cell body for the life span of the oligodendrocyte. Myelin is essential for the rapid conduction of action potentials and, therefore, there may be devastating consequences if oligodendrocytes fail to produce myelin (hypomyelination) or lose their myelin (demyelination) as a consequence of disease.

One disease in which hypomyelination occurs in specific forebrain tracts, but neurons and their axons are spared, is the autosomal recessive disorder phenylketonuria (PKU) (Malamud 1966; Dyer et al. 1996). PKU is caused by a rise in blood phenylalanine (Phe) levels, due to a deficiency in the enzyme phenylalanine hydroxylase (PAH) (Jervis 1953). PAH is expressed primarily in liver and not in brain, and catalyzes the conversion of Phe to tyrosine (Hsieh and Berry 1979). Blood Phe levels normally are about 121 µm; however, in untreated individuals (and mice) with PKU, levels may increase to 1200 µm or more. For the past several decades, newborns diagnosed with PKU are placed on a low Phe diet for life. The low Phe diet decreases Phe levels in blood and brain, thereby allowing myelination to proceed (Thompson et al. 1993; Toft et al. 1994). Individuals with PKU that are continuously treated from birth avoid the severe mental retardation that occurs in untreated individuals (Cowie 1971).

It is well documented that although mental retardation is avoided with dietary treatment, significant problems still exist. Individuals treated for PKU from birth may continue to have elevated blood Phe levels, predominantly because the low Phe diet is distasteful and therefore difficult to maintain (MacDonald et al. 1994). If the diet is discontinued or liberalized (i) blood Phe levels rise, (ii) white matter lesions appear and increase in size, and (ii) intelligence decreases (Smith et al. 1990; Thompson et al. 1990, 1993). In summary, high levels of circulating Phe are reported to correlate with white matter pathology in specific forebrain tracts and neurological deterioration at any age despite dietary treatment during childhood.

The molecular mechanisms underlying the neurological deficits observed in individuals with PKU are unknown. However, decreased levels of neurotransmitters, including dopamine, are likely to play a major role in the observed cognitive disabilities (Diamond et al. 1994; Puglisi-Allegra et al. 2000; Pascucci et al. 2002). To date, two distinct theories have been proposed to explain this phenomenon (for review, see Dyer 1999). The ‘tyrosine/dopamine theory’ predicts that cognitive difficulties stem from decreased levels of tyrosine, the precursor of dopamine. Brain tyrosine levels in an experimental rat model for PKU were speculated to be low as a consequence of high blood Phe levels out-competing tyrosine for transport across the blood–brain barrier (Diamond et al. 1994). Since evidence suggests that dopamine synthesis in prefrontal cortex dopaminergic neurons is directly related to tyrosine levels (for review, see Tam and Roth 1997), Diamond and coworkers postulated that low brain tyrosine levels lead to decreased dopamine levels and thereby to cognitive disabilities in individuals with PKU.

The second hypothesis, the ‘myelin/dopamine theory’, takes into account the primary pathologic finding in treated PKU brain, i.e. decreased myelination within specific tracts in the brain (Malamud 1966; Dyer et al. 1996). Myelination induces axonal maturation, i.e. myelin/axonal interactions trigger heavy phosphorylation of neurofilaments, rearrangements of the cytoskeleton, and swelling in the axon beneath the compact myelin lamellae (Colello et al. 1994; Kirkpatrick and Brady 1994; Sanchez et al. 1996). In the myelin/dopamine theory, myelin/axonal interactions are postulated to transduce signals that up-regulate the production of enzymes involved in the dopamine biosynthetic pathway, e.g. tyrosine hydroxylase (TH), which is the key regulatory enzyme in the dopamine synthetic pathway. Alternatively, myelin/axonal contact may trigger signaling pathways that result in the phosphorylation of TH, thereby activating the enzyme and up-regulating dopamine synthesis. Either or both mechanisms may increase neurotransmitter production.

To the best of our knowledge, this is the first study to explore the relationships between Phe, tyrosine, dopamine synthesis, and myelination in frontal cortex and striatum. The genetic mouse model for PKU, which contains the PAHenu2 gene mutation, was considered the most appropriate animal model for our experiments for the following reasons. The PAHenu2 gene mutation results in inactivity of the PAH gene (McDonald and Charlton 1997), which, in turn, leads to elevated blood Phe levels; PKU mouse blood Phe levels are similar to those in individuals with PKU (Shedlovsky et al. 1993). Moreover, the neuropathology in the PKU mouse brain is strikingly similar to that in the human PKU brain, i.e. forebrain structures are hypomyelinated, including subcortical white matter and the corpus callosum in the frontal cortex, and white matter tracts within the striatum (Dyer et al. 1996). Thus, this PKU mouse model is an excellent system in which to determine if the tyrosine/dopamine and/or the myelin/dopamine hypotheses are correct. The data obtained from our time-course study of PKU mice on a low Phe diet suggest that dopamine recovery is not primarily regulated by tyrosine levels, and that a relationship exists between myelination and dopamine recovery.

Mice

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mice
  5. Histologic staining of cerebral hemisphere brain sections
  6. Western blots
  7. Gas chromatography mass spectrometry (GC-MS) quantification of Phe, tyrosine, and dopamine in blood and brain tissues
  8. Statistics
  9. Results
  10. Blood and brain Phe levels drop to near normal levels following placement of PKU mice on a low Phe diet for 4 weeks
  11. Tyrosine levels do not rebound to normal levels following placement of PKU mice on the low Phe diet for 4 weeks
  12. Dopamine levels increase to near normal in frontal cortex and striatum following placement of PKU mice on diet for 4 weeks
  13. Placement of PKU mice on diet for 4 weeks induces myelination in the frontal cortex and striatum
  14. Myelin and axonal proteins increase in PKU mouse frontal cortex and striatum during the low Phe diet time course
  15. Discussion
  16. Acknowledgements
  17. References

PAHenu2 (PKU) mice contain a mutation in the PAH gene that results in the most severe form of the disease in humans, i.e. classic PKU (McDonald et al. 1990). PKU mice were maintained as a breeding colony at The Children's Hospital of Philadelphia. Homozygote (–/–) PKU males were bred to heterozygote (+/–) females. PKU pups were easily distinguished from littermates based upon their small size (PKU pups are about one-half the size of heterozygote siblings) (McDonald 2000) and their coat color (PKU mice have a light gray-brown coat vs. the dark brown coat of heterozygote siblings). Only age-matched male mice were used in our experiments. Results from untreated and treated male PKU mice were compared to results from control heterozygote male mice (control mice); heterozygote mice had normal levels of blood Phe and therefore were unaffected. Untreated PKU mice were maintained on a Ralston Purina mouse chow diet 5015 (Animal Specialities, Quakertown, PA, USA) until 6–8 weeks of age, when they were placed on a low Phe diet. The low Phe diet consisted of chow containing no Phe (TD 90368, Harlan Teklad, Madison, WI, USA), with drinking water supplemented with 1 g/L Phe. The Ralston Purina chow diet contained 4.0 g/kg tyrosine and the TD90368 diet contain 5.0 g/kg tyrosine. No signs of distress or pain were observed in the treated or untreated PKU mice as a result of their being fed either diet. All mice were housed in an American Association for Accreditation of Laboratory Animal Care (AAALAC) approved facility. Experimental procedures met the guidelines of the agencies that have funded this work and were approved by The Children's Hospital of Philadelphia Institutional Animal Care and Usage Committee.

Histologic staining of cerebral hemisphere brain sections

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mice
  5. Histologic staining of cerebral hemisphere brain sections
  6. Western blots
  7. Gas chromatography mass spectrometry (GC-MS) quantification of Phe, tyrosine, and dopamine in blood and brain tissues
  8. Statistics
  9. Results
  10. Blood and brain Phe levels drop to near normal levels following placement of PKU mice on a low Phe diet for 4 weeks
  11. Tyrosine levels do not rebound to normal levels following placement of PKU mice on the low Phe diet for 4 weeks
  12. Dopamine levels increase to near normal in frontal cortex and striatum following placement of PKU mice on diet for 4 weeks
  13. Placement of PKU mice on diet for 4 weeks induces myelination in the frontal cortex and striatum
  14. Myelin and axonal proteins increase in PKU mouse frontal cortex and striatum during the low Phe diet time course
  15. Discussion
  16. Acknowledgements
  17. References

Mice were anesthetized with ketamine and xylazine and perfused with 4% paraformaldehyde in phosphate-buffered saline pH 7.3, after which the brains were removed and placed in 4% paraformaldehyde in phosphate-buffered saline for 3 days at 4°C. Brains were processed in alcohol and chloroform, embedded in paraffin, and then coronally sliced into 8-µm sections. Sections were deparaffinized and stained with Luxol fast blue, hematoxylin, and eosin according to standard protocols. Luxol fast blue is a standard histologic stain that stains myelin dark blue; a deficiency in myelin is obvious when tracts are stained a light blue.

Western blots

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mice
  5. Histologic staining of cerebral hemisphere brain sections
  6. Western blots
  7. Gas chromatography mass spectrometry (GC-MS) quantification of Phe, tyrosine, and dopamine in blood and brain tissues
  8. Statistics
  9. Results
  10. Blood and brain Phe levels drop to near normal levels following placement of PKU mice on a low Phe diet for 4 weeks
  11. Tyrosine levels do not rebound to normal levels following placement of PKU mice on the low Phe diet for 4 weeks
  12. Dopamine levels increase to near normal in frontal cortex and striatum following placement of PKU mice on diet for 4 weeks
  13. Placement of PKU mice on diet for 4 weeks induces myelination in the frontal cortex and striatum
  14. Myelin and axonal proteins increase in PKU mouse frontal cortex and striatum during the low Phe diet time course
  15. Discussion
  16. Acknowledgements
  17. References

Mice were euthanized, the brains removed, and striatum and frontal cortex dissected out on an ice-cold platform. The tissue was then homogenized and then sonicated in lysis buffer (phosphate-buffered saline containing 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 10 µg/mL DNase, pH 7.3). Protein assays were performed using bicinchoninic acid reagent (Pierce, Rockford, IL, USA). Homogenates from striatum (15 µg) and frontal cortex (50 µg) were loaded onto 8–16% gradient sodium lauryl sulfate polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and electrophoresed (Laemmli 1970). The protein bands then were electrophoretically transferred to nitrocellulose (Amersham, Piscataway, NJ, USA) (Towbin et al. 1979). Blots were blocked with 5.0% non-fat dry milk in phosphate-buffered saline and then probed with either anti-myelin basic protein (MBP) mouse IgG (SMI-94 and SMI-99, Sternberger Monoclonals, Inc, Lutherville, MD, USA) 1 : 1000 each, anti-phosphorylated neurofilament mouse IgG (SMI-31, Sternberger Monoclonals, Inc.) 1 : 1000, or anti-TH mouse IgG (Chemicon, Temecula, CA) 1 : 1000. Blots were incubated with the primary antibodies in phosphate-buffered saline containing 0.5% non-fat dry milk and 0.02% sodium azide overnight with gentle agitation on a rocking platform. Following washing, the blots then were incubated for 1 h with goat anti-mouse IgG conjugated to biotin (Jackson Laboratories, West Grove, PA) 1 : 5000 in phosphate-buffered saline containing 0.5% non-fat dry milk. After washing, the blots were incubated for 1 h with streptavidin–horseradish peroxidase (Amersham) 1 : 5000 in phosphate-buffered saline containing 0.5% non-fat dry milk. Following extensive washing, the blots were developed using ECL Western Blotting Detection Reagents and Analysis System (Amersham) and Hyperfilm–MP X-ray film (Amersham). A series of dilutions for striatum and frontal cortex homogenates were performed and immunoblotted for each antibody to establish that the relationship between protein band and intensity was linear over the range of band intensities observed in the test samples. Scion Image and Adobe Photoshop software were used to quantify differences in protein bands between samples.

Gas chromatography mass spectrometry (GC-MS) quantification of Phe, tyrosine, and dopamine in blood and brain tissues

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mice
  5. Histologic staining of cerebral hemisphere brain sections
  6. Western blots
  7. Gas chromatography mass spectrometry (GC-MS) quantification of Phe, tyrosine, and dopamine in blood and brain tissues
  8. Statistics
  9. Results
  10. Blood and brain Phe levels drop to near normal levels following placement of PKU mice on a low Phe diet for 4 weeks
  11. Tyrosine levels do not rebound to normal levels following placement of PKU mice on the low Phe diet for 4 weeks
  12. Dopamine levels increase to near normal in frontal cortex and striatum following placement of PKU mice on diet for 4 weeks
  13. Placement of PKU mice on diet for 4 weeks induces myelination in the frontal cortex and striatum
  14. Myelin and axonal proteins increase in PKU mouse frontal cortex and striatum during the low Phe diet time course
  15. Discussion
  16. Acknowledgements
  17. References

Phe, tyrosine, and dopamine levels were measured by a stable isotope dilution technique using GC-MS. Briefly, [15N]Phe and [3H]tyrosine (Cambridge Isotopes, Andover, MA, USA) were added to whole blood (40 µL) (isotope final concentration of 200 µm dopamine, 20 µm tyrosine) or brain tissue homogenate (100 µg protein) (isotope final concentration of 20 µm dopamine, 2 µm tyrosine). Samples were deproteinated by adding 1 ml of absolute methanol to each blood or brain homogenate sample, incubating the samples at −20°C for 30 min, and centrifugation. The soluble molecules in the supernatant were derivatized with water/ethanol/pyridine (120 : 64 : 16) and ethyl choroformate to obtain N-ethoxycarbonyl ethyl esters (Huang et al. 1993). Analysis of isotopic enrichment was performed with selected ion monitoring on a Hewlett-Packard 5971 mass-selective detector. The method of Rosenblatt et al. (1992) was used to determine isotope abundance (atom percentage excess) and to calculate Phe and tyrosine concentrations. For direct comparison of Phe levels in brain tissue homogenate samples with those in blood samples, we converted mass Phe/unit protein to mass Phe/unit volume. Since about 10% of brain is protein, the conversion factor 100 µg = 1 µL was used. Similar conversions were performed for tyrosine.

To quantify dopamine in striatum and frontal cortex, weighed tissue samples were homogenized in ice-cold 1% perchloric acid containing 1 µm ascorbic acid, 1 mm ZnSO4, and [3H]dopamine (final concentration of 2 µm) (Cambridge Isotopes). Following centrifugation, the pH of the supernatant was adjusted to pH 6–8 with 4 m KOH. Samples were centrifuged again to remove the precipitated KOH, the supernatant was acidified with 200 µL HCl, and then passed over an activated Strata C18-E column (Phenomenex, Torrence, CA, USA). The eluate was dried overnight, derivatized and analyzed by GC-MS as described above.

Blood and brain Phe levels drop to near normal levels following placement of PKU mice on a low Phe diet for 4 weeks

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mice
  5. Histologic staining of cerebral hemisphere brain sections
  6. Western blots
  7. Gas chromatography mass spectrometry (GC-MS) quantification of Phe, tyrosine, and dopamine in blood and brain tissues
  8. Statistics
  9. Results
  10. Blood and brain Phe levels drop to near normal levels following placement of PKU mice on a low Phe diet for 4 weeks
  11. Tyrosine levels do not rebound to normal levels following placement of PKU mice on the low Phe diet for 4 weeks
  12. Dopamine levels increase to near normal in frontal cortex and striatum following placement of PKU mice on diet for 4 weeks
  13. Placement of PKU mice on diet for 4 weeks induces myelination in the frontal cortex and striatum
  14. Myelin and axonal proteins increase in PKU mouse frontal cortex and striatum during the low Phe diet time course
  15. Discussion
  16. Acknowledgements
  17. References

Prior to performing experiments that examined changes in blood and brain Phe levels, it was important to demonstrate that our PKU mice had elevated blood Phe levels. Our data showed that virtually all of the PKU mice had blood Phe levels about eight-fold above control (Table 1). These Phe levels were comparable to Phe levels in humans with PKU (McDonald et al. 1990; Shedlovsky et al. 1993), thereby confirming that the mice were manifesting the metabolic imbalance associated with PKU.

Table 1.  GC-MS analysis of Phe and tyrosine levels (µm) in control and PKU mouse blood a
  +/– Control–/– PKU–/– PKU 1 week on diet–/– PKU 2 weeks on diet–/– PKU 4 weeks on diet
  • a

    Data represent the means 

  • ±

    ±SE. The number of mice used for each data set is indicated in parentheses. ND, not determined.

  • One-way anova analyses: Phe, p < 0.0001; tyrosine, p < 0.0001.

Phe (µm)144.6 ± 8.1 (n = 5)1162.1 ± 94.1 (n = 5)233.1 ± 16.7 (n = 8)207.2 ± 32.1 (n = 8)146.5 ± 32.1 (n = 4)
Tyrosine (µm)57.2 ± 2.9 (n = 7)22.7 ± 1.1 (n = 4)55.1 ± 4.2 (n = 5)NDND

PKU mice then were placed on the low Phe diet for 4 weeks, during which Phe levels in blood and brain tissue were quantified via GC-MS as described in the Materials and Methods section. Following placement on diet for 4 weeks, blood Phe levels dropped to normal (Table 1). The majority of the decrease in blood Phe took place within the first 24–48 h (data not shown), after which a slow, steady decline to normal occurred through the fourth week of the study (Table 1). A similar drop in Phe levels was observed in brain substructures over the 4-week time period (Tables 2 and 3, Fig. 1). By week 4, Phe levels in the frontal cortex had plummeted from about an 11-fold to a two-fold elevation above control (Fig. 1a). In striatum, Phe levels dropped from about a seven-fold to a one-and-a-half-fold elevation above control (Fig. 1b). Interestingly, while Phe levels decreased to normal in blood, Phe remained slightly elevated in brain following placement of PKU mice on diet for 4 weeks.

Table 2.  GC-MS analysis of Phe, tyrosine, and dopamine levels in control and PKU mouse frontal cortex a
  +/– Control–/– PKU–/– PKU 1 week on diet–/– PKU 2 weeks on diet–/– PKU 4 weeks on diet
  • a

    Data represent the means 

  • ±

    ±SE. The number of mice used for each data set is indicated in parentheses.

  • One-way anova analyses: Phe, p < 0.0001; tyrosine, p < 0.0001; dopamine, p = 0.01.

Phe (µm)74.0 ± 8.8 (n = 15)837.5 ± 84.1 (n = 6)225.8 ± 41.3 (n = 12)197.6 ± 31.4 (n = 8)152.7 ± 37.6 (n = 6)
Tyrosine (µm)67.5 ± 2.6 (n = 14)29.2 ± 1.6 (n = 4)36.2 ± 2.1 (n = 8)40.3 ± 2.6 (n = 8)43.7 ± 2.2 (n = 6)
Dopamine (pmoles/mg tissue)18.9 ± 1.3 (n = 15)11.3 ± 0.8 (n = 4)14.2 ± 1.4 (n = 12)14.1 ± 1.2 (n = 6)17.3 ± 1.9 (n = 5)
Table 3.  GC-MS analysis of Phe, tyrosine, and dopamine levels in control and PKU mouse striatum a
  +/– Control–/– PKU–/– PKU 1 week on diet–/– PKU 2 weeks on diet–/– PKU 4 weeks on diet
  • a

    Data represent the means ± SE. The number of mice used for each data set is indicated in parentheses.

  • One-way anova analyses: Phe, p < 0.0001; tyrosine, p < 0.0001; dopamine, p = 0.0004.

Phe (µm)133.3 ± 12.1936 ± 11.2240.3 ± 27.5221.5 ± 25.8193.7 ± 21.8
(n = 10)(n = 4)(n = 6)(n = 8)(n = 6)
Tyrosine (µm)81.0 ± 5.837.7 ± 3.746.6 ± 2.447.7 ± 2.555.5 ± 2.1
(n = 10)(n = 5)(n = 13)(n = 8)(n = 6)
Dopamine (pmoles/mg tissue)40.6 ± 3.0 (n = 9)14.0 ± 1.6 (n = 3)35.8 ± 1.5 (n = 8)32.5 ± 5.6 (n = 4)36.1 ± 2.3 (n = 5)
image

Figure 1. Changes in Phe and tyrosine levels in frontal cortex and striatum of untreated PKU and PKU mice placed on a low Phe diet for 1, 2, and 4 weeks. (a) The mean values for Phe in untreated and treated PKU frontal cortex were expressed as a percentage of the mean value for Phe in control heterozygote frontal cortex (see Table 2 for mean values). The mean values for tyrosine in untreated and treated PKU frontal cortex were expressed as a percentage of the mean value for tyrosine in control heterozygote frontal cortex (see Table 2 for mean values). (b) As described above in (a), Phe and tyrosine in striatum were expressed as a percentage of control (see Table 3 for mean values).

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Although not the focus of this study, it is noteworthy that Phe levels were differentially regulated in distinct PKU mouse brain structures prior to and during dietary treatment. The normal concentration of Phe in the frontal cortex of control mice was about one-half that in blood and in striatum (Tables 1–3). In untreated PKU mice, Phe was elevated about 11-fold in frontal cortex, about seven-fold in striatum, and about 13-fold in hind cortex (hind cortex data not shown) compared to Phe levels in respective structures in control heterozygote mice (Tables 2 and 3). As mentioned above, Phe levels also were different in striatum and frontal cortex in PKU mice placed on the diet for 4 weeks (Tables 2 and 3, Fig. 1). Moreover, since tyrosine levels were dissimilar in frontal cortex and striatum in PKU and control mice (Tables 2 and 3), these data suggest that distinct brain regions have different demands for specific amino acids.

Tyrosine levels do not rebound to normal levels following placement of PKU mice on the low Phe diet for 4 weeks

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mice
  5. Histologic staining of cerebral hemisphere brain sections
  6. Western blots
  7. Gas chromatography mass spectrometry (GC-MS) quantification of Phe, tyrosine, and dopamine in blood and brain tissues
  8. Statistics
  9. Results
  10. Blood and brain Phe levels drop to near normal levels following placement of PKU mice on a low Phe diet for 4 weeks
  11. Tyrosine levels do not rebound to normal levels following placement of PKU mice on the low Phe diet for 4 weeks
  12. Dopamine levels increase to near normal in frontal cortex and striatum following placement of PKU mice on diet for 4 weeks
  13. Placement of PKU mice on diet for 4 weeks induces myelination in the frontal cortex and striatum
  14. Myelin and axonal proteins increase in PKU mouse frontal cortex and striatum during the low Phe diet time course
  15. Discussion
  16. Acknowledgements
  17. References

Prior to quantifying tyrosine levels in treated PKU mouse brain, it was important to quantify tyrosine levels in control heterozygote and untreated PKU mouse brain and blood. Normal tyrosine levels are shown in Tables 1–3 (see control heterozygote data). Since the normal mouse chow diet was not supplemented with additional tyrosine, and PAH was inactive in PKU mice, blood tyrosine levels in untreated PKU mice were low (about 44% of control) (Table 1). Due to the eight-fold elevation in blood Phe levels in untreated PKU mice, it was surprising that the tyrosine levels in untreated PKU mouse brain frontal cortex and striatum were approximately 1.2- and 1.4-times, respectively, the level of tyrosine in the blood of these mice (Tables 2 and 3). Each of these values was statistically significant (p = 0.01 for tyrosine in frontal cortex vs. tyrosine in blood and p = 0.01 for tyrosine in striatum vs. tyrosine in blood).

In treated PKU mouse frontal cortex, a statistically significant 12% increase in tyrosine was detected in the first week the PKU mice were placed on the low Phe diet (p = 0.02 vs. untreated PKU mouse frontal cortex) (Fig. 1, Table 2). Although tyrosine continued to rise slowly in weeks 2 to 4, each successive increase was not statistically significant from the previous tyrosine level (p = 0.24, p = 0.36, respectively) (Table 2). Even after 4 weeks of diet therapy, there remained a statistically significant difference between tyrosine levels in treated PKU mouse frontal cortex compared to control heterozygote frontal cortex (p < 0.0001). Indeed, tyrosine levels in the 4-week-treated PKU mouse frontal cortex were only 65% of control (Fig. 1).

In the treated PKU mouse striatum, the increase in tyrosine observed in week 1 was not statistically significant compared to untreated PKU striatum (p = 0.07) (Fig. 1, Table 3). A slight increase in tyrosine was observed in week 2. However, the only statistically significant increase in tyrosine occurred in weeks 2 to 4; during this time period, tyrosine increased 10% (p = 0.04 vs. week 1 treated PKU mouse striatum). Tyrosine levels in treated PKU mouse striatum remained statistically significantly different from control heterozygote striatum (p = 0.006) throughout the entire 4-week study. By the fourth week, tyrosine levels in treated PKU mouse striatum were only 68% of control (Fig. 1).

One possible reason why brain tyrosine levels did not increase to control levels might be because blood tyrosine levels remained low in the treated PKU mice; in other words, placement of the PKU mice on the low Phe diet with supplemented tyrosine did not raise blood tyrosine levels. However, this does not appear to be the case, since blood tyrosine rose to near normal levels after 1 week of diet therapy (Table 1). Moreover, the rise in blood tyrosine was verified semiquantitatively by mouse coat color. Untreated PKU mice have light grey-brown fur, whereas treated PKU mice have dark brown fur and are visually indistinguishable from their control heterozygous litter mates (data not shown). (Tyrosine is the precursor of melatin and therefore low tyrosine levels lead to decreased pigmentation and normal tyrosine levels result in normal pigmentation.) Our data therefore indicate that blood tyrosine levels rose to near normal in treated PKU mice. Thus, mechanisms other than low blood tyrosine levels must account for the failure of brain tyrosine levels in the treated PKU mouse to increase above 70% of control.

Dopamine levels increase to near normal in frontal cortex and striatum following placement of PKU mice on diet for 4 weeks

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mice
  5. Histologic staining of cerebral hemisphere brain sections
  6. Western blots
  7. Gas chromatography mass spectrometry (GC-MS) quantification of Phe, tyrosine, and dopamine in blood and brain tissues
  8. Statistics
  9. Results
  10. Blood and brain Phe levels drop to near normal levels following placement of PKU mice on a low Phe diet for 4 weeks
  11. Tyrosine levels do not rebound to normal levels following placement of PKU mice on the low Phe diet for 4 weeks
  12. Dopamine levels increase to near normal in frontal cortex and striatum following placement of PKU mice on diet for 4 weeks
  13. Placement of PKU mice on diet for 4 weeks induces myelination in the frontal cortex and striatum
  14. Myelin and axonal proteins increase in PKU mouse frontal cortex and striatum during the low Phe diet time course
  15. Discussion
  16. Acknowledgements
  17. References

Prior to beginning experiments designed to quantify dopamine in the treated PKU mice, it was important to confirm previous reports that dopamine levels were reduced in untreated PKU rodent brain (Diamond et al. 1994; Puglisi-Allegra et al. 2000). Our data show that dopamine levels in untreated PKU mouse frontal cortex were about 60% of control (p = 0.01) (Table 2), whereas dopamine levels in untreated PKU mouse striatum were about 34% of control (p = 0.0007) (Table 3).

PKU mice then were placed on the low Phe diet for 4 weeks, and dopamine levels in frontal cortex and striatum were quantified via GC-MS as described in the Materials and Methods section. Within frontal cortex, dopamine increased in a biphasic manner to near normal levels (Table 2, Fig. 2). The first increase in dopamine was a modest 15% during the first week of diet treatment. The second increase of an additional 17% took place between weeks 2 and 4 of the diet. Dopamine levels in frontal cortex therefore increased from about 60% to 92% of control. By the end of week 4, no statistically significant difference existed between dopamine levels in the treated PKU frontal cortex and control heterozygote frontal cortex (p = 0.55).

image

Figure 2. Changes in dopamine levels in PKU mouse frontal cortex and striatum during the diet time course study. The mean values for dopamine in untreated and treated PKU frontal cortex were expressed as a percentage of the mean value for dopamine in control heterozygote frontal cortex (see Table 2 values). Similarly, dopamine levels in striatum were expressed as a percentage of control (see Table 3 for mean values).

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A different pattern of dopamine recovery was observed in striatum. A single dramatic leap in dopamine (about 54%) was observed in the treated PKU mouse striatum during the first week; during this time period, dopamine more than doubled in concentration from approximately 34% to 89% of control (Table 3, Fig. 2). Following this striking increase, there was no statistically significant difference between dopamine levels in the treated PKU mouse striatum and the control heterozygote striatum (p = 0.19). The newly established dopamine concentration essentially remained unchanged throughout the last 3 weeks of the study.

In summary, while tyrosine levels remained significantly low in frontal cortex and striatum throughout the time course study, dopamine levels significantly increased in both structures.

Placement of PKU mice on diet for 4 weeks induces myelination in the frontal cortex and striatum

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mice
  5. Histologic staining of cerebral hemisphere brain sections
  6. Western blots
  7. Gas chromatography mass spectrometry (GC-MS) quantification of Phe, tyrosine, and dopamine in blood and brain tissues
  8. Statistics
  9. Results
  10. Blood and brain Phe levels drop to near normal levels following placement of PKU mice on a low Phe diet for 4 weeks
  11. Tyrosine levels do not rebound to normal levels following placement of PKU mice on the low Phe diet for 4 weeks
  12. Dopamine levels increase to near normal in frontal cortex and striatum following placement of PKU mice on diet for 4 weeks
  13. Placement of PKU mice on diet for 4 weeks induces myelination in the frontal cortex and striatum
  14. Myelin and axonal proteins increase in PKU mouse frontal cortex and striatum during the low Phe diet time course
  15. Discussion
  16. Acknowledgements
  17. References

Since tyrosine levels were significantly reduced and Phe levels were slightly elevated in treated PKU mouse brain, it was critical to determine if myelination took place under these conditions. As described in the Materials and Methods section, mouse brain sections were stained with Luxol fast blue, a myelin specific stain, and with hematoxylin and eosin. Our data showed that no new pathology was induced by placing the PKU mouse brain on diet for 4 weeks, i.e. no evidence of immune cell influx or necrosis was detected (Fig. 3). Instead, there clearly was an increase in the amount of myelin in subcortical white matter, corpus callosum, and striatum of treated PKU mice compared to respective structures in untreated PKU mouse brain (Fig. 3). Thus, the newly established levels of Phe and tyrosine in treated PKU mouse brain provided an environment conducive for myelination in the frontal cortex and striatum.

image

Figure 3. Luxol fast blue histologic staining of myelin in untreated and treated PKU mouse brain sections. PKU mice (n = 3) were place on a low Phe diet for 4 weeks. Age-matched untreated (n = 3) and control heterozygote mice (n = 3) (data not shown) were perfused along with the treated PKU mice, and their brains processed and sectioned as described in the Materials and Methods section. (a) Representative section from untreated PKU mouse brain showing pale blue staining (indicative of hypomyelination) in tracts in PKU corpus callosum (arrow) and striatum (arrowhead). (b) Representative section from 4-week-treated PKU mouse brain showing intense blue staining of tracts in PKU corpus callosum (arrow) and striatum (arrowhead). The intense blue stain was similar to that observed in comparable sections from control heterozygote mouse brain, indicating that myelination had taken place in treated PKU mouse brain.

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Myelin and axonal proteins increase in PKU mouse frontal cortex and striatum during the low Phe diet time course

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mice
  5. Histologic staining of cerebral hemisphere brain sections
  6. Western blots
  7. Gas chromatography mass spectrometry (GC-MS) quantification of Phe, tyrosine, and dopamine in blood and brain tissues
  8. Statistics
  9. Results
  10. Blood and brain Phe levels drop to near normal levels following placement of PKU mice on a low Phe diet for 4 weeks
  11. Tyrosine levels do not rebound to normal levels following placement of PKU mice on the low Phe diet for 4 weeks
  12. Dopamine levels increase to near normal in frontal cortex and striatum following placement of PKU mice on diet for 4 weeks
  13. Placement of PKU mice on diet for 4 weeks induces myelination in the frontal cortex and striatum
  14. Myelin and axonal proteins increase in PKU mouse frontal cortex and striatum during the low Phe diet time course
  15. Discussion
  16. Acknowledgements
  17. References

In order to quantify increases in myelination, changes in both myelin and axonal proteins from treated PKU mouse brain frontal cortex and striatum were measured. Axonal proteins also were examined because myelin induces changes in axonal proteins, as described earlier. Thus, changes in the myelin-enriched family of MBP, as well as the neuronal/axonal-enriched proteins phosphorylated neurofilaments and TH were quantified by western blotting as described in Material and Methods.

In frontal cortex of untreated PKU mice, quantities of MBP, phosphorylated neurofilaments, and TH were reduced compared to respective protein levels in control heterozygote frontal cortex (i.e. about 68–77% of control) (Table 4, Fig. 4). MBP increased from about 75% to about 105% in frontal cortex of PKU mice treated 1 week compared to control (Fig. 4b). A similar increase in phosphorylated neurofilaments was detected in 1-week-treated PKU mouse frontal cortex (rose from 75% to 115% of control). However, essentially no change was detected in TH. During the second week of diet, MBP levels rose slightly, phosphorylated neurofilaments levels decreased to control, and TH rose from about 65% to 85% of control. In the third week of diet, MBP increased another 20% to about 130% of control, with phosphorylated neurofilaments and TH remaining essentially unchanged. In the fourth week on diet, MBP levels again rose slightly, phosphorylated neurofilament levels decreased slightly, and TH increased from 80% to 90% of control. In summary, of the three proteins examined within the frontal cortex during the 4-week time course, MBP appeared to most closely mimic the observed increases in dopamine (compare Fig. 4b with Fig. 2).

Table 4.  Western blot analysis of myelin and axonal protein levels in control and PKU mouse frontal cortex a
  +/– Control–/– PKU–/– PKU 1 week on diet–/– PKU 2 weeks on diet–/– PKU 3 weeks on diet–/–PKU 4 weeks on diet
  • a

    Data represent the means ± SE. The number of mice used for each data set is indicated in parentheses.

  • One-way anova analyses: MBP, p < 0.0001; tyrosine hydroxylase, p = 0.0053; phosphorylated neurofilaments, p = 0.0012.

MBP91.2 ± 3.2 (n = 15)69.5 ± 3.3 (n = 17)93.5 ± 4.1 (n = 10)98.6 ± 5.7 (n = 5)117.3 ± 4.5 (n = 8)118.2 ± 8.7 (n = 6)
Tyrosine hydroxylase122.8 ± 3.2 (n = 17)82.4 ± 4.3 (n = 21)79.3 ± 7.1 (n = 10)103.8 ± 8.3 (n = 4)93.6 ± 2.9 (n = 8)109.7 ± 7.0 (n = 7)
Phosphorylated neurofilaments118.4 ± 4.7 (n = 7)90.0 ± 4.9 (n = 7)134.5 ± 0.5 (n = 2)123.5 ± 0.5 (n = 2)121.0 ± 8.2 (n = 6)102.0 ± 5.6 (n = 5)
image

Figure 4. Western blot quantification of MBP, phosphorylated neurofilaments (Phos NF), and TH in frontal cortex of untreated PKU, treated PKU, and control heterozygote mice. (a) Representative bands of MBP, phosphorylated neurofilaments, and TH from blots. (b) Relative amounts of protein in bands (see a) were quantified and the means of each data set were determined as described in the Materials and Methods section (also, see Table 5 for means). The mean values for each protein in untreated and treated PKU frontal cortex were expressed as a percentage of the mean value for each respective protein in control heterozygote frontal cortex.

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A different pattern of protein increases was observed in the striatum of treated PKU mice. In untreated PKU mice, MBP levels were about 74% of control, phosphorylated neurofilaments were about equal to control, and TH was about 78% of control (Table 5, Fig. 5). Following 1 week of diet treatment, MBP levels in striatum jumped to about 112% of control, phosphorylated neurofilaments increased to about 107% of control, and TH rose to 90% of control. In the second through the fourth week of diet, MBP levels oscillated but on average remained about equal to control. Phosphorylated neurofilaments increased to almost 120% of control in the second week and then fell in the third week to its original level. TH levels also remained essentially unchanged in the second and third week. In summary, while MBP and TH both increased in the first week, the large 30% increase in MBP most accurately mirrored the 44% jump in dopamine during this time period (compare Fig. 5b with Fig. 2).

Table 5.  Western blot analysis of myelin and axonal protein levels in control and PKU mouse striatum a
  +/– Control–/– PKU–/– PKU 1 week on diet–/– PKU 2 weeks on diet–/– PKU 3 weeks on diet–/–PKU 4 weeks on diet
  • a

    Data represent the means ± SE. The number of mice used for each data set is indicated in parentheses. ND, not determined.

  • One-way anova analyses: MBP, p = 0.0149; tyrosine hydroxylase, p < 0.0001; phosphorylated neurofilaments, p = 0.0354.

MBP104.0 ± 4.4 (n = 6)76.8 ± 8.1 (n = 9)117.7 ± 13.7 (n = 3)100.0 ± 9.7 (n = 3)114.3 ± 6.3 (n = 4)108.5 ± 5.5 (n = 2)
Tyrosine hydroxylase130.6 ± 2.9 (n = 20)101.6 ± 4.2 (n = 16)118.8 ± 5.2 (n = 6)116.0 ± 8.6 (n = 5)95.8 ± 7.8 (n = 5)ND
Phosphorylated neurofilaments125.8 ± 5.3 (n = 6)123.2 ± 3.1 (n = 5)133.5 ± 2.5 (n = 2)149.0 ± 15.1 (n = 2)127.5 ± 3.2 (n = 4)ND
image

Figure 5. Western blot quantification of MBP, phosphorylated neurofilaments (Phos NF), and TH in frontal cortex of untreated PKU, treated PKU, and control heterozygote mice. (a) Representative bands of MBP, phosphorylated neurofilaments, and TH from blots. (b) Relative amounts of protein in bands (see a) were quantified and the means of each data set were determined as described in the Materials and Methods section (also, see Table 5 for means). The mean values for each protein in untreated and treated PKU striatum were expressed as a percentage of the mean value for each respective protein in control heterozygote striatum.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mice
  5. Histologic staining of cerebral hemisphere brain sections
  6. Western blots
  7. Gas chromatography mass spectrometry (GC-MS) quantification of Phe, tyrosine, and dopamine in blood and brain tissues
  8. Statistics
  9. Results
  10. Blood and brain Phe levels drop to near normal levels following placement of PKU mice on a low Phe diet for 4 weeks
  11. Tyrosine levels do not rebound to normal levels following placement of PKU mice on the low Phe diet for 4 weeks
  12. Dopamine levels increase to near normal in frontal cortex and striatum following placement of PKU mice on diet for 4 weeks
  13. Placement of PKU mice on diet for 4 weeks induces myelination in the frontal cortex and striatum
  14. Myelin and axonal proteins increase in PKU mouse frontal cortex and striatum during the low Phe diet time course
  15. Discussion
  16. Acknowledgements
  17. References

To determine if a relationship between dopamine synthesis and either tyrosine levels or myelination exists, 6–8-week-old male PKU mice were placed on a low Phe diet and levels of dopamine, tyrosine, Phe and myelin were measured during a 4-week time course study. In the course of performing this study, several surprising findings were made concerning the differential regulation of Phe, tyrosine, and dopamine levels in frontal cortex and striatum of PKU mouse brain. The differential regulation of the amino acids Phe and tyrosine in brain initially was not a focus of this study. However, the aberrant levels of these amino acids in PKU brain made a significant contribution to the development of our conclusions that (i) tyrosine levels are not a key regulator of dopamine synthesis, and (ii) a relationship appears to exist between myelination and dopamine production. Therefore, possible mechanisms involving blood–brain barrier transporter proteins that control the flux of Phe and tyrosine into and out of the brain also are discussed herein.

In order to determine if a relationship exists between tyrosine levels and dopamine synthesis in the treated PKU mouse brain, it was necessary to quantify tyrosine in blood and brain tissues in control heterozygous, untreated PKU, and treated PKU mice. Based upon previous studies showing that high levels of Phe out-compete tyrosine for transport across the blood–brain barrier (Choi and Pardridge 1986; Brenton and Gardiner 1988; Pardridge 1998), it was anticipated that brain tyrosine levels would be less than blood tyrosine levels in the untreated PKU mouse. However, we unexpectedly found that tyrosine levels in frontal cortex and striatum were approximately 1.2- and 1.4-times, respectively, the level of tyrosine in the blood of untreated PKU mice. Thus, the eight-fold elevation in blood Phe levels in the untreated PKU mouse did not appear to interfere with the movement of tyrosine into the brain.

The basis for the discrepancy between our results and previous studies, with respect to the ability of tyrosine to be transported through the blood–brain barrier in the presence of elevated Phe, may arise from the different lengths of time the blood–brain barrier was exposed to elevated Phe levels. Previous studies administered a bolus of Phe and shortly thereafter Phe and tyrosine flux was measured in vivo into the brain or in vitro through isolated brain capillary vessels (Choi and Pardridge 1986; Brenton and Gardiner 1988; Pardridge 1998). In contrast, the untreated PKU mouse blood–brain barrier was continuously exposed to abnormal Phe and tyrosine levels from shortly after birth into adulthood. In response to the abnormal long-term exposure of the blood–brain barrier to aberrant concentrations of blood Phe and tyrosine, the blood–brain barrier may have altered its expression pattern of amino acid transporters to try to achieve normal amino acid concentrations in the brain. An adaptation process within the blood–brain barrier occurs during normal development, e.g. concentrations of amino acids in brain normally change during the development of the newborn into the adult (Sershen et al. 1987; Gardiner 1990). Therefore, it may be possible that the expression pattern of blood–brain barrier transporter proteins in untreated PKU mice was altered in response to aberrant blood Phe and tyrosine levels.

Our amino acid data in untreated and treated PKU mouse brain versus control heterozygote brain support the possibility that blood–brain barrier transporter protein expression was abnormal in the untreated PKU mice. For example, in control heterozygote mice, striatum and blood Phe levels were approximately equal. However, in untreated PKU mice, Phe levels in striatum were about 80% of blood Phe levels. Phe levels in control heterozygote mouse frontal cortex were about 51% of blood levels, but in untreated PKU mice this ratio was approximately 72%. When a comparison is made between amino acid levels in PKU mouse brain and those in control heterozygote brain structures, two pieces of evidence suggest that the blood–brain barrier transporter proteins were abnormal in PKU mice. First, while blood Phe levels were restored to normal in treated PKU mice, Phe levels remained elevated two-fold in frontal cortex and one-and-a-half-fold in striatum. Second, tyrosine levels did not increase above 70% of normal levels in either brain structure in treated PKU mice despite the fact that tyrosine levels were near normal in blood. These data suggest that the blood–brain barrier attempted to maintain elevated levels of Phe and low levels of tyrosine in the treated PKU mouse brain, i.e. levels that had been ‘normal’ in the untreated PKU brain. It is unknown if Phe and tyrosine levels would eventually have been restored to normal in treated PKU mouse brain, since our study did not continue beyond 4 weeks. Based upon our data, however, futures studies are necessary to gain an understanding of what happens to blood–brain barrier transporter proteins in individuals with PKU following long-term changes in Phe and tyrosine levels subsequent to relaxation or resumption of a low Phe diet.

One cellular system that appears to impact the flux of amino acids through the blood–brain barrier is glial in origin. Evidence suggests that tissue-specific gene expression in brain capillary endothelium is regulated by cells such as astrocytes; glial foot processes cover more than 95% of the endothelium (Pardridge 1991). The activity of L-system-mediated transporters (the transporter system that moves Phe and tyrosine through the blood–brain barrier) can be modulated by several distinct pathways, including factors derived from glia (Chisty et al. 2002). Thus, abnormalities in glia may affect not only the expression of but also the activity of amino acid transporter systems in the blood–brain barrier of PKU mice. Indeed, the primary pathology in the PKU brain is glial in origin, i.e. decreased amounts of myelin produced by oligodendroglia and increased numbers of mixed phenotype glia (Dyer et al. 1996). Since mixed phenotype glia are located within white matter tracts and along the blood vessels (Dyer et al. 2000), it is possible that they influence not only the gene expression of amino acid transporters in the blood–brain barrier, but the activity of these transporter proteins as well.

Brain tyrosine levels remained significantly reduced in the treated PKU mouse brain, and yet under these conditions, dopamine still increased to near normal concentrations in both frontal cortex and striatum. The ‘tyrosine/dopamine’ theory, however, predicted that low brain tyrosine levels would prevent increases in dopamine synthesis. Since this did not occur, why did tyrosine levels fail to regulate dopamine synthesis in the treated PKU brain? The answer may lie in the fact that studies which examined the sensitivity of prefrontal cortex dopaminergic neurons to various tyrosine levels were performed in otherwise normal brain conditions, i.e. Phe levels were normal (for review, see Tam and Roth 1997). If Phe levels had been elevated, the results of these studies may have been different. Indeed, elevated levels of radiolabeled Phe added to isolated enzyme preparations (Ikeda et al. 1967; Katz et al. 1976) and synaptosomal preparations (Katz et al. 1976), PC12 cells (De Pietro and Fernstrom 1999), and bovine chromaffin cells (Fukami et al. 1990) resulted in the synthesis of DOPA from the radioactive Phe. Importantly, the elevated levels of Phe used in these studies were well within the range found in individuals with PKU. TH and PAH have a very high degree of amino acid homology (Ledley et al. 1987; Fitzpatrick 2000), further strengthening the likelihood of Phe being a substrate for TH in dopaminergic neurons. Thus, in PKU brain, when tyrosine levels are low and Phe levels are high, an unlimited supply of substrate for dopamine synthesis appears to exist, i.e. Phe.

Significant increases in MBP and dopamine levels were observed in frontal cortex and striatum during the first week the PKU mice were placed on diet. The approximate levels of Phe and tyrosine at the 1-week time point then should be good estimates for the concentrations of Phe and tyrosine that were conducive to the observed recovery in these brain structures. In striatum at the 1-week time point, levels of Phe fell to about 240 µm (about 1.8-fold above control), and in frontal cortex Phe decreased to about 225 µm (about a three-fold elevation above control). Tyrosine only rose to 53% and 58% of control in frontal cortex and striatum, respectively, during the first week of the study. These data re-emphasize the point that low brain tyrosine levels do not impede MBP/myelination and dopamine synthesis in the treated PKU brain.

Evidence regarding the molecular mechanism by which Phe induces pathology in the PKU brain has been previously reported. Elevated levels of Phe, in the range detected in untreated PKU mice and humans (about 1200 µm), act as a moderate non-competitive inhibitor of HMG-CoA reductase, the key regulatory enzyme in the cholesterol biosynthetic pathway (Shefer et al. 2000). Since cholesterol is a major lipid in the myelin membrane and has been implicated in signaling pathways regulating cytoskeleton in the myelin sheath (Lintner and Dyer 2000), a moderate inhibition of HMG-CoA reductase is likely to have a major impact on the ability of oligodendrocytes to elaborate myelin. Indeed, exposure of cultured immature oligodendrocytes to about 1200 µm Phe resulted in the development of large numbers of mixed phenotype glia (Dyer et al. 1996). Thus, based upon these and additional studies (Dyer et al. 2000), it was postulated that the mixed phenotype glia detected in the hypomyelinated brain structures in the PKU brain are oligodendrocytes that have switched to a non-myelinating phenotype. Indeed, mixed phenotype glia are process-bearing, do not produce membrane sheaths, and express both myelin proteins (including MBP) and glial fibrillary acid protein (Dyer et al. 2000). These data offer a plausible explanation for what on the surface appears to be conflicting histologic and western blot analysis data. Histologic analysis showed a paucity of myelin in the frontal cortex (e.g. subcortical white matter and myelin in corpus callosum) whereas western blot data indicated that frontal cortex and striatum of untreated PKU mice contained about 75% of the normal amount of MBP.

MBP and dopamine increases took place at similar times in the treated PKU mouse frontal cortex and striatum. The fact that the recovery patterns were different in both brain structures suggests that a relationship exists between the two events. In frontal cortex, MBP and dopamine showed a biphasic pattern of increases in week 1 and weeks 3–4, whereas in striatum they both rose to near normal levels in a single leap during week 1 of the study. The reason for the biphasic recovery in frontal cortex is unknown and may be due to different maturation times of (i) oligodendrocytes subpopulations, and/or (ii) subpopulations of non-myelinating oligodendrocytes (mixed phenotype glia) switching to myelinating oligodendrocytes (Dyer and Philibotte 1995). Evidence for adult progenitor oligodendrocytes playing a role in myelination of the PKU brain was not previously found (Dyer et al. 2000). In any case, the data suggest that increases in MBP are associated with myelination since newly formed myelin was verified by histologic examination of treated PKU mouse brain sections. In addition, initial increases in MBP were associated with up-regulation of phosphorylated neurofilaments, further confirming the presence of newly formed myelin (Colello et al. 1994; Kirkpatrick and Brady 1994; Sanchez et al. 1996).

In frontal cortex, increases in TH consistently lagged 1 week behind increases in MBP/myelination. These data suggest that myelination up-regulated TH synthesis. However, the observed increase in dopamine in the first week of the study (when tyrosine levels were 50% of control) occurred without an up-regulation in TH protein levels. Thus, another mechanism must account for the increased synthesis of dopamine during this period of time. One possible mechanism may be that myelin/axonal contact triggered signaling pathways that phosphorylated TH, thereby increasing the activity of the existing TH (Richtand et al. 1985). This mechanism may be feasible since myelin/axonal contact is known to transduce signals that phosphorylate neurofilaments (Colello et al. 1994; Kirkpatrick and Brady 1994; Sanchez et al. 1996). Clearly, future studies are needed to investigate this possibility.

It is possible that alternative mechanisms may account for the up-regulation of dopamine in PKU mice that were placed on the low Phe diet. An elegant study by Ikeda et al. (1967) demonstrated that TH, purified from bovine adrenal glands and from canine brain caudate nuclei, readily converted phenylalanine into DOPA. This study also demonstrated that concentrations of Phe greater than 10−3 m had an inhibitory effect on dopamine production; 2 × 10−3 m Phe inhibited DOPA synthesis by about 50%. Thus, an alternative mechanism may be that high brain Phe levels inhibit the conversion of tyrosine into DOPA, thereby resulting in low dopamine levels. When Phe levels fall, the inhibition of TH is released and dopamine synthesis is restored. However, this does not appear to be a viable mechanism since Phe levels were approximately 10−3 m in the untreated PKU mouse brain; as shown by Ikeda et al. (1967), maximum synthesis of DOPA from Phe still takes place at 10−3 m Phe. Moreover, the amount of Phe available for interaction with TH is actually less than the total amount of Phe in the brain due to the sequestration of Phe in various pools within cells (Shefer et al. 2000; Smith and Kang 2000). Thus, it seems unlikely that dopamine synthesis is regulated by the inhibition of TH via high Phe levels in untreated PKU mouse brain.

An approximate 20% reduction in the local rate of leucine incorporation into cerebral protein (lCPSleu) in the PKU mouse brain versus control has been reported (Smith and Kang 2000). Thus, another potential mechanism regulating dopamine synthesis may be that decreased protein synthesis leads to decreased levels of enzymes that produce neurotransmitters, thereby leading to decreased neurotransmitters (Pascucci et al. 2002). Interestingly, brain regions rich in neuronal cell bodies (e.g. pyramidal cell layer of the CA2 region of the hippocampus) had the highest rates of lCPSleu (about 90% of control), and brain regions rich in myelin or synapses showed the lowest lCPSleu (about 80% of control) (Smith and Kang 2000). These observations may provide a key insight into the mechanism underlying the lCPSleu decrease in PKU mouse brain. Myelin is a highly metabolically active, extremely large membrane (for review, see Holtzman et al. 1996; Madison et al. 1996; Dyer 1999). Thus, in the untreated PKU mouse brain, significantly less protein synthesis may be expected to take place compared to control brain simply because a very large metabolically active membrane is not present. Moreover, since myelin induces neuronal maturation, the decrease in protein synthesis also may impact neurons/axons. Thus, the observed global decrease in protein synthesis detected in PKU mouse brain (Smith and Kang 2000) may not be surprising.

In summary, our data suggest that the tyrosine/dopamine theory is not applicable to the recovering treated PKU brain. In addition to failing to take into account the above discussed role of Phe as substrate for TH and the possible aberrant expression pattern of blood–brain barrier amino acid transporters, the tyrosine/dopamine theory ignores the primary neuropathologic findings in untreated PKU brain. Our data instead support the hypothesis that myelination influences dopamine synthesis. It is reasonable to expect that the observed pathology, i.e. hypomyelination with increased numbers of mixed phenotype glia, has an effect on brain function since pathologic findings generally can be correlated with clinical symptoms. Although the influence of myelin on axonal maturation and action potential conduction has been previously established, the actual molecular mechanisms by which axonal function is enhanced by signals emanating from myelin are unknown. Since (i) dopamine is not the only neurotransmitter that is significantly reduced in the untreated PKU brain, i.e. norepinephrine and serotonin also are reduced (Diamond et al. 1994; Puglisi-Allegra et al. 2000; Pascucci et al. 2002), and (ii) myelin induces maturation of axons regardless of the neurotransmitters that they produce, it may be reasonable to speculate that myelin/axonal interactions elicit signals that up-regulate all neurotransmitters. Moreover, in diseases in which loss of myelin occurs, such PKU when Phe levels rise and multiple sclerosis, loss of myelin may lead to a down-regulation of neurotransmitter production.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mice
  5. Histologic staining of cerebral hemisphere brain sections
  6. Western blots
  7. Gas chromatography mass spectrometry (GC-MS) quantification of Phe, tyrosine, and dopamine in blood and brain tissues
  8. Statistics
  9. Results
  10. Blood and brain Phe levels drop to near normal levels following placement of PKU mice on a low Phe diet for 4 weeks
  11. Tyrosine levels do not rebound to normal levels following placement of PKU mice on the low Phe diet for 4 weeks
  12. Dopamine levels increase to near normal in frontal cortex and striatum following placement of PKU mice on diet for 4 weeks
  13. Placement of PKU mice on diet for 4 weeks induces myelination in the frontal cortex and striatum
  14. Myelin and axonal proteins increase in PKU mouse frontal cortex and striatum during the low Phe diet time course
  15. Discussion
  16. Acknowledgements
  17. References