Distribution of monoamine oxidase proteins in human brain: implications for brain imaging studies.

Positron emission tomography (PET) imaging of monoamine oxidases (MAO-A: [(11)C]harmine, [(11)C]clorgyline, and [(11)C]befloxatone; MAO-B: [(11)C]deprenyl-D2) has been actively pursued given clinical importance of MAOs in human neuropsychiatric disorders. However, it is unknown how well PET outcome measures for the different radiotracers are quantitatively related to actual MAO protein levels. We measured regional distribution (n=38) and developmental/aging changes (21 hours to 99 years) of both MAOs by quantitative immunoblotting in autopsied normal human brain. MAO-A was more abundant than MAO-B in infants, which was reversed as MAO-B levels increased faster before 1 year and, unlike MAO-A, kept increasing steadily to senescence. In adults, regional protein levels of both MAOs were positively and proportionally correlated with literature postmortem data of MAO activities and binding densities. With the exception of [(11)C]befloxatone (binding potential (BP), r=0.61, P=0.15), correlations between regional PET outcome measures of binding in the literature and MAO protein levels were good (P<0.01) for [(11)C]harmine (distribution volume, r=0.86), [(11)C]clorgyline (λk3, r=0.82), and [(11)C]deprenyl-D2 (λk3 or modified Patlak slope, r=0.78 to 0.87), supporting validity of the latter imaging measures. However, compared with in vitro data, the latter PET measures underestimated regional contrast by ∼2-fold. Further studies are needed to address cause of the in vivo vs. in vitro nonproportionality.

Distribution of vesicular monoamine transporter 2 protein in human brain: implications for brain imaging studies.

The choice of reference region in positron emission tomography (PET) human brain imaging of the vesicular monoamine transporter 2 (VMAT2), a marker of striatal dopamine innervation, has been arbitrary, with cerebellar, whole cerebral, frontal, or occipital cortices used. To establish whether levels of VMAT2 are in fact low in these cortical areas, we measured VMAT2 protein distribution by quantitative immunoblotting in autopsied normal human brain (n=6). Four or five species of VMAT2 immunoreactivity (75, 55, 52, 45, 35 kDa) were detected, which were all markedly reduced in intensity in nigrostriatal regions of patients with parkinsonian conditions versus matched controls (n=9 to 10 each). Using the intact VMAT2 immunoreactivity, cerebellar and cerebral neocortices had levels of the transporter >100-fold lower than the VMAT2-rich striatum and with no significant differences among the cortical regions. We conclude that human cerebellar and cerebral cortices contain negligible VMAT2 protein versus the striatum and, in this respect, all satisfy a criterion for a useful reference region for VMAT2 imaging. The slightly lower PET signal for VMAT2 binding in occipital (the currently preferred reference region) versus cerebellar cortex might not therefore be explained by differences in VMAT2 protein itself but possibly by other imaging variables, for example, partial volume effects.

Brain serotonin transporter in human methamphetamine users.

RATIONALE: Research on methamphetamine (MA) toxicity primarily focuses on the possibility that some of the behavioural problems in human MA users might be caused by damage to brain dopamine neurones. However, animal data also indicate that MA can damage brain serotonin neurones, and it has been suggested that cognitive problems and aggression in MA users might be explained by serotonergic damage. As information on the brain serotonin system in human MA users is fragmentary, our objective was to determine whether protein levels of serotonin transporter (SERT), a key marker of serotonin neurones, are decreased in brain of chronic MA users. METHODS: SERT immunoreactivity was measured using an immunoblotting procedure in autopsied brain of 16 chronic MA users testing positive for the drug in blood and brain and matched controls. RESULTS: SERT levels were non-significantly decreased (-14% to -33%) in caudate, putamen and thalamus (normal in hippocampus), and, unlike the robust striatal dopamine reduction, there was marked overlap between control and MA user ranges. Concentrations of SERT were significantly decreased (-23% to -39%) in orbitofrontal and occipital cortices (normal in frontopolar and temporal cortices). CONCLUSIONS: Our data suggest that MA might modestly damage brain serotonin neurones and/or inhibit SERT protein expression, with cerebral cortex being more affected than sub-cortical regions. The SERT reduction in orbitofrontal cortex complements other data suggesting involvement of this area in MA-related behaviour. Decreased brain SERT could also be related to the clinical finding that treatment with a selective serotonin re-uptake inhibitor might increase relapse to MA.

Preferential loss of serotonin markers in caudate versus putamen in Parkinson's disease.

Interest in serotonergic involvement in Parkinson's disease (PD) has focussed recently on the possibility that the remaining serotonin neurons innervating striatum (caudate and putamen) might release dopamine as a 'false transmitter'--an action that could have both beneficial and harmful (e.g. promotion of levodopa-induced dyskinesias) consequences. Evidence for a brain serotonergic disturbance in PD is derived in large part from findings of decreased binding of different radioligands to the serotonin transporter (SERT), one 'marker' of serotonin neurons. However, it is not known whether the reported changes in SERT binding reflect actual changes in levels of SERT protein or whether concentrations of all serotonin markers are similarly and markedly decreased in the two striatal subdivisions. We measured levels of SERT immunoreactivity, and for comparison, protein levels of tryptophan hydroxylase (TPH; the marker synthetic enzyme) using a Western blot procedure, as well as concentrations of serotonin, its metabolite 5-hydroxyindoleacetic acid (5-HIAA), and dopamine by HPLC in post-mortem striatum of patients with PD and normal controls. Whereas concentrations of dopamine were severely decreased (caudate, -80%; putamen, -98%) and showed little (caudate) or no (putamen) overlap between individual control and patient values, levels of all four serotonin markers were less markedly reduced (-30% to -66%) with some patients having distinctly normal levels. Unlike the preferential loss of dopamine in putamen, the caudate was affected more than putamen by loss of all serotonin markers: serotonin (-66% versus -51%), 5-HIAA (-42% versus -31%), SERT (-56% versus -30%) and TPH (-59% versus -32%). Striatal serotonin concentration was similar in the subset of patients reported to have had dyskinesias versus those not reported to have had this drug complication. Previous findings of decreased SERT binding are likely explained by loss of SERT protein. Reduced striatal levels of all of the key serotonergic markers (neurotransmitter and metabolite, transporter protein, synthesizing enzyme protein) provide strong evidence for a serotonergic disturbance in PD, but with some patients affected much more than others. The more marked caudate reduction suggests that raphe neurons innervating this area are more susceptible to 'damage' than those innervating putamen and that any functional impairment caused by striatal serotonin loss might primarily involve the caudate. Questions related to the, as yet undetermined, clinical consequences in PD of a striatal serotonin deficiency (caudate: cognitive impairment?) and preservation (putamen: levodopa-induced dyskinesias?) should be addressed in prospective brain imaging and pharmacological studies.

Regional distribution of serotonin transporter protein in postmortem human brain: is the cerebellum a SERT-free brain region?

INTRODUCTION: The primary approach in assessing the status of brain serotonin neurons in human conditions such as major depression and exposure to the illicit drug ecstasy has been the use of neuroimaging procedures involving radiotracers that bind to the serotonin transporter (SERT). However, there has been no consistency in the selection of a "SERT-free" reference region for the estimation of free and nonspecific binding, as occipital cortex, cerebellum and white matter have all been employed. OBJECTIVE AND METHODS: To identify areas of human brain that might have very low SERT levels, we measured, by a semiquantitative Western blotting procedure, SERT protein immunoreactivity throughout the postmortem brain of seven normal adult subjects. RESULTS: Serotonin transporter could be quantitated in all examined brain areas. However, the SERT concentration in cerebellar cortex and white matter were only at trace values, being approximately 20% of average cerebral cortex and 5% of average striatum values. CONCLUSION: Although none of the examined brain areas are completely free of SERT, human cerebellar cortex has low SERT binding as compared to other examined brain regions, with the exception of white matter. Since the cerebellar cortical SERT binding is not zero, this region will not be a suitable reference region for SERT radioligands with very low free and nonspecific binding. For SERT radioligands with reasonably high free and nonspecific binding, the cerebellar cortex should be a useful reference region, provided other necessary radioligand assumptions are met.