A new approach to biochemical evaluation of brain dopamine metabolism

1. Dopaminergic neurotransmission in brain is receiving increased attention because of its known involvement in Parkinson's disease and new methods for the treatment of this disorder and because of hypotheses relating several psychiatric disorders to abnormalities in brain dopaminergic systems. 2. Chemical assessment of brain dopamine metabolism has been attempted by measuring levels of its major metabolite, homovanillic acid (HVA), in cerebrospinal fluid, plasma, or urine. Because HVA is derived in part from dopamine formed in noradrenergic neurons, plasma levels and urinary excretion rates of HVA do not adequately reflect solely metabolism of brain dopamine. 3. Using debrisoquin, the peripheral contributions of HVA to plasma or urinary HVA can be diminished, but the extent of residual HVA formation in noradrenergic neurons is unknown. By measuring the levels of methoxy-hydroxyphenylglycol (MHPG) in plasma or of urinary norepinephrine metabolites (total MHPG in monkeys; the sum of total MHPG and vanillyl mandelic acid (VMA) in humans) along with HVA, it is possible to estimate the degree of impairment by debrisoquin of HVA formation from noradrenergic neuronal dopamine and thereby better assess brain dopamine metabolism. 4. This method was applied to a monkey before and after destruction of the nigrostriatal pathway by the administration of MPTP.     

Convection-Enhanced Delivery of AAV2-PrPshRNA in Prion-Infected Mice

Prion disease is caused by a single pathogenic protein (PrP^Sc), an abnormal conformer of the normal cellular prion protein PrP^C. Depletion of PrP^C in prion knockout mice makes them resistant to prion disease. Thus, gene silencing of the Prnp gene is a promising effective therapeutic approach. Here, we examined adeno-associated virus vector type 2 encoding a short hairpin RNA targeting Prnp mRNA (AAV2-PrP-shRNA) to suppress PrP^C expression both in vitro and in vivo. AAV2-PrP-shRNA treatment suppressed PrP levels and prevented dendritic degeneration in RML-infected brain aggregate cultures. Infusion of AAV2-PrP-shRNA-eGFP into the thalamus of CD-1 mice showed that eGFP was transported to the cerebral cortex via anterograde transport and the overall PrP^C levels were reduced by ∼70% within 4 weeks. For therapeutic purposes, we treated RML-infected CD-1 mice with AAV2-PrP-shRNA beginning at 50 days post inoculation. Although AAV2-PrP-shRNA focally suppressed PrP^Sc formation in the thalamic infusion site by ∼75%, it did not suppress PrP^Sc formation efficiently in other regions of the brain. Survival of mice was not extended compared to the untreated controls. Global suppression of PrP^C in the brain is required for successful therapy of prion diseases.     

Ultrasound-Enhanced Drug Transport and Distribution in the Brain

Drug delivery in the brain is limited by slow drug diffusion in the brain tissue. This study tested the hypothesis that ultrasound can safely enhance the permeation of drugs in the brain. In vitro exposure to ultrasound at various frequencies (85 kHz, 174 kHz, and 1 MHz) enhanced the permeation of tritium-labeled molecules with molecular weight up to 70 kDa across porcine brain tissue. A maximum enhancement of 24-fold was observed at 85 kHz and 1,200 J/cm². In vivo exposure to 1-MHz ultrasound further demonstrated the ability of ultrasound to facilitate molecule distribution in the brain of a non-human primate. Finally, ultrasound under conditions similar to those used in vivo was shown to cause no damage to plasmid DNA, siRNA, adeno-associated virus, and fetal rat cortical neurons over a range of conditions. Altogether, these studies demonstrate that ultrasound can increase drug permeation in the brain in vitro and in vivo under conditions that did not cause detectable damage.     

Carbidopa-Based Modulation of the Functional Effect of the AAV2-hAADC Gene Therapy in 6-OHDA Lesioned Rats

Progressively blunted response to L-DOPA in Parkinson’s disease (PD) is a critical factor that complicates long-term pharmacotherapy in view of the central importance of this drug in management of the PD-related motor disturbance. This phenomenon is likely due to progressive loss of one of the key enzymes involved in the biosynthetic pathway for dopamine in the basal ganglia: aromatic L-amino acid decarboxylase (AADC). We have developed a gene therapy based on an adeno-associated virus encoding human AADC (AAV2-hAADC) infused into the Parkinsonian striatum. Although no adverse clinical effects of the AAV2-hAADC gene therapy have been observed so far, the ability to more precisely regulate transgene expression or transgene product activity could be an important long-term safety feature. The present study was designed to define pharmacological regulation of the functional activity of AAV2-hAADC transgene product by manipulating L-DOPA and carbidopa (AADC inhibitor) administration in hemi-parkinsonian rats. Thirty days after unilateral striatal infusion of AAV2-hAADC, animals displayed circling behavior and acceleration of dopamine metabolism in the lesioned striatum after administration of a low dose of L-DOPA (5 mg/kg) co-administered with 1.25 mg/kg of carbidopa. This phenomenon was not observed in control AAV2-GFP-treated rats. Withdrawal of carbidopa from a daily L-DOPA regimen decreased the peripheral L-DOPA pool, resulting in almost total loss of L-DOPA-induced behavioral response in AAV2-hAADC rats and a significant decline in striatal dopamine turnover. The serum L-DOPA level correlated with the magnitude of circling behavior in AAV2-hAADC rats. Additionally, AADC activity in homogenates of lesioned striata transduced by AAV2-AADC was 10-fold higher when compared with AAV2-GFP-treated control striata, confirming functional transduction. Our data suggests that the pharmacological regulation of circulating L-DOPA might be effective in the controlling of function of AAV2-hAADC transgene product in PD gene therapy.     

Effect of MPTP-induced parkinsonism in monkeys on the urinary excretion of HVA and MHPG during debrisoquin administration

During debrisoquin administration to three monkeys there were significant reductions in excretion rates of HVA, the major dopamine metabolite, and MHPG, the major norepinephrine metabolite. Excretion rates of HVA were highly correlated to those of MHPG. The regression line relating HVA and MHPG excretion suggests that a portion of HVA (about 25%) is derived from a source independent of norepinephrine metabolites. There was a striking reduction of this portion of HVA excretion after MPTP-induced destruction of dopaminergic nigrostriatal neurons. These results support the view that the rate of HVA formation in brain dopaminergic neurons can be estimated from the relationship of urinary excretion rates of HVA and MHPG before and during debrisoquin treatment.     

An In Vivo Microdialysis Study of Striatal 6-[18F]Fluoro-L-m-Tyrosine Metabolism

In vivo brain microdialysis was used to monitor 6-[¹⁸F]fluoro-L-m-tyrosine (FMT) uptake and metabolism in the striatum of conscious freely moving rats for 3 hours after FMT injection (25 mg/kg, iv). Microdialysate collected 20 to 120 min post-dose, contained FMT at a concentration (0.2 to 0.3 nM) approximately ten-fold below that of its metabolite [¹⁸F]fluoro-3-hydroxyphenylacetic acid (FPAC; 3.2 to 3.3 nM). D-amphetamine (2.5 mg/kg, i.p.) injected 120 min after significantly increased microdialysate FPAC (3.27 ± 0.31 nM to 4.51 ± 0.45 nM) in control but not reserpinized rats. Taken together these data demonstrate FMT is heavily metabolized following its entry into the striatum yielding FPAC which appears to be stored, at least in part, in reserpine sensitive cytoplasmic vesicles. Presynaptic retention of FPAC may contribute to the preferential accumulation of FMT positron emission tomography (PET) signaling in dopaminergic brain areas.     

Recommended safe practices for using the neurotoxin MPTP in animal experiments

The metabolism and distribution of the parkinsonian syndrome inducing neurotoxin MPTP has been studied in non-human primates and mice housed in controlled environmental chambers. 14C6-MPTP was prepared and injected at concentrations normally employed for lesioning experiments (30 mg/kg in mice, 0.3 mg/kg in monkeys). All interior surfaces of the chambers which could be reached by animals or their excreta were contaminated with radiolabeled metabolites. Vapor born unmetabolized MPTP was negligible, although significant amounts of MPTP were found in the excreta of mice (less than or equal to 15% injected dose) and small amounts from rhesus monkeys (less than 2%). Procedures to minimize contact with animal fur, bedding and excreta should protect investigators working with MPTP over extended periods. Permanganate oxidation effectively detoxifies solutions of MPTP. MPTP, MPP+, common synthetic intermediates, and the products of MPTP's oxidation are not mutagenic as measured by a Salmonella-microsome assay.     

Hemiparkinsonism in monkeys after unilateral internal carotid artery infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Infusion of MPTP (0.2-0.8 mg/kg) into the right internal carotid artery of monkeys produces toxin-induced injury to the right nigrostriatal pathway with sparing of other dopaminergic neurones on the infused side and with negligible or little injury to the opposite, untreated side. There are contralateral limb dystonic postures, rigidity, and bradykinesia, but the animals are able to eat and maintain health without drug treatment. Spontaneous motor activity is attended by circling towards the injured side, whereas treatment with L-DOPA/-carbidopa or apomorphine stimulates circling towards the intact side. Dopamine and dopamine metabolite levels are normal in the left caudate and putamen, but markedly depressed on the right (MPTP-treated) side. This animal hemiparkinsonian model will be useful in studies of volitional movement control, drug treatments of Parkinson's disease, and functional efficacy of brain tissue implants.     

Automated Segmentation Tool for Brain Infusions

This study presents a computational tool for auto-segmenting the distribution of brain infusions observed by magnetic resonance imaging. Clinical usage of direct infusion is increasing as physicians recognize the need to attain high drug concentrations in the target structure with minimal off-target exposure. By co-infusing a Gadolinium-based contrast agent and visualizing the distribution using real-time using magnetic resonance imaging, physicians can make informed decisions about when to stop or adjust the infusion. However, manual segmentation of the images is tedious and affected by subjective preferences for window levels, image interpolation and personal biases about where to delineate the edge of the sloped shoulder of the infusion. This study presents a computational technique that uses a Gaussian Mixture Model to efficiently classify pixels as belonging to either the high-intensity infusate or low-intensity background. The algorithm was implemented as a distributable plug-in for the widely used imaging platform OsiriX®. Four independent operators segmented fourteen anonymized datasets to validate the tool’s performance. The datasets were intra-operative magnetic resonance images of infusions into the thalamus or putamen of non-human primates. The tool effectively reproduced the manual segmentation volumes, while significantly reducing intra-operator variability by 67±18%. The tool will be used to increase efficiency and reduce variability in upcoming clinical trials in neuro-oncology and gene therapy.