Abstract Objective Parkinson disease (PD) is defined by the accumulation of misfolded α‐synuclein (α‐syn) in Lewy bodies and Lewy neurites. It affects multiple cortical and subcortical neuronal populations. The majority of people with PD develop dementia, which is associated with Lewy bodies in neocortex and referred to as Lewy body dementia (LBD). Other neuropathologic changes, including amyloid β (Aβ) and tau accumulation, occur in some LBD cases. We sought to quantify α‐syn, Aβ, and tau accumulation in neocortical, limbic, and basal ganglia regions. Methods We isolated insoluble protein from fresh frozen postmortem brain tissue samples for eight brains regions from 15 LBD, seven Alzheimer disease (AD), and six control cases. We measured insoluble α‐syn, Aβ, and tau with recently developed sandwich ELISAs. Results We detected a wide range of insoluble α‐syn accumulation in LBD cases. The majority had substantial α‐syn accumulation in most regions, and dementia severity correlated with neocortical α‐syn. However, three cases had low neocortical levels that were indistinguishable from controls. Eight LBD cases had substantial Aβ accumulation, although the mean Aβ level in LBD was lower than in AD. The presence of Aβ was associated with greater α‐syn accumulation. Tau accumulation accompanied Aβ in only one LBD case. Interpretation LBD is associated with insoluble α‐syn accumulation in neocortical regions, but the relatively low neocortical levels in some cases suggest that other changes contribute to impaired function, such as loss of neocortical innervation from subcortical regions. The correlation between Aβ and α‐syn accumulation suggests a pathophysiologic relationship between these two processes.

Objective Interpretation of diffusion MRI in the living brain requires validation against gold standard histological measures. We compared diffusion values of the nigrostriatal tract to PET and histological results in non-human primates (NHPs) with varying degrees of unilateral nigrostriatal injury induced by MPTP, a toxin selective for dopaminergic neurons. Methods Sixteen NHPs had MRI and PET scans of three different presynaptic radioligands and blinded video-based motor ratings before and after unilateral carotid artery infusion of variable doses of MPTP. Diffusion measures of connections between midbrain and striatum were calculated. Then animals were euthanized to quantify striatal dopamine concentration, stereologic measures of striatal tyrosine hydroxylase (TH) immunostained fiber density and unbiased stereologic counts of TH stained nigral cells. Results Diffusion measures correlated with MPTP dose, nigral TH-positive cell bodies and striatal TH-positive fiber density but did not correlate with in vitro nigrostriatal terminal field measures or in vivo PET measures of striatal uptake of presynaptic markers. Once nigral TH cell count loss exceeded 50% the stereologic terminal field measures reached a near zero floor effect but the diffusion measures continued to correlate with nigral cell counts. Conclusion Diffusion measures in the nigrostriatal tract correlate with nigral dopamine neurons and striatal fiber density, but have the same relationship to terminal field measures as a previous report of striatal PET measures of presynaptic neurons. These diffusion measures have the potential to act as non-invasive index of the severity of nigrostriatal injury. Diffusion imaging of the nigrostriatal tract could potentially have diagnostic value in humans with Parkinson disease or related disorders.

OBJECTIVE:People with Parkinson disease (PD) frequently develop dementia, which is associated with neocortical deposition of alpha-synuclein (α-syn) in Lewy bodies and Lewy neurites. In addition, neuronal loss and deposition of aggregated α-syn also occur in multiple subcortical nuclei that project to neocortical, limbic, and basal ganglia regions. Therefore, we quantified regional deficits in innervation from these PD-affected subcortical nuclei, by measuring the neurotransmitters and neurotransmitter transporter proteins originating from projections of dopaminergic neurons in substantia nigra pars compacta, serotonergic neurons in dorsal raphé nuclei, noradrenergic neurons in locus coeruleus, and cholinergic neurons in nucleus basalis of Meynert.METHODS:High-performance liquid chromatography and novel enzyme-linked immunosorbent assays were performed to quantify dopaminergic, serotonergic, noradrenergic, and cholinergic innervation in postmortem brain tissue. Eight brain regions from 15 PD participants (with dementia and Braak stage 6 α-syn deposition) and six age-matched controls were tested.RESULTS:PD participants compared to controls had widespread reductions of dopamine transporter in caudate, amygdala, hippocampus, inferior parietal lobule (IPL), precuneus, and visual association cortex (VAC) that exceeded loss of dopamine, which was only significantly reduced in caudate and amygdala. In contrast, PD participants had comparable deficits of both serotonin and serotonin transporter in caudate, middle frontal gyrus, IPL, and VAC. PD participants also had significantly reduced norepinephrine levels for all eight brain regions tested. Vesicular acetylcholine transporter levels were only quantifiable in caudate and hippocampus and did not differ between PD and control groups.INTERPRETATION:These results demonstrate widespread deficits in dopaminergic, serotonergic, and noradrenergic innervation of neocortical, limbic, and basal ganglia regions in advanced PD with dementia.

ObjectiveWe evaluated the efficacy of the potent antioxidant C3 to salvage nigrostriatal neuronal function after 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) exposure in nonhuman primates. C3 is a first‐in‐class functionalized water‐soluble fullerene that reduces oxygen radical species associated with neurodegeneration in in vitro studies. However, C3 has not been evaluated as a neuroprotective agent in a Parkinson model in vivo.MethodsMacaque fascicularis monkeys were used in a double‐blind, placebo‐controlled study design. MPTP‐lesioned primates were given systemic C3 (n = 8) or placebo (n = 7) for 2 months starting 1 week after MPTP. Outcomes included in vivo behavioral measures of motor parkinsonism using a validated nonhuman primate rating scale, kinematic analyses of peak upper extremity velocity, positron emission tomography imaging of 6‐[18F]fluorodopa (FD; reflects dopa decarboxylase) and [11C]dihydrotetrabenazine (DTBZ; reflects vesicular monoamine transporter type 2), ex vivo quantification of striatal dopamine, and stereologic counts of tyrosine hydroxylase–immunostained neurons in substantia nigra.ResultsAfter 2 months, C3‐treated monkeys had significantly improved parkinsonian motor ratings, greater striatal FD and DTBZ uptake, and higher striatal dopamine levels. None of the C3‐treated animals developed any toxicity.InterpretationSystemic treatment with C3 reduced striatal injury and improved motor function despite administration after the MPTP injury process had begun. These data strongly support further development of C3 as a promising therapeutic agent for Parkinson disease. Ann Neurol 2014;76:393–402

A single unilateral intracarotid infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into non-human primates causes injury to the nigrostriatal pathway including nigral cell bodies, axons and striatal terminal fields. In this model, motor parkinsonism correlates well with the loss of nigral dopaminergic cell bodies but only correlates with in vitro measures of nigrostriatal terminal fields when nigral cell loss does not exceed 50%. The goals of this study are to determine the relationship of motor parkinsonism with the degree of injury to nigrostriatal axons, as reflected by in vitro fiber length density measures, and compare in vivo with in vitro measures of striatal terminal fields. We determined axon integrity by measuring fiber length density with tyrosine hydroxylase (TH) immunohistology and dopamine transporter (DAT) density with DAT immunohistology. We then calculated the terminal arbor size and compared these measures with previously published data of quantified in vivo positron emission tomography (PET) measures of presynaptic dopaminergic neurons, autoradiographic measures of DAT and vesicular monoamine transporter type 2 (VMAT2), striatal dopamine, nigral cell counts, and parkinsonian motor ratings in the same animals. Our data demonstrate that in vivo and in vitro measures of striatal terminal fields correlate with each other regardless of the method of measurement. PET-based in vivo striatal measures accurately reflect in vitro measures of DAT and VMAT2. Terminal arbor size and other terminal field measures correlate with nigral TH immunoreactive (TH-ir) cell counts only when nigral TH-ir cell loss does not exceed 50%. Fiber length density was the only striatal measure that linearly correlated with motor ratings (Spearman: r=−0.81, p<0.001, n=16).

ObjectiveMolecular imaging and clinical endpoints are frequently discordant in Parkinson disease clinical trials, raising questions about validity of these imaging measures to reflect disease severity. We compared striatal uptake for 3 positron emission tomography (PET) tracers with in vitro measures of nigral cell counts and striatal dopamine in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐treated monkeys.MethodsSixteen macaques had magnetic resonance imaging and baseline PETs using 6‐[18F]fluorodopa (FD), [11C]dihydrotetrabenazine (DTBZ), and 2beta‐[11C]carbomethoxy‐3beta‐(4‐fluorophenyl)tropane (CFT). MPTP (0–0.31mg/kg) infused unilaterally via the internal carotid artery produced stable hemiparkinsonism by 3 weeks. After 8 weeks, PETs were repeated and animals were euthanized for striatal dopamine measurements and unbiased counts of tyrosine hydroxylase–stained nigral cells.ResultsStriatal uptake for each radiotracer (FD, DTBZ, CFT) correlated with stereologic nigral cell counts only for nigral loss <50% (r2 = 0.84, r2 = 0.86, r2 = 0.87, p < 0.001 respectively; n = 10). In contrast, striatal uptake correlated with striatal dopamine over the full range of dopamine depletion (r2 = 0.95, r2 = 0.94, r2 = 0.94, p < 0.001; n = 16). Interestingly, indices of striatal uptake of FD, DTBZ, and CFT correlated strongly with each other (r2 = 0.98, p < 0.001).InterpretationTracer uptake correlated with nigral neurons only when nigral loss was <50%. This along with previous work demonstrating that nigral cell counts correlate strongly with parkinsonism ratings may explain discordant results between neuroimaging and clinical endpoints. Furthermore, strong correlations among striatal uptake for these tracers support lack of differential regulation of decarboxylase activity (FD), vesicular monoamine transporter type 2 (DTBZ), and dopamine transporter (CFT) within 2 months after nigrostriatal injury. ANN NEUROL 2013;73:390–396

ObjectiveDevelopment of an effective therapy to slow the inexorable progression of Parkinson disease requires a reliable, objective measurement of disease severity. In the present study, we compare presynaptic positron emission tomography (PET) tracer uptake in the substantia nigra (SN) to cell loss and motor impairment in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐treated nonhuman primates.MethodsPresynaptic PET tracers 6‐[18F]‐fluorodopa (FD), [11C]‐2β‐methoxy‐3β‐4‐fluorophenyltropane (CFT), and [11C]‐dihydrotetrabenazine (DTBZ) were used to measure specific uptake in the SN and striatum before and after a variable dose of MPTP in nonhuman primates. These in vivo PET‐based measures were compared with motor impairment, as well as postmortem tyrosine hydroxylase‐positive cell counts and striatal dopamine concentration.ResultsWe found the specific uptake of both CFT and DTBZ in the SN had a strong, significant correlation with dopaminergic cell counts in the SN (R2 = 0.77, 0.53, respectively, p < 0.001), but uptake of FD did not. Additionally, both CFT and DTBZ specific uptake in the SN had a linear relationship with motor impairment (rs = −0.77, −0.71, respectively, p < 0.001), but FD uptake did not.InterpretationOur findings demonstrate that PET‐measured binding potentials for CFT and DTBZ for a midbrain volume of interest targeted at the SN provide faithful correlates of nigral neuronal counts across a full range of lesion severity. Because these measures correlate with both nigral cell counts and parkinsonian ratings, we suggest that these SN PET measures are relevant biomarkers of nigrostriatal function. Ann Neurol 2013;74:602–610

Objective: Nigrostriatal reserve refers to the threshold of neuronal injury to dopaminergic cell bodies and their terminal fields required to produce parkinsonian motor deficits. Inferential studies have estimated striatal dopamine reserve to be at least 70%. Knowledge of this threshold is critical for planning interventions to prevent symptom onset or reverse nigrostriatal injury sufficient to restore function in people with Parkinson disease. In this study, we determine the nigrostriatal reserve in a non-human primate model that mimics the motor manifestations of Parkinson disease.Methods: Fifteen macaque monkeys received unilateral randomized doses of the selective dopaminergic neuronal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. We compared blinded validated ratings of parkinsonism to in vitro measures of striatal dopamine and unbiased stereologic counts of nigral neurons after tyrosine hydroxylase immunostaining.Results: The percent of residual cell counts in lesioned nigra correlated linearly with the parkinsonism score at 2 months (r = -0.87, p<0.0001). The parkinsonism score at 2 months correlated linearly with the percent residual striatal dopamine (r = residual striatal dopamine (r = -0.77, p = 0.016) followed by a flooring effect once nigral cell loss exceeded, p = 0.016) followed by a flooring effect once nigral cell loss exceeded 50%. A reduction of about 14 to 23% of nigral neuron counts or 14% to 37% of striatal dopamine was sufficient to induce mild parkinsonism.Conclusions: The nigral cell body and terminal field injury needed to produce parkinsonian motor manifestations may be much less than previously thought. (c) 2012 Elsevier Inc. All rights reserved.

Radioligands for DAT and VMAT2 are widely used presynaptic markers for assessing dopamine (DA) nerve terminals in Parkinson disease (PD). Previous in vivo imaging and postmortem studies suggest that these transporter sites may be regulated as the numbers of nigrostriatal neurons change in pathologic conditions. To investigate this issue, we used in vitro quantitative autoradioradiography to measure striatal DAT and VMAT2 specific binding in postmortem brain from 14 monkeys after unilateral internal carotid artery infusion of 1-Methyl-4-Phenyl-1,2,3,6-tetrahydropyridine (MPTP) with doses varying from 0 to 0.31 mg/kg. Quantitative estimates of the number of tyrosine hydroxylase (TH)-immunoreactive (ir) neurons in substantia nigra (SN) were determined with unbiased stereology, and quantitative autoradiography was used to measure DAT and VMAT2 striatal specific binding. Striatal VMAT2 and DAT binding correlated with striatal DA (rs = 0.83, rs = 0.80, respectively, both with n = 14, p<0.001) but only with nigra TH-ir cells when nigral cell loss was 50% or less (r = 0.93, n = 8, p = 0.001 and r = 0.91, n = 8, p = 0.002 respectively). Reduction of VMAT2 and DAT striatal specific binding sites strongly correlated with each other (r = 0.93, n = 14, p<0.0005). These similar changes in DAT and VMAT2 binding sites in the striatal terminal fields of the surviving nigrostriatal neurons demonstrate that there is no differential regulation of these two sites at 2 months after MPTP infusion.

Levodopa, when combined with a decarboxylase inhibitor, essentially delivers dopamine directly to the brain, with no net effect on brain blood vessels. For future neuroimaging studies of Parkinson disease and Tourette syndrome, we sought to rapidly produce a biologically relevant levodopa concentration in plasma and then maintain that concentration long enough to assess motor, cognitive, emotional, and neuroimaging responses, while minimizing side effects in levodopa-naive individuals. Based on available pharmacokinetic data and a two-compartment model, we designed a decreasing-exponential-rate infusion to meet these goals. This report gives results of double-blind levodopa and placebo infusions in six healthy subjects. Mean plasma levodopa concentrations were within 3% of their 1200ng/mL target at 20 and 40min into the infusion, and within 20% between ∼12 and 90min. Levodopa significantly reduced serum prolactin and raised serum growth hormone concentrations. Volunteers had no significant side effects.

Background: Previous work in this laboratory has demonstrated antidepressant effects with neural transplants of embryonic stem (ES) cells (“N2-5HT”) that have been engineered to differentiate into high proportions of serotonergic (5HT) and dopaminergic (DA) neurons. Clinically and neuroanatomically, the pathoetiology of depression and anxiety are intimately associated with corticolimbic function. Here, we sought to further characterize N2-5HT cells engrafted into the anterior cingulate cortex (ACCx) and assess graft effects in animal models of anxiety and depression. Method: The ACCx of 24 adult rats were engrafted with N2-5HT cells, 14 control subjects received feeder cells, and 10 subjects underwent sham surgery. Behavior was examined using Forced Swim, Social Interaction and the Elevated Plus Maze. Immunohistochemical reactions used to characterize the engrafted cells included antiTH, -5HT, -DAT, -AADC, and –DBH. Functional integration was assessed with electron microscopy, bromo-deoxyuridine (BrdU) labeling, anterograde tracing from the basolateral amygdala (BLA), and up-regulation of c-fos. Results: Large numbers of 5HT and DA neurons were found integrated within ACCx up-regulating c-fos and interacting with amygdalofugal fibers. Increased numbers of BrdU-labeled cells were seen in the dentate gyrus. Transplants produced robust anxiolytic effects in addition to previously seen antidepressant effects. Conclusions: N2-5HT cells functionally integrate within the corticolimbic system producing significant effects in models of anxiety and depression. Locally, they may provide elevated levels of 5HT and DA, and are associated with afferents from the BLA. Increased neurogenesis within the dentate gyrus suggests modulation of function influence by these grafts across corticolimbic regions.

Plasma measurements of levodopa and its major metabolites including dopamine and 3-O-methyldopa have been limited by cumbersome methods and poor sensitivity within relatively narrow ranges of plasma levels. We now report a modification of an HPLC method that permits concomitant measurements of a wide range of concentrations of levodopa, dopamine (DA), carbidopa, 3-O-methyldopa (3-OMD) and 3,4-dihydroxyphenyl acetic acid (DOPAC) from one HPLC injection. The recoveries ranged from 77 to 107% with an intra-day precision around 5% (CV) and inter-day CV's about 10–20%. This validated method will simplify pharmacokinetic studies of levodopa and its metabolites for mechanistic studies or therapeutic clinical monitoring which play a crucial role in development of strategies to prolong motor benefits from individual doses and reduce involuntary movements called dykinesias.