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The article presents an abstract of the medical research paper "Participation of Par-4 in Degeneration of Dopaminergic Neurons in Models of Parkinson's Disease," which will be presented in the 30th meeting of the American Society for Neurochemistry, to be held in New Orleans, Louisiana. The research examines the role of Par-4, a 38 kDa protein, in oxidative stress and mitochondrial dysfunction.

Identification of Parkinson's disease at the earliest possible stage of the disease may provide the best opportunity for the use of disease modifying treatments. However, diagnosing the disease during the pre-symptomatic period remains an unmet goal. To that end, we used pharmacological MRI (phMRI) to assess the function of the cortico-basal ganglia circuit in a non-human primate model of dopamine deficiency to determine the possible relationships between phMRI signals with behavioral, neurochemical, and histological measurements. Animals with unilateral treatments with the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), that expressed stable, long-term hemiparkinsonism were challenged with the dopaminergic receptor agonist, apomorphine, and structure-specific phMRI blood oxygen level-dependent (BOLD) activation responses were measured. Behavioral, histopathological, and neurochemical measurements were obtained and correlated with phMRI activation of structures of the cortico-basal ganglia system. Greater phMRI activations in the basal ganglia and cortex were associated with slower movement speed, decreased daytime activity, or more pronounced parkinsonian features. Animals showed decreased stimulus-evoked dopamine release in the putamen and substantia nigra pars compacta and lower basal glutamate levels in the motor cortex on the MPTP-lesioned hemisphere compared to the contralateral hemisphere. The altered neurochemistry was significantly correlated with phMRI signals in the motor cortex and putamen. Finally, greater phMRI activations in the caudate nucleus correlated with fewer tyrosine hydroxylase-positive (TH + ) nigral cells and decreased TH + fiber density in the putamen. These results reveal the correlation of phMRI signals with the severity of the motor deficits and pathophysiological changes in the cortico-basal ganglia circuit., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: To determine if the intranasal delivery of neuroactive compounds is a viable, long-term treatment strategy for progressive, chronic neurodegenerative disorders, such as Parkinson's disease (PD), intranasal methodologies in preclinical models comparable to humans are needed., New Method: We developed a methodology to evaluate the repeated intranasal delivery of neuroactive compounds on the non-human primate (NHP) brain, without the need for sedation. We evaluated the effects of the neuroactive peptide, DNSP-11 following repeated intranasal delivery and dose-escalation over the course of 10-weeks in Rhesus macaques. This approach allowed us to examine striatal target engagement, safety and tolerability, and brain distribution following a single 125 I-labeled DNSP-11 dose., Results: Our initial data support that repeated intranasal delivery and dose-escalation of DNSP-11 resulted in bilateral, striatal target engagement based on neurochemical changes in dopamine (DA) metabolites-without observable, adverse behavioral effects or weight loss in NHPs. Furthermore, a 125 I-labeled DNSP-11 study illustrates diffuse rostral to caudal distribution in the brain including the striatum-our target region of interest., Comparison With Existing Methods: The results of this study are compared to our experiments in normal and 6-OHDA lesioned rats, where DNSP-11 was repeatedly delivered intranasally using a micropipette with animals under light sedation., Conclusions: The results from this proof-of-concept study support the utility of our repeated intranasal dosing methodology in awake Rhesus macaques, to evaluate the effects of neuroactive compounds on the NHP brain. Additionally, results indicate that DNSP-11 can be safely and effectively delivered intranasally in MPTP-treated NHPs, while engaging the DA system., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A major consequence of Parkinson's disease (PD) involves the loss of dopaminergic neurons in the substantia nigra (SN) and a subsequent loss of dopamine (DA) in the striatum. We have shown that glial cell line-derived neurotrophic factor (GDNF) shows robust restorative and protective effects for DA neurons in rats, non-human primates and possibly in humans. Despite GDNF's therapeutic potential, its clinical value has been questioned due to its limited diffusion to target areas from its large size and chemical structure. Several comparatively smaller peptides are thought to be generated from the prosequence. A five amino-acid peptide, dopamine neuron stimulating peptide-5 (DNSP-5), has been proposed to demonstrate biological activity relevant to neurodegenerative disease. We tested the in vitro effects of DNSP-5 in primary dopaminergic neurons dissected from the ventral mesencephalon of E14 Sprague Dawley rat fetuses. Cells were treated with several doses (0.03, 0.1, 1.0, 10.0 ng/mL) of GDNF, DNSP-5, or an equivalent volume of citrate buffer (vehicle). Morphological features of tyrosine hydroxylase positive neurons were quantified for each dose. DNSP-5 significantly increased (p < 0.001) all differentiation parameters compared to citrate vehicle (at one or more dose). For in vivo studies, a unilateral DNSP-5 treatment (30 μg) was administered directly to the SN. Microdialysis in the ipsilateral striatum was performed 28 days after treatment to determine extracellular levels of DA and its primary metabolites (3,4-dihydroxyphenylacetic acid and homovanillic acid). A single treatment significantly increased (~66%) extracellular DA levels compared to vehicle, while DA metabolites were unchanged. Finally, the protective effects of DNSP-5 against staurosporine-induced cytotoxicity were investigated in a neuronal cell line showing substantial protection by DNSP-5. Altogether, these studies strongly indicate biological activity of DNSP-5 and suggest that DNSP-5 has neurotrophic-like properties that may be relevant to the treatment of neurodegenerative diseases like PD., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Using an automated test panel, age-associated declines in learning, remembering and performing a novel visuomotor task were assessed in 497 normal adults ranging from 18 to 95 years old. As predicted, task performance times slowed with increasing age in the cross-sectional portion of the study. However in the subsequent longitudinal study, while motor learning was significantly slower in adults over 62 years old, motor memory was pristinely preserved in normal adults from 18 to 95 years old. When tested 2 years after the first training session and without intervening rehearsal, mean performance times were retained and continued to improve by 10% in young adults and 13% in aged adults, reflecting long lasting preservation of motor memories. While the maximum lifetime of an unpracticed, novel motor memory in humans is not known, the present study suggests that new motor memories can be retained for at least 2 years without rehearsal in normal aged adults. This age-resistant component of motor memory stands in contrast to the well-known decrements in other motor and cognitive processes with human aging.

The most common treatment for Parkinson's disease (PD) aims at pharmacologically augmenting striatal dopamine (DA) using the DA precursor levodopa. Such treatment provides symptomatic relief, but does not slow or halt continued degeneration of nigral dopaminergic neurons. Considerable effort has been devoted to the search for neurotrophic factors with survival-promoting activities on dopaminergic neurons that could potentially be of therapeutic value in the treatment of PD. One such candidate is glial cell line-derived neurotrophic factor (GDNF).

The potential of dopaminergic treatments to improve upper limb motor movements was tested in 7 aged rhesus monkeys using L-3,4-dihydroxyphenylalanine (L-dopa) or the selective dopamine uptake inhibitor 1-2(bis[4-fluorophenyl] methoxy]ethyl)-4-(3-phenylpropyl) piperazine hydrochloride (GBR-12909). Six young monkeys were studied for comparison. L-Dopa or GBR-12909 improved upper limb motor performance by up to 40% in the aged animals. At this point their performance was comparable to that of young adults. Dopaminergic therapy could be useful in elderly humans experiencing declines in upper limb motor functions.

Functional magnetic resonance imaging (fMRI) was used to analyze blood oxygen level-dependent (BOLD) responses in the nigrostriatal system (caudate nucleus, putamen and substantia nigra) of awake rhesus monkeys to systemic apomorphine administration. The study (1) measured BOLD responses as an index of neuronal activity in the three structures following injections of the mixed D1/D2 agonist, and (2) assessed the effects of isoflurane anesthesia on the fMRI responses. Compared to control saline injections, 0.1 mg/kg apomorphine significantly activated the caudate nucleus (P < or = 0.005), putamen (P < or = 0.001) and substantia nigra (P < or = 0.005). The responses were consistent with activation of GABAergic neurons in these three structures seen in other animal models. Isoflurane gas measurably blunted the response to apomorphine, so that a significant apomorphine activation was only seen in the substantia nigra of anesthetized animals. Even there, the mean MR signal change was reduced from 9.8% in awake monkeys to 2.3% in anesthetized animals. The data support the hypothesis that fMRI can be used to study the effects of drugs that alter basal ganglia activity in awake rhesus monkeys.

Dysfunction and death of midbrain dopaminergic neurons underlies the clinical features of Parkinson's disease (PD). Increasing evidence suggests roles for oxidative stress and a form of cell death called apoptosis in the pathogenesis of PD. We recently identified a 38-kd protein called prostate apoptosis response-4 (Par-4), which is rapidly induced in cultured neurons after exposure to apoptotic insults, and appears to play a necessary role in the cell death process. We now report that Par-4 levels increase dramatically in midbrain dopaminergic neurons of monkeys and mice exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The increase in Par-4 levels occurs in both neuronal cell bodies in the substantia nigra and their axon terminals in the striatum, and precedes loss of tyrosine hydroxylase immunoreactivity and cell death. In the monkey model, Par-4 levels were also increased in several brain regions (red nucleus, lateral geniculate nucleus, and cerebral cortex) in which functional alterations have previously been documented in PD patients and MPTP-treated monkeys. Exposure of cultured human dopaminergic neural cells to the complex I inhibitor rotenone, or to Fe2+, resulted in Par-4 induction, mitochondrial dysfunction, and subsequent apoptosis. Blockade of Par-4 induction by antisense treatment prevented rotenone- and Fe2+-induced mitochondrial dysfunction and apoptosis demonstrating a critical role for Par-4 in the cell death process. The data suggest that Par-4 may be involved in the neurodegenerative process in PD.

As part of our studies of age-associated changes in motor functions, we have designed an automated movement assessment panel (MAP) to evaluate upper limb and hand movements. Here we describe two versions of the MAP, one for human testing and one for nonhuman primates, and methods for conducting parallel tests in rhesus monkeys and human volunteers. The results are reported from a battery of tests on young adult rhesus monkeys (n = 10, 5-8 years old), young adult human subjects (n = 10, 18-22 years old) and ten aged human subjects (n = 10, 66-68 years old) to demonstrate the capability of the MAP in quantifying arm and hand movement times. The performance times on the two simplest tasks tested were consistent from trial to trial, demonstrating that a stable behavioral baseline could be established for evaluating changes in motor functions over time and assessing treatments for improving motor functions. Motor learning was seen in the more complex movement tasks tested, indicating their usefulness in analyzing this behavior. Finally, age-associated changes in performance times were robustly delineated by the four tasks evaluated in the human subjects.

Functional MRI (fMRI) was used to study striatal sensitivity to levodopa in hemiparkinsonian rhesus monkeys. Responses consistent with increased neuronal activity were seen in areas whose normal dopaminergic input from the substantia nigra pars compacta had been ablated by MPTP. Sites of increased activity following levodopa included the lateral putamen, the ventral region of the caudate head, septal areas, and midlateral amygdala in the MPTP-lesioned hemisphere. Increased activity was also observed in the same areas in the nonlesioned hemisphere, but was less pronounced in spatial extent and magnitude, suggesting either subclinical contralateral damage and/or functional adaptations in the contralateral dopamine systems. The increases in neuronal activity following levodopa treatment were temporally correlated with increases in striatal dopamine levels. Chronic levodopa treatment reduced behavioral responsiveness to levodopa and abolished the fMRI response. These results suggest that fMRI can detect changes in dopamine receptor-mediated neuronal sensitivity to dopaminergic agents.

Principal component analysis (PCA) is one of several structure-seeking multivariate statistical techniques, exploratory as well as inferential, that have been proposed recently for the characterization and detection of activation in both PET and fMRI time series data. In particular, PCA is data driven and does not assume that the neural or hemodynamic response reaches some steady state, nor does it involve correlation with any pre-defined or exogenous experimental design template. In this paper, we present a generalized linear systems framework for PCA based on the singular value decomposition (SVD) model for representation of spatio-temporal fMRI data sets. Statistical inference procedures for PCA, including point and interval estimation will be introduced without the constraint of explicit hypotheses about specific task-dependent effects. The principal eigenvectors capture both the spatial and temporal aspects of fMRI data in a progressive fashion; they are inherently matched to unique and uncorrelated features and are ranked in order of the amount of variance explained. PCA also acts as a variation reduction technique, relegating most of the random noise to the trailing components while collecting systematic structure into the leading ones. Features summarizing variability may not directly be those that are the most useful. Further analysis is facilitated through linear subspace methods involving PC rotation and strategies of projection pursuit utilizing a reduced, lower-dimensional natural basis representation that retains most of the information. These properties will be illustrated in the setting of dynamic time-series response data from fMRI experiments involving pharmacological stimulation of the dopaminergic nigro-striatal system in primates.

Multispectral automated segmentation of MR images of the brains of 10 young (5-8 years), 10 middle-aged (12-17 years), and 11 old (21-27 years) female rhesus monkeys revealed age-associated changes in brain volume and composition. Total brain parenchymal volume (expressed as fraction of intracranial volume-%ICV) decreased at a linear rate of 0.3+/-0.04% ICV/year. Up to age approximately 15 years, this loss was almost entirely due to gray matter loss, with a compensatory increase in cerebrospinal fluid (CSF), and possibly some white matter. Brain tissue composition, expressed as the gray matter/white matter volume ratio confirmed that gray matter loss exceeded white matter loss, but the rate of decline in the gray/white ratio began to slow after approximately 15 years. Comparison of these age-associated changes in rhesus brain with those in humans suggest that the brain aging in rhesus is a good model of human brain aging, but occurs approximately 3-fold faster., (Copyright 1999 Elsevier Science B.V.)

Microdialysis measurements of dopamine (DA) and DA metabolites were carried out in the putamen and substantia nigra of unilateral 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned rhesus monkeys that received intraventricular injections of vehicle or glial-derived neurotrophic factor (GDNF, 300 microg) 3 weeks prior to the microdialysis studies. Following behavioral measures in the MPTP-lesioned monkeys, they were anesthetized with isoflurane and placed in a stereotaxic apparatus. Magnetic resonance imaging (MRI)-guided sterile stereotaxic procedures were used for implantations of the microdialysis probes. Basal extracellular levels of DA and the DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were found to be decreased by >95% in the right putamen of the MPTP-lesioned monkeys as compared to normal animals. In contrast, basal DA levels were not significantly decreased, and DOPAC and HVA levels were decreased by only 65% and 30%, respectively, in the MPTP-lesioned substantia nigra. Significant reductions in d-amphetamine-evoked DA release were also observed in the MPTP-lesioned substantia nigra and putamen of the monkeys as compared to normal animals. A single intraventricular administration of GDNF into one group of MPTP-lesioned monkeys elicited improvements in the parkinsonian symptoms in these animals at 2-3 weeks post-administration. In addition, d-amphetamine-evoked overflow of DA was significantly increased in the substantia nigra but not the putamen of MPTP-lesioned monkeys that had received GDNF. Moreover, post-mortem brain tissue studies showed increases in whole tissue levels of DA and DA metabolite levels primarily within the substantia nigra in MPTP-lesioned monkeys that had received GDNF. Taken together, these data support that single ventricular infusions of GDNF produce improvements in motoric behavior in MPTP-lesioned monkeys that correlate with increases in DA neuronal function that are localized to the substantia nigra and not the putamen., (Copyright 1999 Elsevier Science B.V.)

Dopamine neurons in the substantia nigra of the midbrain are the primary neuronal population affected by 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) toxicity, which produces the pathological and behavioral features of Parkinson's disease in nonhuman primates and man. We have identified another injury site in magnetic resonance imaging (MRI) brain scans in 13 of 37 rhesus monkeys taken 10-12 months after administration of this neurotoxin via the right carotid artery. Focal lesions, ranging in volume from 6.75 to 60 mm3 in the rostral globus pallidus region, were seen on the right side of the brain in these 13 animals in addition to the midbrain effects. While no significant differences were seen between globus pallidus lesioned and nonlesioned animals in the severity of MPTP-induced parkinsonian symptoms, the response to levodopa was muted in pallidal-lesioned animals. To confirm the role of neurotoxicity in producing the lesions, brain scans from an additional 12 monkeys were evaluated during the acute period following exposure to either MPTP (n = 6) or saline (n = 6). Focal lesions in the rostral globus pallidus were seen as early as 2-4 h following a carotid artery infusion in two of six MPTP recipients, but no evidence of injury was seen in saline recipients. The globus pallidus includes important components of the neural circuitry regulating motor functions. The present results indicate that in addition to midbrain dopamine neurons, a focal region of the rostral globus pallidus is selectively vulnerable to MPTP toxicity., (Copyright 1999 Academic Press.)

Considerable effort has been devoted to the search for molecules that might exert trophic influences on midbrain dopamine neurons, and potentially be of therapeutic value in the treatment of Parkinson's disease. One such candidate is glial cell line-derived neurotrophic factor (GDNF). GNDF is distantly related to the transforming growth factor-beta superfamily and is widely expressed in many neuronal and non-neuronal tissues. GDNF uses a multisubunit receptor system in which GFRalpha-1 and Ret function as the ligand-binding and signalling components, respectively. In addition to its effects on cultured fetal midbrain dopamine neurons, GDNF promotes recovery of the injured nigrostriatal dopamine system and improves motor functions in rodent and nonhuman primate models of Parkinson's disease. Intraventricular, intrastriatal and intranigral routes of administration are efficacious in both models. In parkinsonian nonhuman primates, GDNF treatment improves bradykinesia, rigidity and postural instability. In this model, adult midbrain dopamine neurons stimulated by GDNF show increased cell size, neuritic extent, and expression of phenotypic markers. The neurorestorative effects of a single administration of GDNF last for at least a month and can be maintained in rhesus monkeys by monthly injections. GDNF also induces neuroprotective changes in dopamine neurons, which are active within hours following trophic factor administration in rodents. The powerful neuroprotective and neurorestorative properties of GDNF seen in preclinical studies suggest that trophic factors may play an important role in treating Parkinson's disease.

The present study determined the topographical distribution profile for [125I]-glial cell line-derived neurotrophic factor in unlesioned and MPTP-lesioned (unilateral intracarotid injection) rhesus monkeys following an intraventricular injection. Autoradiographic analysis showed that following a bolus intraventricular injection, there was widespread distribution of [125I]-glial cell line-derived neurotrophic factor throughout the ventricular system (walls of lateral, third, and fourth ventricles and aqueduct), with some accumulation at the lateral ventricle injection site, possibly associated with the ependymal cell layer. In both unlesioned and MPTP-lesioned monkeys, there was labelling of the cerebral cortex, substantia nigra/ventral tegmental area and sequestration of [125I]-glial cell line-derived neurotrophic factor adjacent to the hippocampal formation, globus pallidus, ventral to and in the substantia nigra. However, [125I]-glial cell line-derived neurotrophic factor did not appear to diffuse readily or accumulate in the caudate-putamen even though there was some penetration away from the ventricular walls. Throughout the brain, there was also substantial non-parenchymal labelling of [125I]-glial cell line-derived neurotrophic factor, possibly associated with extracellular matrix components, meninges and vasculature due to the heparin binding properties of glial cell line-derived neurotrophic factor. In addition to the extensive loss of tyrosine hydroxylase immunoreactivity within the substantia nigra, there was also decreased accumulation of [125I]-glial cell line-derived neurotrophic factor and reduced glial cell line-derived neurotrophic factor immunoreactivity ipsilateral to the lesion. Microscopic analysis showed that glial cell line-derived neurotrophic factor immunoreactivity was associated with upper cortical layers including a high density of immunoreactivity at the surface of the cortex (meningeal, pial layer, vasculature) and around the ventricular walls (with some cellular labelling and labelling of vasculature). Moderate staining was observed in nigral cells contralateral to the MPTP-lesion, whereas only minimal levels of that glial cell line-derived neurotrophic factor immunoreactivity were detected ipsilateral to the lesion. This study shows that intraventricularly injected glial cell line-derived neurotrophic factor accumulates not only around the ventricular walls, but also in specific brain regions in which sub-populations of cells are more readily accessible than others. The presence of cells labelled with [125I] and immunopositive for glial cell line-derived neurotrophic factor in the substantia nigra indicates that these cells are a target for the trophic factor following intraventricular administration. Thus, the behavioral improvement observed in MPTP-lesioned monkeys following an intraventricular injection of glial cell line-derived neurotrophic factor is likely the result of activation of nigral cells.

Glial cell line-derived neurotrophic factor (GDNF) injected intranigrally protects midbrain dopamine neurons against 6-hydroxydopamine (6-OHDA) toxicity. The timing between GDNF administration and exposure to 6-OHDA is critical in achieving optimal protection. When injected 6 hr before an intranigral injection of 6-OHDA, GDNF provides complete protection as measured by the number of surviving neurons in the substantia nigra of adult rats. The surviving neuronal population decreases by approximately 50% with 12 and 24 hr separating GDNF and 6-OHDA administrations. In controls with 6-OHDA lesions, there is <10% survival of nigral dopamine neurons. No significant increase in survival is seen with either concurrent injections of GDNF and 6-OHDA or 1 hr GDNF pretreatment. Based on HPLC measurements, striatal and midbrain dopamine levels are at least twofold higher on the lesioned side in animals receiving GDNF 6 hr before a 6-OHDA lesion compared with vehicle recipients. Protein synthesis is necessary for GDNF-induced neuroprotective effects because cycloheximide pretreatment that inhibits protein synthesis also blocks neuroprotection.