In-vitro and in-vivo performance studies of a porous infusion catheter designed for intraparenchymal delivery of therapeutic agents of varying size.

BACKGROUND: Limitations have previously existed for the use of brain infusion catheters with extended delivery port designs to achieve larger distribution volumes using convection-enhanced delivery (CED), due to poor transmittance of materials and uncontrolled backflow. The goal of this study was to evaluate a novel brain catheter that has been designed to allow for extended delivery and larger distribution volumes with limited backflow of fluid. It was characterized using a broad range of therapeutic pore sizes both for transmittance across the membranes to address possible occlusion and for distribution in short term infusion studies, both in-vitro in gels and in-vivo in canines. METHODS: Brain catheters with pore sizes of 10, 12, 15, 20 and 30 µm were evaluated using three infusates prepared in 0.9% sterile saline with diameters approximating 2, 5, and 30 nm, respectively. Magnevist™ was chosen as the small molecule infusate to mimic low-molecular weight therapeutics. Galbumin™ served as a surrogate for an assortment of proteins used for brain cancer and Parkinson's disease. Gadoluminate™ was used to assess the distribution of large therapeutics, such as adeno-associated viral particles and synthetic nanoparticles. The transmittance of the medium and large tracer particles through catheters of different pore size (15, 20 and 30 µm) was measured by MRI and compared with the measured concentration of the control. Infusions into 0.2% agarose gels were performed in order to evaluate differences in transmittance and distribution of the small, medium, and large tracer particles through catheters with different pore sizes (10, 12, 15, 20 and 30 µm). In-vivo infusions were performed in the canine in order to evaluate the ability of the catheter to infuse the small, medium, and large tracer particles into brain parenchyma at high flow rates through catheters with different pore sizes (10, 15, and 20 µm). Two catheters were stereotactically inserted into the brain for infusion, one per hemisphere, in each animal (N = 6). RESULTS: The transmittance of Galbumin and Gadoluminate across the catheter membrane surface was 100% to within the accuracy of the measurements. There was no evidence of any blockage or retardation of any of the infusates. Catheter pore size did not appear to significantly affect transmittance or distribution in gels of any of the molecule sizes in the range of catheter pore sizes tested. There were differences in the distributions between the different tracer molecules: Magnevist produced relatively large distributions, followed by Gadoluminate and Galbumin. We observed no instances of uncontrolled backflow in a total of 12 in-vivo infusions. In addition, several of the infusions resulted in substantial amounts remaining in tissue. We expect the in-tissue distributions to be substantially improved in the larger human brain. COMPARISON WITH EXISTING METHODS: The new porous brain catheter performed well in terms of both backflow and intraparenchymal infusion of molecules of varying size in the canine brain under CED flow conditions. CONCLUSIONS: Overall, the data presented in this report support that the novel porous brain catheter can deliver therapeutics of varying sizes at high infusion rates in the brain parenchyma, and resist backflow that can compromise the efficacy of CED therapy. Additional work is needed to further characterize the brain catheter, including animal toxicity studies of chronically implanted brain catheters to lay the foundation for its use in the clinic.

Surgical methodology and protocols for preventing implanted cerebral catheters from becoming obstructed during and after neurosurgery.

BACKGROUND: Convection Enhanced Delivery (CED) into targeted brain areas has been tested in animal models and clinical trials for the treatment of various neurological diseases. NEW METHOD: We used a series of techniques, to in effect, maintain positive pressure inside the catheter relative to the outside, that included a hollow stylet, a high volume bolus of solution to clear the line, a low and slow continuous flow rate during implantation, and heat sealing the catheter at the time of implantation. RESULTS: 120 catheters implanted into brain parenchyma of 89 adult female rhesus monkeys across four sets of experiments. After experiencing a high delivery failure rate - non patent catheters - (19 %) because of tissue entrapment and debris and/or blood clots in the catheter tip, we developed modifications, including increasing the bolus infusion volume from 10 to 20 µl such that by the third experiment, the failure rate was 8 % (1 of 12 implants). Increasing the bolus volume to 100 µl and maintaining positive pressure in the catheter during preparation and implantation yielded a failure rate of 0 % (0/12 implants) by the fourth experiment. COMPARISON WITH EXISTING METHODS: We provide a retrospective analysis to reveal how several different manipulations affect catheter patency and how post-op MRI examination is essential for assessing catheter patency in situ. CONCLUSIONS: The results of the present study identified that the main cause of the catheter blockages were clots that rendered the catheter non-patent. We resolved this by modifying the surgical procedures that prevented these clots from forming.

Costs of Implementing Quality in Research Practice.

Using standardized guidelines in preclinical research has received increased interest in light of recent concerns about transparency in data reporting and apparent variation in data quality, as evidenced by irreproducibility of results. Although the costs associated with supporting quality through a quality management system are often obvious line items in laboratory budgets, the treatment of the costs associated with quality failure is often overlooked and difficult to quantify. Thus, general estimations of quality costs can be misleading and inaccurate, effectively undervaluing costs recovered by reducing quality defects. Here, we provide examples of quality costs in preclinical research and describe how we have addressed misconceptions of quality management implementation as only marginally beneficial and/or unduly burdensome. We provide two examples of implementing a quality management system (QMS) in preclinical experimental (animal) research environments - one in Europe, the German Mouse Clinic, having established ISO 9001 and the other in the United States, the University of Kentucky (UK), having established Good Laboratory Practice-compliant infrastructure. We present a summary of benefits to having an effective QMS, as may be useful in guiding discussions with funders or administrators to promote interest and investment in a QMS, which ultimately supports shared, mutually beneficial outcomes.

The effects of huntingtin-lowering: what do we know so far?

Therapies targeting mutant huntingtin DNA, mRNA, and protein have a chance at becoming the first disease-modifying treatments for Huntington's disease, a fatal inherited neurodegenerative disorder for which only symptom management treatments are available today. This review focuses on evidence addressing several key questions pertinent to huntingtin-lowering, ranging from the functions of wild-type huntingtin (wtHTT) that may be disrupted by huntingtin-lowering treatments through the various ways huntingtin can be lowered, the tolerability of wtHTT-lowering in mice and primates, what has been found in the Ionis Pharmaceutical safety trial of a huntingtin-lowering therapy, and to the question of how much mutant huntingtin may need to be lowered for a therapy to be clinically effective. We conclude that adverse consequences of lowering wtHTT in animals appear to be brain region-specific, and/or dependent upon the animal's stage of development and the amount by which huntingtin is lowered. Therefore, safe approaches to huntingtin-lowering in patients may be to lower huntingtin only moderately, or lower huntingtin only in the most affected brain regions, or lower huntingtin allele-selectively, or all of the above. Many additional questions about huntingtin-lowering remain open, and will only be answered by upcoming clinical trials, such as whether the delivery approaches currently planned will be adequate to get the treatment to the necessary brain regions, and whether non-allele-selective huntingtin-lowering will be safe in the long run. Meantime, there is a role for preclinical research to address key knowledge gaps, including the effects of non-allele-selective huntingtin-lowering on protein trafficking and viability at the cellular level, the tolerability of wtHTT-lowering in the corticostriatal connections of the primate brain, and the effects of this lowering on the functioning of neurotransmitter systems and the transport of neurotrophic factors to the striatum.

GDNF revisited: A novel mammalian cell-derived variant form of GDNF increases dopamine turnover and improves brain biodistribution.

Parkinson's disease (PD) is a disorder affecting dopamine neurons for which there is no cure. Glial cell line-derived neurotrophic factor (GDNF) and the closely related protein neurturin are two trophic factors with demonstrated neuroprotective and neurorestorative properties on dopamine neurons in multiple animal species. However, GDNF and neurturin Phase-2 clinical trials have failed to demonstrate a significant level of improvement over placebo controls. Insufficient drug distribution in the brain parenchyma has been proposed as a major contributing factor for the lack of clinical efficacy in the Phase-2 trial patients. To address this issue, a novel mammalian cell-derived variant form of GDNF (GDNFv) was designed to promote better tissue distribution by reducing its heparin binding to the extracellular matrix and key amino acids were substituted to enhance its chemical stability. Administration of this fully glycosylated GDNFv in the normal rat striatum increased dopamine turnover and produced significantly greater brain distribution than E. coli-produced wildtype GDNF (GDNFwt). Intrastriatal GDNFv also protected midbrain dopamine neuron function in 6-hydroxydopamine-lesioned rats. Studies conducted in normal adult rhesus macaques support that GDNFv was well tolerated in all animals and demonstrated a greater volume of distribution than GDNFwt in the brain following intrastriatal infusion. Importantly, favorable physiological activity of potential therapeutic value was maintained in this variant trophic factor with significant target activation in GDNFv recipients as indicated by dopamine turnover modulation. These data suggest that GDNFv may be a promising drug candidate for the treatment of PD. Additional studies are needed in non-human primates with dopamine depletion. This article is part of the Special Issue entitled 'Drug Repurposing: old molecules, new ways to fast track drug discovery and development for CNS disorders'.

Objectively measuring effects of electro-acupuncture in parkinsonian rhesus monkeys.

Acupuncture has increasingly been used as an alternative therapy for treatment of Parkinson's disease (PD). However, the efficacy of acupunture for PD still remains unclear. The present study was designed to objectively and safely monitor anti-parkinsonian effects of electroacupuncture (EA) and brain activity in nonhuman primates modeling human PD. Six middle-aged rhesus monkeys were extensively studied by a computerized behavioral testing battery and by pharmacological MRI (phMRI) scans with specific dopaminergic drug stimulations. All animals were evaluated for behavior and phMRI responses under normal, parkinsonian, parkinsonian with EA treatment and parkinsonian after EA treatment conditions. Stable parkinsonian features were observed in all animals prior to entering the EA study and positive responses to levodopa (L-dopa) challenge were also seen in all animals. The results demonstrated that chronic EA treatments could significantly improve the movement speed and the fine motor performance time during the period of EA treatments, and the effectiveness of EA could be detected even 3 months after the EA treatment. The phMRI data revealed that chronic EA treatments could alter neuronal activity in the striatum, primary motor cortex (M1), cingulate gyrus and global pallidus externa (GPe) in the ipsilateral hemisphere to MPTP lesions. As seen in the changes of parkinsonian features, the residual effects of phMRI responses to apomorphine (APO) challenge could also be found in the aforementioned areas. The results strongly suggest that anti-parkinsonian effects of EA can be objectively assessed, and the method used in the present study could be translated into the human clinic with some minor modifications.

Continuous intranigral infusion is not associated with observable behavioral deficits or marked pathology: a preclinical safety study.

OBJECTIVE A better understanding of the effects of chronically delivering compounds to the substantia nigra and nearby areas is important for the development of new therapeutic approaches to treat alpha-synucleinopathies, like Parkinson's disease. Whether chronic intranigral delivery of an infusate could be achieved without causing motor dysfunction or marked pathology remains unclear. The authors evaluated the tolerability of continuously delivering an infusate directly into the rhesus monkey substantia nigra via a programmable pump coupled to a novel intraparenchymal needle-tip catheter surgically implanted using MRI-guided techniques. METHODS The MRI contrast agent gadopentetate dimeglumine (Magnevist, 5 mM) was used to noninvasively evaluate catheter patency and infusion volume associated with 2 flow rates sequentially tested in each of 3 animals: 0.1 µl/min for 14 days into the right substantia nigra and 0.1 µl/min for 7 days plus 0.2 µl/min for an additional 7 days into the left substantia nigra. Flow rate tolerability was assessed via clinical observations and a microscopic examination of the striatum and midbrain regions. RESULTS Evaluation of postsurgical MRI indicated that all 6 catheters remained patent throughout the study and that the volume of distribution achieved in the left midbrain region at a rate of up to 0.2 µl/min (2052 ± 168 mm3) was greater than that achieved in the right midbrain region at a constant rate of 0.1 µl/min (1225 ± 273 mm3) by nearly 2-fold. Both flow rates provided sufficient infusate coverage of the rhesus (and possibly the human) midbrain region. There were no indications of observable deficits in behavior. Histopathological evaluations confirmed that all catheter tips were placed in or near the pars compacta region of the substantia nigra in all animals. There was no evidence of infection at any of the 6 catheter sites. Mild to moderate microglial reactions were observed at most catheter track sites and were comparable between the 2 infusion rates. Finally, there was neither observable decrease of tyrosine hydroxylase staining in the striatum nor detectable necrosis of neurons in the pars compacta region of the substantia nigra in any of the animals. CONCLUSIONS The data from this study support the feasibility of using a pump-and-catheter system for chronic intranigral infusion and lay the foundation for using this approach to treat Parkinson's disease or other related degenerative diseases that would benefit from targeted drug delivery to the substantia nigra or to other brainstem regions.

Onset Time and Durability of Huntingtin Suppression in Rhesus Putamen After Direct Infusion of Antihuntingtin siRNA.

One possible treatment for Huntington's disease involves direct infusion of a small, interfering RNA (siRNA) designed to reduce huntingtin expression into brain tissue from a chronically implanted programmable pump. Here, we studied the suppression of huntingtin mRNA achievable with short infusion times, and investigated how long suppression may persist after infusion ceases. Rhesus monkeys received 3 days of infusion of Magnevist into the putamen to confirm catheter patency and fluid distribution. After a 1-week washout period, monkeys received radiolabeled siRNA targeting huntingtin. After 1 or 3 days of siRNA delivery, monkeys were either terminated, or their pumps were shut off and they were terminated 10 or 24 days later. Results indicate that the onset of huntingtin mRNA suppression in the rhesus putamen occurs rapidly, achieving a plateau throughout the putamen within 4 days. Conversely, loss of huntingtin suppression progresses slowly, persisting an estimated 27-39 days in the putamen and surrounding white matter. These findings indicate the rapid onset and durability of siRNA-mediated target gene suppression observed in other organs also occurs in the brain, and support the use of episodic delivery of siRNA into the brain for treatment of Huntington's disease and possibly other neurodegenerative diseases.

Decreased mitochondrial bioenergetics and calcium buffering capacity in the basal ganglia correlates with motor deficits in a nonhuman primate model of aging.

Altered mitochondrial function in the basal ganglia has been hypothesized to underlie cellular senescence and promote age-related motor decline. We tested this hypothesis in a nonhuman primate model of human aging. Six young (6-8 years old) and 6 aged (20-25 years old) female Rhesus monkeys (Macaca mulatta) were behaviorally characterized from standardized video records. Additionally, we measured mitochondrial bioenergetics along with calcium buffering capacity in the substantia nigra and putamen (PUT) from both age groups. Our results demonstrate that the aged animals had significantly reduced locomotor activity and movement speed compared with younger animals. Moreover, aged monkeys had significantly reduced ATP synthesis capacity (in substantia nigra and PUT), reduced pyruvate dehydrogenase activity (in PUT), and reduced calcium buffering capacity (in PUT) compared with younger animals. Furthermore, this age-related decline in mitochondrial function in the basal ganglia correlated with decline in motor function. Overall, our results suggest that drug therapies designed to enhance altered mitochondrial function may help improve motor deficits in the elderly.

Pharmacologic MRI (phMRI) as a tool to differentiate Parkinson's disease-related from age-related changes in basal ganglia function.

The prevalence of both parkinsonian signs and Parkinson's disease (PD) per se increases with age. Although the pathophysiology of PD has been studied extensively, less is known about the functional changes taking place in the basal ganglia circuitry with age. To specifically address this issue, 3 groups of rhesus macaques were studied: normal middle-aged animals (used as controls), middle-aged animals with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism, and aged animals (>20 years old) with declines in motor function. All animals underwent the same behavioral and pharmacologic magnetic resonance imaging (phMRI) procedures to measure changes in basal ganglia function in response to dopaminergic drug challenges consisting of apomorphine administration followed by either a D1 (SCH23390) or a D2 (raclopride) receptor antagonist. Significant functional changes were predominantly seen in the external segment of the globus pallidus (GPe) in aged animals and in the striatum (caudate nucleus and putamen) in MPTP-lesioned animals. Despite significant differences seen in the putamen and GPe between MPTP-lesioned versus aged animals, a similar response profile to dopaminergic stimulations was found between these 2 groups in the internal segment of the GP. In contrast, the pharmacologic responses seen in the control animals were much milder compared with the other 2 groups in all the examined areas. Our phMRI findings in MPTP-lesioned parkinsonian and aged animals suggest that changes in basal ganglia function in the elderly may differ from those seen in parkinsonian patients and that phMRI could be used to distinguish PD from other age-associated functional alterations in the brain.

Reversible cognitive, motor, and driving impairments in severe hypothyroidism.

BACKGROUND: Hypothyroidism has been associated with cognitive and motor impairments that are likely to constitute hazards in the operation of motor vehicles and a public safety risk. However, there is a paucity of data that would provide an evidence basis for recommendations to hypothyroid patients. The purpose of this study was to determine the specific neurological and psychological deficits consequent to hypothyroidism and whether they are of sufficient magnitude to impede the safe operation of motor vehicles. METHODS: Repeated measurements were obtained in euthyroid, hypothyroid, and euthyroid hormone replaced states of thyroid cancer outpatients, at an academic medical center, who underwent thyroid hormone withdrawal preparation for radioiodine scanning. Study design used a within-subjects longitudinal "A-B-A" with each subject tested at three visits in the same sequence: euthyroid, hypothyroid, and euthyroid for a total of 32 subjects. Data on clinical status and cognitive performance were collected using standard instruments, including ThyDQoL and ThySRQ measures, National Adult Reading Test, Boston Naming Test, Mini-Mental State Exam, Wechsler Adult Intelligence Test-Revised, Letter Fluency FAS, and Beck Depression Inventory. Fine-motor function was measured with an automated assessment panel, and driving performance on a commercial driving simulator. RESULTS: In severe hypothyroidism (median thyrotropin 83.2 mIU/L), fine-motor performance of hands and reaction times in emergency braking tests were slowed, as well as subjective slowing reported on structured clinical scales. Depression was present, typified by vegetative and mood alterations, but lacking reported guilt and lowered self-esteem seen in other types of depression. Cognitive impairment was characterized by declines on speeded executive tests. In contrast, episodic memory performance improved over time regardless of thyroid hormone status. Braking times increased in hypothyroidism by 8.5%, equivalent to reports of effects from a blood alcohol level of 0.082 g/100 mL (above the U.S. legal driving limit). CONCLUSIONS: Transient profound hypothyroidism is characterized by reversible depression, decreased fine-motor performance, slowed reaction times, and decreased processing speed. These data represent new empirical evidence that support the recommendation that complex activities requiring rapid responses, such as operating motor vehicles, should be avoided during hypothyroidism. This has broader implications regarding functional impairments and risk to public health.

Six-month partial suppression of Huntingtin is well tolerated in the adult rhesus striatum.

Huntington's disease is caused by expression of a mutant form of Huntingtin protein containing an expanded polyglutamine repeat. One possible treatment for Huntington's disease may be to reduce expression of mutant Huntingtin in the brain via RNA interference. Unless the therapeutic molecule is designed to be allele-specific, both wild-type and mutant protein will be suppressed by an RNA interference treatment. A key question is whether suppression of wild-type as well as mutant Huntingtin in targeted brain regions can be tolerated and result in a net benefit to patients with Huntington's disease. Whether Huntingtin performs essential functions in the adult brain is unclear. Here, we tested the hypothesis that the adult primate brain can tolerate moderately reduced levels of wild-type Huntingtin protein for an extended period of time. A serotype 2 adeno-associated viral vector encoding for a short hairpin RNA targeting rhesus huntingtin messenger RNA (active vector) was bilaterally injected into the striatum of four adult rhesus monkeys. Four additional animals received a comparable vector encoding a scrambled control short hairpin RNA (control vector). General health and motor behaviour were monitored for 6 months. Upon termination, brain tissues were sampled and assessed blindly for (i) huntingtin messenger RNA knockdown; (ii) Huntingtin protein expression; and (iii) neuropathological changes. Reduction in wild-type huntingtin messenger RNA levels averaging ∼30% was measured in the striatum of active vector recipients 6 months post-injection. A widespread reduction in Huntingtin protein levels was also observed by immunohistochemistry in these animals, with an average protein reduction of ∼45% relative to controls measured by western blot analysis in the putamen of active vector recipients. As with control vector recipients, no adverse effects were observed behaviourally, and no neurodegeneration was found on histological examination of active vector recipients. Our results suggest that long-term partial suppression of wild-type Huntingtin may be safe, and thus if a comparable level of suppression of mutant Huntingtin is beneficial, then partial suppression of both wild-type and mutant Huntingtin may result in a net benefit in patients with heterozygous Huntington's disease.

Widespread suppression of huntingtin with convection-enhanced delivery of siRNA.

Huntington's disease is an autosomal dominant neurodegenerative disease caused by a toxic gain of function mutation in the huntingtin gene (Htt). Silencing of Htt with RNA interference using direct CNS delivery in rodent models of Huntington's disease has been shown to reduce pathology and promote neuronal recovery. A key translational step for this approach is extension to the larger non-human primate brain, achieving sufficient distribution of small interfering RNA targeting Htt (siHtt) and levels of Htt suppression that may have therapeutic benefit. We evaluated the potential for convection enhanced delivery (CED) of siHtt to provide widespread and robust suppression of Htt in nonhuman primates. siHtt was infused continuously for 7 or 28 days into the nonhuman primate putamen to analyze effects of infusion rate and drug concentration on the volume of effective suppression. Distribution of radiolabeled siHtt and Htt suppression were quantified by autoradiography and PCR, respectively, in tissue punches. Histopathology was evaluated and Htt suppression was also visualized in animals treated for 28 days. Seven days of CED led to widespread distribution of siHtt and significant Htt silencing throughout the nonhuman primate striatum in an infusion rate and dose dependent manner. Htt suppression at therapeutic dose levels was well tolerated by the brain. A model developed from these results predicts that continuous CED of siHtt can achieve significant coverage of the striatum of Huntington's disease patients. These findings suggest that this approach may provide an important therapeutic strategy for treating Huntington's disease.

Improved methods for electroacupuncture and electromyographic recordings in normal and parkinsonian rhesus monkeys.

Although acupuncture has been widely and routinely used in healthcare in the USA, its use has been based more on empirical observation than on scientific knowledge. Therefore, there is a great need for better understanding the underlying mechanism(s) of action. A great body of evidence supports that nonhuman primates are a candidate for studying human diseases. However, the use of nonhuman primates in neurophysiological, neuroimaging and neurochemical studies is extremely challenging, especially under fully conscious, alert conditions. In the present study, we developed a protocol for safely performing acupuncture, electroacupuncture (EA) and electromyography (EMG) in both normal nonhuman primates and animals with parkinsonian-like symptoms. Four normal and four hemiparkinsonian middle-aged rhesus monkeys were extensively trained, behaviorally monitored, and received both EA and EMG for several months. The results demonstrated that (1) all rhesus monkeys used in the study could be trained for procedures including EA and EMG; (2) all animals tolerated the procedures involving needle/electrode insertion; (3) EA procedures used in the study did not adversely alter the animal's locomotor activities; rather, MPTP-treated animals showed a significant improvement in movement speed; and (4) EMG detected significant differences in muscle activity between the arms with and without MPTP-induced rigidity. Our results support that rhesus monkeys can be used as an experimental animal model to study EA and that EMG has the potential to be used to objectively assess the effects of antiparkinsonian therapies. The results also indicate that animals, especially those with parkinsonian-like symptoms, could benefit from long-term EA stimulations.

Aging-related gene expression in hippocampus proper compared with dentate gyrus is selectively associated with metabolic syndrome variables in rhesus monkeys.

Age-dependent metabolic syndrome (MetS) is a well established risk factor for cardiovascular disease, but it also confers major risk for impaired cognition in normal aging or Alzheimer's disease (AD). However, little is known about the specific pathways mediating MetS-brain interactions. Here, we performed the first studies quantitatively linking MetS variables to aging changes in brain genome-wide expression and mitochondrial function. In six young adult and six aging female rhesus monkeys, we analyzed gene expression in two major hippocampal subdivisions critical for memory/cognitive function [hippocampus proper, or cornu ammonis (CA), and dentate gyrus (DG)]. Genes that changed with aging [aging-related genes (ARGs)] were identified in each region. Serum variables reflecting insulin resistance and dyslipidemia were used to construct a quantitative MetS index (MSI). This MSI increased with age and correlated negatively with hippocampal mitochondrial function (state III oxidation). More than 2000 ARGs were identified in CA and/or DG, in approximately equal numbers, but substantially more ARGs in CA than in DG were correlated selectively with the MSI. Pathways represented by MSI-correlated ARGs were determined from the Gene Ontology Database and literature. In particular, upregulated CA ARGs representing glucocorticoid receptor (GR), chromatin assembly/histone acetyltransferase, and inflammatory/immune pathways were closely associated with the MSI. These results suggest a novel model in which MetS is associated with upregulation of hippocampal GR-dependent transcription and epigenetic coactivators, contributing to decreased mitochondrial function and brain energetic dysregulation. In turn, these MSI-associated neuroenergetic changes may promote inflammation, neuronal vulnerability, and risk of cognitive impairment/AD.

Dopamine neuron stimulating actions of a GDNF propeptide.

BACKGROUND: Neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), have shown great promise for protection and restoration of damaged or dying dopamine neurons in animal models and in some Parkinson's disease (PD) clinical trials. However, the delivery of neurotrophic factors to the brain is difficult due to their large size and poor bio-distribution. In addition, developing more efficacious trophic factors is hampered by the difficulty of synthesis and structural modification. Small molecules with neurotrophic actions that are easy to synthesize and modify to improve bioavailability are needed. METHODS AND FINDINGS: Here we present the neurobiological actions of dopamine neuron stimulating peptide-11 (DNSP-11), an 11-mer peptide from the proGDNF domain. In vitro, DNSP-11 supports the survival of fetal mesencephalic neurons, increasing both the number of surviving cells and neuritic outgrowth. In MN9D cells, DNSP-11 protects against dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA)-induced cell death, significantly decreasing TUNEL-positive cells and levels of caspase-3 activity. In vivo, a single injection of DNSP-11 into the normal adult rat substantia nigra is taken up rapidly into neurons and increases resting levels of dopamine and its metabolites for up to 28 days. Of particular note, DNSP-11 significantly improves apomorphine-induced rotational behavior, and increases dopamine and dopamine metabolite tissue levels in the substantia nigra in a rat model of PD. Unlike GDNF, DNSP-11 was found to block staurosporine- and gramicidin-induced cytotoxicity in nutrient-deprived dopaminergic B65 cells, and its neuroprotective effects included preventing the release of cytochrome c from mitochondria. CONCLUSIONS: Collectively, these data support that DNSP-11 exhibits potent neurotrophic actions analogous to GDNF, making it a viable candidate for a PD therapeutic. However, it likely signals through pathways that do not directly involve the GFRalpha1 receptor.

Pharmacological MRI (phMRI) monitoring of treatment in hemiparkinsonian rhesus monkeys.

There is a great need for the development of noninvasive, highly sensitive, and widely available imaging methods that can potentially be used to longitudinally monitor treatment of Parkinson's disease (PD). Here we report the monitoring of GDNF-induced functional changes of the basal ganglia in hemiparkinsonian monkeys via pharmacological MRI measuring the blood oxygenation level-dependent (BOLD) response to a direct dopamine agonist (apomorphine, APO). After testing BOLD responsiveness to APO in their normal state, two additional scans were taken with the same dose of APO stimulation after induced parkinsonism. Then all animals were chronically treated with GDNF for 18 weeks by a programmable pump and catheter system. The catheter was surgically implanted into the right putamen and connected to the pump via flexible polyurethane tubing, phMRI scans were taken at both 6 and 18 weeks while they received 22.5 microg of GDNF per day. In addition, behavioral changes were monitored throughout the entire study. The primary finding of this study was that APO-evoked activations in the DA denervated putamen were attenuated by the chronic intraputamenal infusion of GDNF accompanied by improvements of parkinsonian features, movement speed, and APO-induced rotation compared to data collected before the chronic GDNF treatment. The results suggest that phMRI methods in combination with administration of a selective DA agonist may be useful for monitoring neurorestorative therapies in PD patients in the future.

Development of a stable, early stage unilateral model of Parkinson's disease in middle-aged rhesus monkeys.

An important issue raised in testing new neuroprotective/restorative treatments for Parkinson's disease (PD) is the optimal stage in the disease process to initiate therapy. Current palliative treatments are effective in the early disease stages raising ethical concerns about substituting an experimental treatment for a proven therapy. Thus, we have endeavored to create a stable 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) nonhuman primate model of early PD. The new model was created by controlling for dose and route administration of MPTP (unilateral intracarotid infusion), and age of the animals (middleaged, 16-19 years old) in 27 female rhesus monkeys. All animals showed stable parkinsonian features lasting for up to 12-month as per behavioral evaluation. Compared with late-stage PD animals, postmortem analysis demonstrated that more dopaminergic neurons remained in the substantia nigra pars compacta, and more fibers were found in the striatum. In addition, tissue levels of striatal dopamine and its metabolites were also higher. Our results support that a milder but stable PD model can be produced in middle-aged rhesus monkeys.

Motor memory preservation in aged monkeys mirrors that of aged humans on a similar task.

We studied long-term motor memory preservation in rhesus monkeys tested on a task similar to that employed in humans. First, motor speed and rate of motor decline was measured in 23 animals ranging from 4 to 26 years old. The task for the animals consisted of removing a food reward from a curved rod within the inner chamber of an automated panel. Young animals performed twice as fast as the aged animals. Second, young (n=6) and aged (n=10) animals were re-tested 1 year later on the same task with no intervening practice. We anticipated a decline in motor speed of 144 ms/year, instead the average performance time recorded during the repeat session improved significantly by 17% in the aged animals. This finding mirrors that of a longitudinal study conducted in humans using a similar test panel and supports that, while initial performance times of a novel motor task decline with age, motor memory traces are preserved over an extended time interval, even without continued practice. The data also support that the rhesus monkey could be used as a model to study the mechanisms by which long-term retention of motor memory occurs in aging.

Amperometric measures of age-related changes in glutamate regulation in the cortex of rhesus monkeys.

l-glutamate (glutamate) is the principal excitatory neurotransmitter of the central nervous system and is involved in altered neural function during aging and in neurodegenerative diseases. Relatively little is known about the mechanisms of glutamate signaling in the primate brain, in part, because there is an absence of a method capable of rapidly measuring glutamate in either a non-clinical or a clinical setting. We have addressed this paucity of information by measuring extracellular glutamate at 1 Hz in the pre-motor and motor cortices of young, middle-aged, and aged monkeys using a minimally invasive amperometric recording method. In the motor cortex, mean resting glutamate levels were five times higher in the aged group compared to the young group while the pre-motor cortex showed an increasing trend in resting glutamate levels that was not statistically significant. In addition, we measured rapid, phasic glutamate release after local pressure-ejection of nanoliter volumes of either isotonic 70 mM potassium (to stimulate glutamate release) or 1 mM glutamate (to study glutamate uptake) into the pre-motor and motor cortex. In the pre-motor cortex, we measured reproducible glutamate uptake signals that had a significantly decreased (47%) rate of glutamate uptake in aged animals compared to young animals. However, following a 70 mM potassium delivery, we did not observe any consistent changes in evoked release between young versus aged animals. Using these non-clinical microelectrodes to measure glutamate signaling in the brain, our results support the hypothesis that the glutamatergic system undergoes reorganization with aging of the central nervous system.