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International audience; Due to the implication of docosahexaenoic acid (DHA) in neurogenesis, synaptogenesis, neurite outgrowth and to its high incorporation into the brain, this n-3 long chain polyunsaturated fatty acid (LCPUFA) is considered as crucial in the development and maintenance of the learning memory performance throughout life. In the present chapter we aimed at reviewing data investigating the relation between DHA and cognition during the perinatal period, young adult- and adulthood and neurodegenerative diseases such as Alzheimer disease (AD). In Humans, dietary DHA supplementation from the perinatal period to adulthood does not reveal a clear and consistent memory improvement whereas it is the case in animal studies. The positive effects observed in animal models may have been enhanced by using n-3 PUFA deficient animal models as controls. In animal models of AD, a general consensus on the beneficial effects of n-3 LCPUFA in attenuating cognitive impairment was established. These studies make DHA a potential suitable micronutrient for the maintenance of cognitive performance at all periods of life.

Aggregates of hyperphosphorylated tau protein are found in a group of diseases called tauopathies, which includes Alzheimer's disease. The causes and consequences of tau hyperphosphorylation are routinely investigated in laboratory animals. Mice are the models of choice as they are easily amenable to transgenic technology; consequently, their tau phosphorylation levels are frequently monitored by Western blotting using a panel of monoclonal/polyclonal anti-tau antibodies. Given that mouse secondary antibodies can recognize endogenous mouse immunoglobulins (Igs) and the possible lack of specificity with some polyclonal antibodies, non-specific signals are commonly observed. Here, we characterized the profiles of commonly used anti-tau antibodies in four different mouse models: non-transgenic mice, tau knock-out (TKO) mice, 3xTg-AD mice, and hypothermic mice, the latter a positive control for tau hyperphosphorylation. We identified 3 tau monoclonal antibody categories: type 1, characterized by high non-specificity (AT8, AT180, MC1, MC6, TG-3), type 2, demonstrating low non-specificity (AT270, CP13, CP27, Tau12, TG5), and type 3, with no non-specific signal (DA9, PHF-1, Tau1, Tau46). For polyclonal anti-tau antibodies, some displayed non-specificity (pS262, pS409) while others did not (pS199, pT205, pS396, pS404, pS422, A0024). With monoclonal antibodies, most of the interfering signal was due to endogenous Igs and could be eliminated by different techniques: i) using secondary antibodies designed to bind only non-denatured Igs, ii) preparation of a heat-stable fraction, iii) clearing Igs from the homogenates, and iv) using secondary antibodies that only bind the light chain of Igs. All of these techniques removed the non-specific signal; however, the first and the last methods were easier and more reliable. Overall, our study demonstrates a high risk of artefactual signal when performing Western blotting with routinely used anti-tau antibodies, and proposes several solutions to avoid non-specific results. We strongly recommend the use of negative (i.e., TKO) and positive (i.e., hypothermic) controls in all experiments.

We investigated the effect of MPTP-induced lesion of the substantia nigra pars compacta (SNpc) dopaminergic neurons on GABAB receptors in the basal ganglia of mice and monkeys using receptor autoradiography and in situ hybridization. The extent of the lesion was measured with striatal catecholamine content, striatal binding of 125I-RTI-121 to dopamine transporter (DAT), and DAT expression in the SNpc. GABAB receptors in mice brain were evaluated using 3H-CGP54626 and its expression was measured with oligonucleotides probes targeting the mRNAs of GABAB(1a+b), GABAB(1a), GABAB(1b), GABAB(2) subunits. In monkeys, 125I-CGP64213 and selective probes for GABAB(1a+b) and GABAB(2) mRNAs were used. In mice, dopamine content, 125I-RTI-121 binding, and DAT expression were reduced by 44%, 40%, and 39% after a dose of 40 mg/kg of MPTP and 74%, 70%, and 34% after 120 mg/kg of MPTP, respectively. In monkeys, dopamine content and DAT expression were decreased by more than 90% and 80%, respectively. In the striatum and the subthalamic nucleus, GABAB receptors were unchanged following MPTP in both species. In the SNpc of mice, MPTP (120 mg/kg) induced a significant decrease of 3H-CGP54626 binding (−10%) and of the expression of GABAB(1a+b) mRNA (−13%). The decrease of the expression of GABAB(1a+b) mRNA was correlated with dopamine content, 125I-RTI-121 binding and DAT expression. In MPTP-treated monkeys, 125I-CGP64213 binding (−40%), GABAB(1a+b) mRNA (−69%) and GABAB(2) mRNA (−66%) were also significantly decreased in the SNpc. Our results suggest that MPTP-induced denervation is associated with a decrease of GABAB receptors restricted to the SNpc. These observations may be relevant to the pathophysiology of motor disorders involving dysfunction of the basal ganglia such as Parkinson disease. Synapse 40:225–234, 2001. © 2001 Wiley-Liss, Inc.

The MPTP monkey is a well-characterized animal model of parkinsonism and provides an exceptional tool for the study of dyskinesias induced by dopamine-like agents. Several such agents have been tested during the past 15 years, and it has been found that the duration of action of these compounds is the most reliable variable with which to predict their dyskinesiogenic profile. It is proposed that L-dopa-induced dyskinesias represent a form of pathological learning caused by chronic pulsatile (nonphysiological) stimulation of dopamine receptors, which activates a cascade of molecular and biochemical events. These events include defective regulation of Fos proteins that belong to the deltaFosB family, increased expression of neuropeptides, and defective GABA- and glutamate-mediated neurotransmission in the output structures of the basal ganglia.

The effect of chronic treatment with the D2 dopamine agonist U91356A or L-DOPA therapy on the regulation of preproenkephalin (PPE) mRNA was investigated in the caudate-putamen of previously drug-naive cynomolgus monkeys Macaca fascicularis rendered parkinsonian by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In MPTP monkeys, pulsatile treatment with either L-DOPA or U91356A relieved parkinsonian symptoms but caused progressive sensitization to treatment and, as expected, induced choreic dyskinesias. In contrast, U91356A given in a continuous mode led to partial behavioral tolerance without appearance of dyskinesias. Using in situ hybridization histochemistry, lesioning was shown to produce elevation of PPE mRNA levels in the lateral and medial parts of the putamen and in the lateral part of the caudate nucleus compared to control animals at the three rostrocaudal regions analyzed. In general, no change of PPE mRNA levels were observed in the medial caudate after MPTP lesioning with or without L-DOPA or U91356A treatments in the three rostrocaudal regions measured except for an increase in the caudal part of L-DOPA-treated MPTP monkeys. In the putamen and lateral caudate nucleus, elevated PPE mRNA expression by MPTP generally was not corrected (or only partially corrected) by chronic L-DOPA treatment except for the rostral medial putamen where correction to control values was observed. In general, pulsatile administration of U91356A partially corrected the lesion-induced elevation of PPE mRNA levels in the putamen and lateral caudate nucleus whereas the correction was more pronounced and widespread when MPTP monkeys received the continuous administration of this drug. These results indicate that the mode of administration of a D2 dopamine receptor agonist, such as U91356A, although at a roughly equivalent dosage influences the extent of inhibition of the expression of PPE in the denervated striatum of monkeys. In addition, the general lack of correction of the MPTP-induced increase of PPE mRNA in the striatum of L-DOPA-treated monkeys compared to the decreases observed with the D2 agonist treatments suggest that the D1 agonist component of L-DOPA therapy opposes the D2 agonist activity. Hence, D1 receptor agonist activity would stimulate PPE mRNA expression whereas D2 receptor agonists inhibit the expression of this peptide. Increases in PPE expression in the striatum may be implicated in the induction of dyskinesias since both groups of treated MPTP monkeys displaying dyskinesias had elevated striatal PPE mRNA levels whereas the MPTP monkeys with the lowest striatal PPE mRNA levels developed tolerance without dyskinesias.

The effect of a chronic D2 dopamine receptor agonist (U91356A) treatment on dopamine receptor gene expression in the brain of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys was investigated using quantitative in situ hybridization histochemistry. U91356A was administered to MPTP-monkeys for 27 days in a pulsatile (n=3) or continuous (n=3) schedule. Animals treated in a pulsatile mode showed progressive sensitization and developed dyskinesia; whereas with the continuous mode behavioural tolerance was observed but no dyskinesia developed. Untreated MPTP as well as naive control animals were also studied. The efficacy and uniformity of the MPTP effect was assessed by measures of dopamine concentrations by high performance liquid chromatography with electrochemical detection in the relevant brain areas. D1 and D2 receptor messenger RNAs levels were examined by in situ hybridization histochemistry using human complementary RNA probes. Intense specific labelling for D1 and D2 receptor messenger RNAs was measured in the caudate and putamen with a rostrocaudal gradient for D2 receptors and a lower density in the cortex for D1 receptors messenger RNA. D1 receptor mRNA levels in rostral striatum and cortex decreased whereas D2 receptor messenger RNA in caudal striatum increased in MPTP-monkeys compared to control animals. Continuous administration of U91356A reversed the MPTP-induced increase of D2 receptor messenger RNA, whereas the pulsatile administration did not significantly correct these messenger RNA changes. U91356A treatment whether continuous or pulsatile partially corrected the D1 receptor messenger RNA lesion-induced decrease in the striatum, whereas no correction was observed in the cortex. All MPTP-monkeys were extensively and similarly denervated suggesting that the D1 and D2 receptor expression changes following U91356A administration were treatment related. Our data show a lesion-induced imbalance of D1 (decrease) and D2 (increase) receptor messenger RNAs in the striatum of MPTP-monkeys. The response of these receptors to D2 agonist treatment showed receptor selectivity and was influenced by the time-course of drug delivery. Hence chronic continuous but not pulsatile administration of U91356A reversed the striatal D2 receptor messenger RNA increase.

Essential tremor (ET) is the most prevalent adult-onset movement disorder. Despite its health burden, no clear pathognomonic sign has been identified to date because of the rarity of clinicopathological studies. Moreover, treatment options are still scarce and have not significantly changed in the last 30 years, underscoring the urgent need to develop new treatment avenues. In the recent years, leucine-rich repeat (LRR) and immunoglobulin (Ig) domain-containing Nogo receptor-interacting proteins 1 and 2 (LINGO1 and LINGO2, respectively) have been increasingly regarded as possible ET modulators due to emerging genetic association studies linking LINGO with ET. We have investigated LINGO protein and messenger RNA (mRNA) expression in the cerebellum of patients with ET, patients with Parkinson's disease (PD), and a control group using Western immunoblotting and in situ hybridization. Protein levels of LINGO1, but not LINGO2, were significantly increased in the cerebellar cortex of ET patients compared with controls, particularly in individuals with longer disease duration. Compared with controls, LINGO1 protein levels were increased in the cerebellar white matter of PD and ET patients but, for the latter, only when disease duration exceeded 20 years. However, no alteration in LINGO1 mRNA was observed between groups in either the cerebellar cortex or the white matter. We observed alterations in LINGO expression in diseased brain that seemed to progress along with the disease, being initiated in the cerebellar cortex before reaching the white matter. Because LINGO up-regulation has been identified as a potential pathological response to ongoing neurodegenerative processes, the present data suggest that LINGO1 is a potential drug target for ET.

Defects in p21-activated kinase (PAK) are suspected to play a role in cognitive symptoms of Alzheimer's disease (AD). Dysfunction in PAK leads to cofilin activation, drebrin displacement from its actin-binding site, actin depolymerization/severing, and, ultimately, defects in spine dynamics and cognitive impairment in mice. To determine the role of PAK in AD, we first quantified PAK by immunoblotting in homogenates from the parietal neocortex of subjects with a clinical diagnosis of no cognitive impairment (n = 12), mild cognitive impairment (n = 12), or AD (n = 12). A loss of total PAK, detected in the cortex of AD patients (-39% versus controls), was correlated with cognitive impairment (r(2) = 0.148, p = 0.027) and deposition of total and phosphorylated tau (r(2) = 0.235 and r(2) = 0.206, respectively), but not with Aβ42 (r(2) = 0.056). Accordingly, we found a decrease of total PAK in the cortex of 12- and 20-month-old 3xTg-AD mice, an animal model of AD-like Aβ and tau neuropathologies. To determine whether PAK dysfunction aggravates AD phenotype, 3xTg-AD mice were crossed with dominant-negative PAK mice. PAK inactivation led to obliteration of social recognition in old 3xTg-AD mice, which was associated with a decrease in cortical drebrin (-25%), but without enhancement of Aβ/tau pathology or any clear electrophysiological signature. Overall, our data suggest that PAK decrease is a consequence of AD neuropathology and that therapeutic activation of PAK may exert symptomatic benefits on high brain function.

We evaluated the integrity and function of the blood-brain barrier in 3xTg-AD mice aged 3-18 months and in APP/PS1 mice aged 8-months to determine the impacts of changes in amyloid and tau proteins on the brain vascular changes. The vascular volume (Vvasc) was sub-normal in 3xTg-AD mice aged from 6 to 18 months, but not in the APP/PS1 mice. The uptakes of [(3)H]-diazepam by the brains of 3xTg-AD, APP/PS1 and their age-matched control mice were similar at all the times studied, suggesting that the simple diffusion of small solutes is unchanged in transgenic animals. The uptake of d-glucose by the brains of 18-month old 3xTg-AD mice, but not by those of 8-month old APP/PS1 mice, was reduced compared to their age-matched controls. Accordingly, the amount of Glut-1 protein was 1.4 times lower in the brain capillaries of 18 month-old 3xTg-AD mice than in those of age-matched control mice. We conclude that the brain vascular volume is reduced early in 3xTg-AD mice, 6 months before the appearance of pathological lesions, and that this reduction persists until they are at least 18 months old. The absence of alterations in the BBB of APP/PS1 mice suggests that hyperphosphorylated tau proteins contribute to the vascular changes that occur in AD.

Intravenous immunoglobulin (IVIg) is currently evaluated in clinical trials for the treatment of various disorders of the central nervous system. To assess its capacity to reach central therapeutic targets, the brain bioavailability of IVIg must be determined. We thus quantified the passage of IVIg through the blood–brain barrier (BBB) of C57Bl/6 mice using complementary quantitative and qualitative methodologies. As determined by enzyme-linked immunosorbent assay, a small proportion of systemically injected IVIg was detected in the brain of mice (0.009±0.001% of injected dose in the cortex) whereas immunostaining revealed localization mainly within microvessels and less frequently in neurons. Pharmacokinetic analyses evidenced a low elimination rate constant (0.0053 per hour) in the cortex, consistent with accumulation within cerebral tissue. In situ cerebral perfusion experiments revealed that a fraction of IVIg crossed the BBB without causing leakage. A dose-dependent decrease of brain uptake was consistent with a saturable blood-to-brain transport mechanism. Finally, brain uptake of IVIg after a subchronic treatment was similar in the 3xTg-AD mouse model of Alzheimer disease compared with nontransgenic controls. In summary, our results provide evidence of BBB passage and bioavailability of IVIg into the brain in the absence of BBB leakage and in sufficient concentration to interact with the therapeutic targets.

Background Amyloid precursor protein (APP), a key molecule in Alzheimer’s disease (AD), is metabolized in two alternative cleavages, generating either the amyloidogenic peptides involved in AD pathology or the soluble form of APP (sAPPα). The level of amyloidogenic peptides in human cerebrospinal fluid (CSF) is considered to be a biomarker of AD, whereas the level of sAPPα in CSF as a biomarker has not been clearly established. sAPPα has neurotrophic and neuroprotective properties. Stimulating its formation and secretion is a promising therapeutic target in AD research. To this end, very sensitive tests for preclinical and clinical research are required. Methods The tests are based on homogenous time-resolved fluorescence and require no washing steps. Results We describe two new rapid and sensitive tests for quantifying mouse and human sAPPα. These 20 μl-volume tests quantify the levels of: i) endogenous mouse sAPPα in the conditioned medium of mouse neuron primary cultures, as well as in the CSF of wild-type mice, ii) human sAPPα in the CSF of AD mouse models, and iii) human sAPPα in the CSF of AD and non-AD patients. These tests require only 5 μl of conditioned medium from 5 × 104 mouse primary neurons, 1 μl of CSF from wild-type and transgenic mice, and 0.5 μl of human CSF. Conclusions The high sensitivity of the mouse sAPPα test will allow high-throughput investigations of molecules capable of increasing the secretion of endogenous sAPPα in primary neurons, as well as the in vivo validation of molecules of interest through the quantification of sAPPα in the CSF of treated wild-type mice. Active molecules could then be tested in the AD mouse models by quantifying human sAPPα in the CSF through the progression of the disease. Finally, the human sAPPα test could strengthen the biological diagnosis of AD in large clinical investigations. Taken together, these new tests have a wide field of applications in preclinical and clinical studies.

Dopamine D1 and D2 receptors as well as the GABA/benzodiazepine receptor complex in the striatum and the globus pallidus (internal: GPi and external: GPe) were studied by autoradiography using [3H]SCH 23390, [3H]spiperone, and [3H]flunitrazepam ([3H]FNZ) respectively, in five groups of cynomolgus monkeys. These included (i) untreated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-monkeys; (ii) MPTP monkeys treated chronically with levodopa injections; (iii) MPTP monkeys treated chronically with injections of the novel D2 agonist U91356A; (iv) MPTP monkeys treated chronically with U91356A delivered through an osmotic mini-pump; and (5) naive controls. Animals treated in a pulsatile mode with U91356A or levodopa injections showed progressive sensitization to their respective drug and developed choreic dyskinesia. In contrast, animals treated in a continuous mode with U91356A showed behavioral tolerance but did not develop dyskinesia. A trend for a down-regulation of putaminal D2 receptors was observed following D2 agonist stimulation with U913356A. Striatal [3H]FNZ binding was significantly decreased only in animals treated in a continuous mode with U91356A. The dopamine receptor decrease in the striatum could be implicated with the development of tolerance but cannot explain the appearnce of dyskinesia. Denervation by MPTP was associated with a decrease of the GPe/GPi [3H]FNZ binding ratio which reflects an imbalance of striatal output pathways; this ratio was not reversed by any of the treatments although changes were observed in the GPe and GPi. Indeed, pulsatile U91356A treatment restored the decreased [3H]FNZ binding in the GPe near control values and levodopa showed a similar tendency. A significant increase of [3H]FNZ binding in the GPi only of dyskinetic monkeys, namely those treated with pulsatile U91356A or levodopa was seen compared to untreated MPTP or naive controls. This GABAA receptor up-regulation might lead to a supersensitive state of the GPi to gabaergic input which may be involved in the mechanism underlying the development of dopaminomimetic-induced dyskinesia.

Background: Intravenous immunoglobulin preparation (IVIg) is composed of >98% human IgG obtained from the plasma of healthy donors. It is approved for the treatment of immunodeficiency and autoimmune diseases and is under study in Alzheimer disease (AD) as well. Understanding the mechanisms of action of IVIg in AD would allow a more rational use of this product and may lead to the development of replacement therapies. Methods: To this aim, we investigated the effects of IVIg treatment in 3xTg-AD mice, which reproduce both As and tau pathologies. 3xTg-AD mice and non-transgenic controls were injected twice weekly with IVIg (or vehicle) for 1 or 3 months. Results: IVIg did not induce a significant immune response and was detected in the serum (ELISA: 22.2 mg/mL 6 0.7, mean 6 SEM), on splenocytes (flow cytometry: mean fluorescence index: 28.2 6 1.2 versus 7.5 6 0.3. Mean 6 SEM in IVIg and vehicle 3xTg-AD mice, respectively. p

Intravenous immunoglobulin (IVIg) is a blood-derived product, used for the treatment of immunodeficiency and autoimmune diseases. Since a range of immunotherapies have recently been proposed as a therapeutic strategy for Parkinson’s disease (PD), we investigated the effects of an IVIg treatment in a neurotoxin-induced animal model of PD. Mice received four injections of MPTP (15 mg/kg) at 2-hour intervals followed by a 14-day IVIg treatment, which induced key immune-related changes such as increased regulatory T-cell population and decreased CD4+/CD8+ ratio. The MPTP treatment induced significant 80% and 84% decreases of striatal dopamine concentrations (P P P

Dietary lipids modify brain fatty acid profile, but evidence of their direct effect on neuronal function is sparse. The enthorinal cortex (EC) neurons connecting to the hippocampus play a critical role in learning and memory. Here, we have exposed mice to diets based on canola:soybean oils (40 : 10, g/kg) or safflower : corn oils (25 : 25, g/kg) to investigate the relationship between the lipid profile of brain fatty acids and the intrinsic properties of EC neurons. Consumption of canola : soybean oil-enriched diet led to the increase of the monounsaturated fatty acid oleic acid and to a decrease of arachidonic acid in ethanolamine glycerophospholipids of the white matter. We also found an important rise in docosahexaenoic acid (DHA) within ethanolamine glycerophospholipids and phosphatidylserine of gray matter. The canola:soybean oil treatment led to a shorter duration of action potential (-21%), a reduction in the duration of postsynaptic response (-21%) and increased firing activity (+43%). Data from additional experiments with animals fed DHA alone or DHA with canola oil suggested that dietary monounsaturated fatty acid may have contributed to these effects on EC neuron physiology. Since neuronal function within the enthorhinal-hippocampal loop is critical to learning and memory processes, the present data may provide a functional basis for the beneficial cognitive effects of canola oil-based diets.

International audience; The accumulation of amyloid-β peptide (Aβ) in the brain is a critical hallmark of Alzheimer's disease. This high cerebral Aβ concentration may be partly caused by impaired clearance of Aβ across the blood-brain barrier (BBB). The low-density lipoprotein receptor-related protein-1 (LRP-1) and the ATP-binding cassette (ABC) protein ABCB1 (P-glycoprotein) are involved in the efflux of Aβ across the BBB. We hypothesized that other ABC proteins, such as members of the G subfamily, are also involved in the BBB clearance of Aβ. We therefore investigated the roles of ABCG2 (BCRP) and ABCG4 in the efflux of [3H] Aβ1-40 from HEK293 cells stably transfected with human ABCG2 or mouse abcg4. We showed that ABCG2 and Abcg4 mediate the cellular efflux of [3H] Aβ1-40. In addition, probucol fully inhibited the efflux of [3H] Aβ1-40 from HEK293-abcg4 cells. Using the in situ brain perfusion technique, we showed that GF120918 (dual inhibitor of Abcb1 and Abcg2) strongly enhanced the uptake (Clup, μl/g/s) of [3H] Aβ1-40 by the brains of Abcb1-deficient mice, but not by the brains of Abcb1/Abcg2-deficient mice, suggesting that Abcg2 is involved in the transport of Aβ at the mouse BBB. Perfusing the brains of Abcb1/Abcg2- and Abca1-deficient mice with [3H] Aβ1-40 plus probucol significantly increased the Clup of Aβ. This suggests that a probucol-sensitive transporter that is different from Abca1, Abcb1, and Abcg2 is involved in the brain efflux of Aβ. We suggest that this probucol-sensitive transporter is Abcg4. We conclude that Abcg4 acts in concert with Abcg2 to efflux Aβ from the brain across the BBB.

Besides memory deficits, Alzheimer's disease (AD) patients suffer from neuropsychiatric symptoms, including alterations in social interactions, which are subject of a growing number of investigations in transgenic models of AD. Yet the biological mechanisms underlying these behavioural alterations are poorly understood. Here, a social interaction paradigm was used to assess social dysfunction in the triple-transgenic mouse model of AD (3xTg-AD). We observed that transgenic mice displayed dimorphic behavioural abnormalities at different ages. Social disinhibition was observed in 18 months old 3xTg-AD males compared to age and sex-matched control mice. In 3xTg-AD females, social disinhibition was present at 12 months followed by reduced social interactions at 18 months. These dimorphic behavioural alterations were not associated with alterations in AD neuropathological markers such as Aβ or tau levels in the frontal cortex. However, patch-clamp recordings revealed that enhanced social interactions coincided temporally with an increase in both excitatory and inhibitory basal synaptic inputs to layer 2-3 pyramidal neurons in the prefrontal cortex. These findings uncover a novel pattern of occurrence of psychiatric-like symptoms between sexes in an AD model. Our results also reveal that functional alterations in synapse activity appear as a potentially significant substrate underlying behavioural correlates of AD.

BackgroundMounting evidence supports a significant role of inflammation in Parkinson's disease (PD) pathophysiology, with several inflammatory pathways being suggested as playing a role in the dopaminergic degeneration seen in humans and animal models of the disease. These include tumor necrosis factor, prostaglandins and oxidative-related stress components. However, the role of innate immunity has not been established in PD.MethodsBased on the fact that the myeloid differentiation primary response gene (88) (MyD88) is the most common adaptor protein implicated in toll-like receptor (TLR) signaling, critical in the innate immune response, we undertook a study to investigate the potential contribution of this specific pathway to MPTP-induced brain dopaminergic degeneration using MyD88 knock out mice (MyD88-/-), following our observations that the MyD88-dependent pathway was critical for MPTP dopaminergic toxicity in the enteric nervous system. Post-mortem analyses assessing nigrostriatal dopaminergic degeneration and inflammation were performed using HPLC, western blots, autoradiography and immunofluorescence.ResultsOur results demonstrate that MyD88-/- mice are as vulnerable to MPTP-induced dopamine and DOPAC striatal depletion as wild type mice. Furthermore, MyD88-/- mice show similar striatal dopamine transporter and tyrosine hydroxylase loss, as well as dopaminergic cell loss in the substantia nigra pars compacta in response to MPTP. To evaluate the extent of the inflammatory response created by the MPTP regimen utilized, we further performed bioluminescence imaging using TLR2-luc/gfp transgenic mice and microglial density analysis, which revealed a modest brain microglial response following MPTP. This was accompanied by a significant astrocytic reaction in the striatum, which was of similar magnitude both in wild type and MyD88-/- mice.ConclusionsOur results suggest that subacute MPTP-induced dopaminergic degeneration observed in the central nervous system is MyD88-independent, in contrast to our recent observations that this pathway, in the same cohort of animals, is critical in the loss of dopaminergic neurons in the enteric nervous system.

Transactive response DNA binding protein 43 (TDP-43) plays a central role in the neuropathology of frontotemporal lobar degeneration and amyotrophic lateral sclerosis, but the relationship between TDP-43 abnormalities and Alzheimer disease (AD) remains unclear. To determine whether TDP-43 can serve as a neuropathologic marker of AD, we performed biochemical characterization and quantification of TDP-43 in homogenates from parietal neocortex of subjects with aclinical diagnosis of no cognitive impairment (NCI, n = 12), mild cognitive impairment (MCI, n = 12), or AD (n = 12). Immunoblots revealed increased detergent-insoluble TDP-43 in the cortex of 0, 3, and 6 of the 12 individuals with NCI, MCI, or AD, respectively. Detergent-insoluble TDP-43 was positively correlated with the accumulation of soluble Aβ42, amyloid plaques, and paired helical filamenttau. In contrast, phospho-TDP-43 was decreased in the cytosolic fraction and detergent-soluble membrane/nuclear fraction from AD patients and correlated with antemortem cognitive function.Immunofluorescence analysis confirmed that the frequencies of individuals with TDP-43 or phospho-TDP-43 cytoplasmic inclusions were higher in AD than in NCI, with MCI at an intermediate level. These data indicate that abnormalities of TDP-43 occur in an important subset of MCI and AD patients and that they correlate with the clinical and neuropathologic features of AD.

n-3 PUFA are receiving growing attention for their therapeutic potential in central nervous system (CNS) disorders. We have recently shown that long-term treatment with DHA alters the physiology of entorhinal cortex (EC) neurons. In the present study, we investigated by patch-clamp the effect of another major dietaryn-3 PUFA, α-linolenic acid (LNA), on the intrinsic properties of EC neurons. Mice were chronically exposed to isoenergetic diets deficient inn-3 PUFA or enriched in either DHA or LNA on an equimolar basis. GC analyses revealed an increase in DHA (34 %) and a decrease in arachidonic acid (AA, − 23 %) in brain fatty acid concentrations after consumption of the DHA-enriched diet. Dietary intake of LNA similarly affected brain fatty acid profiles, but at a lower magnitude (DHA: 23 %, AA: − 13 %). Compared to then-3 PUFA-deficient diet, consumption of DHA, but not LNA, induced membrane hyperpolarisation ( − 60 to − 70 mV), increased cellular capacitance (32 %) and spontaneous excitatory postsynaptic current frequency (50 %). We propose that the inefficiency of LNA to modulate cellular capacitance was related to its inability to increase the brain DHA:AA ratio over the threshold necessary to up-regulate syntaxin-3 (46 %) and translocate drebrin (40 % membrane:cytosol ratio). In summary, our present study shows that the increase in brain DHA content following chronic administration of LNA was not sufficient to alter the passive and synaptic properties of EC neurons, compared to direct dietary intake of DHA. These diverging results have important implications for the therapeutic use ofn-3 PUFA in CNS disease, favouring the use of preformed DHA.

Background APP expression misregulation can cause genetic Alzheimer's disease (AD). Recent evidences support the hypothesis that polymorphisms located in microRNA (miRNA) target sites could influence the risk of developing neurodegenerative disorders such as Parkinson's disease (PD) and frontotemporal dementia. Recently, a number of single nucleotide polymorphisms (SNPs) located in the 3'UTR of APP have been found in AD patients with family history of dementia. Because miRNAs have previously been implicated in APP expression regulation, we set out to determine whether these polymorphisms could affect miRNA function and therefore APP levels. Results Bioinformatics analysis identified twelve putative miRNA bindings sites located in or near the APP 3'UTR variants T117C, A454G and A833C. Among those candidates, seven miRNAs, including miR-20a, miR-17, miR-147, miR-655, miR-323-3p, miR-644, and miR-153 could regulate APP expression in vitro and under physiological conditions in cells. Using luciferase-based assays, we could show that the T117C variant inhibited miR-147 binding, whereas the A454G variant increased miR-20a binding, consequently having opposite effects on APP expression. Conclusions Taken together, our results provide proof-of-principle that APP 3'UTR polymorphisms could affect AD risk through modulation of APP expression regulation, and set the stage for further association studies in genetic and sporadic AD.

Neuroprotectin D1 (NPD1) is a stereoselective mediator derived from the omega-3 essential fatty acid docosahexaenoic acid (DHA) with potent inflammatory resolving and neuroprotective bioactivity. NPD1 reduces Aβ42 peptide release from aging human brain cells and is severely depleted in Alzheimer's disease (AD) brain. Here we further characterize the mechanism of NPD1's neurogenic actions using 3xTg-AD mouse models and human neuronal-glial (HNG) cells in primary culture, either challenged with Aβ42 oligomeric peptide, or transfected with beta amyloid precursor protein (βAPP)(sw) (Swedish double mutation APP695(sw), K595N-M596L). We also show that NPD1 downregulates Aβ42-triggered expression of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) and of B-94 (a TNF-α-inducible pro-inflammatory element) and apoptosis in HNG cells. Moreover, NPD1 suppresses Aβ42 peptide shedding by down-regulating β-secretase-1 (BACE1) while activating the α-secretase ADAM10 and up-regulating sAPPα, thus shifting the cleavage of βAPP holoenzyme from an amyloidogenic into the non-amyloidogenic pathway. Use of the thiazolidinedione peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone, the irreversible PPARγ antagonist GW9662, and overexpressing PPARγ suggests that the NPD1-mediated down-regulation of BACE1 and Aβ42 peptide release is PPARγ-dependent. In conclusion, NPD1 bioactivity potently down regulates inflammatory signaling, amyloidogenic APP cleavage and apoptosis, underscoring the potential of this lipid mediator to rescue human brain cells in early stages of neurodegenerations.

Alzheimer's disease (AD) affects cognitive modalities that are known to be regulated by adult neurogenesis, such as hippocampal- and olfactory-dependent learning and memory. However, the relationship between AD-associated pathologies and alterations in adult neurogenesis has remained contentious. In the present study, we performed a detailed investigation of adult neurogenesis in the triple transgenic (3xTg) mouse model of AD, a unique model that generates both amyloid plaques and neurofibrillary tangles, the hallmark pathologies of AD. In both neurogenic niches of the brain, the hippocampal dentate gyrus and forebrain subventricular zone, we found that 3xTg mice had decreased numbers of (i) proliferating cells, (ii) early lineage neural progenitors, and (iii) neuroblasts at middle age (11months old) and old age (18months old). These decreases correlated with major reductions in the addition of new neurons to the respective target areas, the dentate granule cell layer and olfactory bulb. Within the subventricular zone niche, cytological alterations were observed that included a selective loss of subependymal cells and the development of large lipid droplets within the ependyma of 3xTg mice, indicative of metabolic changes. Temporally, there was a marked acceleration of age-related decreases in 3xTg mice, which affected multiple stages of neurogenesis and was clearly apparent prior to the development of amyloid plaques or neurofibrillary tangles. Our findings indicate that AD-associated mutations suppress neurogenesis early during disease development. This suggests that deficits in adult neurogenesis may mediate premature cognitive decline in AD.

Cystamine has shown significant neuroprotective properties in preclinical studies of Parkinson's disease (PD) and Huntington's disease (HD). Cysteamine, its FDA-approved reduced form, is scheduled to be tested for clinical efficacy in HD patients. Here, we studied the key cystamine metabolites, namely cysteamine, hypotaurine and taurine, as well as cysteine, in order to identify which one is more distinctively responsible for the neuroprotective action of cystamine. After a single administration of cystamine (10, 50 or 200 mg/kg), naïve mice were perfused with phosphate-buffered saline (PBS) at 1, 3, 12, 24 or 48 h post-injection and brain and plasma samples were analyzed by two distinct HPLC methods. Although plasma levels remained under the detection threshold, significant increases in cysteamine brain levels were detected with the 50 and 200 mg/kg doses in mice perfused 1 and 3 h following cystamine injection. To further assess cysteamine as the candidate molecule for pre-clinical and clinical trials in PD, we evaluated its capacity to cross the blood brain barrier. Using an in situ cerebral perfusion technique, we determined that the brain transport coefficient (Clup) of cysteamine (259 μM) was 0.15 ± 0.02 μL/g/s and was increased up to 0.34 ± 0.07 μL/g/s when co-perfused in the presence of cysteine. Taken together, these results strongly suggest that cysteamine is the neuroactive metabolite of cystamine and may further support its therapeutic use in neurodegenerative diseases, particularly in HD and PD.

Preclinical data suggest that cystamine stands as a promising neuroprotective agent against Huntington's and Parkinson's diseases. To decipher the mechanisms of action of cystamine, we investigated the effects of various doses of cystamine (10, 50, and 200 mg/kg) on the regulation of the brain-derived neurotrophic factor (BDNF), its receptor tropomyosin-receptor-kinase B (TrkB) and on the heat shock protein 70 (Hsp70) brain mRNA expression in relation to the time after administration. We have determined that the lower cystamine dose is the most efficient to promote putative neuroprotective effects. Indeed, an acute administration of 10 mg/kg of cystamine increased the expression of BDNF mRNA in the substantia nigra compacta (SNc), although it did not significantly influence TrkB or Hsp70 mRNA. Higher cystamine doses resulted in the absence of activation of any of these markers or led to non-specific effects. We have also substantiated the neuroprotective effect of a 21-day treatment of 10 mg/kg/day of cystamine in young adult mice against MPTP-induced loss of tyrosine hydroxylase-striatal fiber density, nigral dopamine cells and nigral Nurr1 mRNA expression. The neuroprotective action of cystamine in the same animals was associated with an up-regulation of BDNF in the SNc. Taken together, these results strengthen the neuroprotective potential of cystamine in the treatment of Parkinson's disease and point towards the up-regulation of BDNF as an important mechanism of action.

Combined evidence from neuroimaging and neuropathological studies shows that signs of vascular pathology and brain hypoperfusion develop early in Alzheimer's disease (AD). To investigate the functional implication of these abnormalities, we have studied the cerebrovascular volume and selected markers of blood-brain barrier (BBB) integrity in 11-month-old 3 x Tg-AD mice, using the in situ brain perfusion technique. The cerebrovascular volume of distribution of two vascular space markers, [3H]-inulin and [14C]-sucrose, was significantly lower (-26% and -27%, respectively; p < 0.01) in the brain of 3 x Tg-AD mice compared to non-transgenic littermates. The vascular volume reduction was significant in the hippocampus (p < 0.01), but not in the frontal cortex and cerebellum. However, the brain transport coefficient (Clup) of [14C]-D-glucose (1 microM) and [3H]-diazepam was similar between 3xTg-AD mice and controls, suggesting no difference in the functional integrity of the BBB. We also report a 32% increase (p < 0.001) in the thickness of basement membranes surrounding cortical microvessels along with a 20% increase (p < 0.05) of brain collagen content in 3xTg-AD mice compared to controls. The present data indicate that the cerebrovascular space is reduced in a mouse model of Abeta and tau accumulation, an observation consistent with the presence of cerebrovascular pathology in AD.

Aging and metabolism-related disorders are risk factors for Alzheimer disease (AD). Because sirtuins may increase the life span through regulation of cellular metabolism, we compared the concentration of sirtuin 1 (SIRT1) in the brains of AD patients (n = 19) and controls (n = 22) using Western immunoblots and in situ hybridization. We report a significant reduction of SIRT1 (messenger RNA [mRNA], -29%; protein, -45%) in the parietal cortex of AD patients, but not in the cerebellum. Further analyses in a second cohort of 36 subjects confirmed that cortical SIRT1 was decreased in AD but not in individuals with mild cognitive impairment. SIRT1 mRNA and its translated protein correlated negatively with the duration of symptoms (mRNA, r2 = -0.367; protein, r2 = -0.326) and the accumulation of paired helical filament tau (mRNA, r2 = -0.230; protein, r2 = -0.119), but weakly with insoluble amyloid-beta 42 (mRNA, r2= -0.090; protein, r2 = -0.072). A significant relationship between SIRT1 levels and global cognition scores proximate to death was also found (r2= +0.09, p = 0.049). In contrast, cortical SIRT1 levels remained unchanged in a triple-transgenic animal model of AD. Collectively, our results indicate that loss of SIRT1 is closely associated with the accumulation of amyloid-beta and tau in the cerebral cortex of persons with AD.

Analyses of brain phospholipid fatty acid profiles reveal a selective deficiency and enrichment in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), respectively. In order to account for this difference in brain fatty acid levels, we hypothesized that EPA is more rapidly beta-oxidized upon its entry into the brain. Wild-type C57BL/6 mice were perfused with either (14)C-EPA or (14)C-DHA via in situ cerebral perfusion for 40s, followed by a bicarbonate buffer to wash out the residual radiolabeled polyunsaturated fatty acid (PUFA) in the capillaries. (14)C-PUFA-perfused brains were extracted for chemical analyses of neutral lipid and phospholipid fatty acids. Based on the radioactivity in aqueous, total lipid, neutral lipid and phospholipid fractions, volume of distribution (V(D), microl/g) was calculated. The V(D) between (14)C-EPA- and (14)C-DHA-perfused samples was not statistically different for total lipid, neutral lipids or total phospholipids. However, the V(D) of (14)C-EPA in the aqueous fraction was 2.5 times higher than that of (14)C-DHA (p=0.025), suggesting a more extensive beta-oxidation than DHA. Furthermore, radiolabeled palmitoleic acid, a fatty acid that can be synthesized de novo, was detected in brain phospholipids from (14)C-EPA but not from (14)C-DHA-perfused mice suggesting that beta-oxidation products of EPA were recycled into endogenous fatty acid biosynthetic pathways. These findings suggest that low levels of EPA in brain phospholipids compared to DHA may be the result of its rapid beta-oxidation upon uptake by the brain.

To investigate potential dietary risk factors of Alzheimer's disease (AD), triple transgenic (3xTg-AD) mice were exposed from 4 to 13 months of age to diets with a low n-3:n-6 polyunsaturated fatty acid (PUFA) ratio incorporated in either low-fat (5% w/w) or high-fat (35% w/w) formulas and compared with a control diet. The n-3:n-6 PUFA ratio was decreased independently of the dietary treatments in the frontal cortex of 3xTg-AD mice compared to non-transgenic littermates. Consumption of a high-fat diet with a low n-3:n-6 PUFA ratio increased amyloid-beta (Abeta) 40 and 42 concentrations in detergent-insoluble extracts of parieto-temporal cortex homogenates from 3xTg-AD mice. Low n-3:n-6 PUFA intake ratio increased insoluble tau regardless of total fat consumption, whereas high-fat diet incorporating a low n-3:n-6 PUFA ratio also increased soluble tau compared to controls. Moreover, the high-fat diet decreased cortical levels of the postsynaptic marker drebrin, while leaving presynaptic proteins synaptophysin, SNAP-25 and syntaxin 3 unchanged. Overall, these results suggest that high-fat consumption combined with low n-3 PUFA intake promote AD-like neuropathology.

L-Dopa therapy in Parkinson's disease (PD) is counfounded by the development of involuntary movements such as L-Dopa-induced dyskinesias (LIDs). In this study GABA(A) receptor autoradiography was assessed using [(3)H]flunitrazepam binding to the benzodiazepine site of the GABA(A) receptor and [(35)S]t-butylbicyclophosphorothionate (TBPS) binding to the chloride channel of GABA(A) receptors in the substantia nigra reticulata (SNr) and subthalamic nucleus (STN). L-Dopa-treated parkinsonian monkeys experiencing LIDs were compared to animals in which LIDs was prevented by adjunct treatments with CI-1041, a selective antagonist of the NR1A/2B subtype of NMDA receptor, or low doses of the dopamine D2 receptor agonist, cabergoline. Our results demonstrated a decrease of GABA(A) receptor specific binding in the posterior part of the SNr in dyskinetic monkeys compared to nondyskinetic animals, while no modulation has been observed in the STN. These results provide evidence for the first time that pharmacological treatments preventing LIDs in nonhuman primate model of PD are associated with normalization of GABA(A) receptor-mediated signalling in the SNr.

L-Dopa-induced dyskinesias (LIDs), the disabling abnormal involuntary movements induced by chronic use of L-Dopa, limit the quality of life in Parkinson's disease (PD) patients. Modulation of group II metabotropic glutamate receptors (mGluR2/3) in the basal ganglia, a brain region critically involved in motor control, is considered as an alternative approach in therapy of PD. In this study, receptor binding autoradiography of [3H]LY341495, a mGluR2/3 selective radioligand, was used to investigate possible changes in mGluR2/3 in the basal ganglia of L-Dopa-treated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys having developed LIDs compared to animals in which LIDs were prevented by adjunct treatments with CI-1041, a selective antagonist of the NR1A/2B subtype of NMDA receptor, or low doses of the dopamine D2 receptor agonist, cabergoline. Our study is the first to provide evidence of: (1) the similar localization of [3H]LY341495 specific binding to mGluR2/3 in the primate basal ganglia as compared to receptor distribution measured by immunohistochemistry in human and rat as well as this ligand binding in intact rat brain; (2) no change of [3H]LY341495 specific binding in basal ganglia after nigrostriatal denervation by MPTP; and (3) a widespread reduction of [(3)H]LY341495 specific binding to mGluR2/3 in the caudate nucleus (-17% to -31%), putamen (-12% to -45%) and globus pallidus (-56 to -59%) of non-dyskinetic animals treated with L-Dopa+cabergoline as compared to controls, MPTP monkeys treated with saline, L-Dopa alone (dyskinetic) or L-Dopa+CI-1041 (non-dyskinetic). This study is the first to propose a close interaction between mGluR2/3 and dopamine D2 receptors activation in the basal ganglia.

The striatum harbors a population of dopaminergic interneurons that increases in number in animal models of Parkinson's disease (PD), presumably to compensate for dopamine (DA) depletion. The purpose of the present study was to determine the fate of striatal dopaminergic neurons in parkinsonian monkeys in which striatal DA depletion had been alleviated by systemic administration of l-dopa. The number of striatal dopaminergic neurons, visualized with tyrosine hydroxylase (TH) immunohistochemistry, was measured in three groups of cynomolgus (Macaca fascicularis) monkeys: (1) normal untreated monkeys; (2) monkeys rendered parkinsonian following systemic injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), but otherwise untreated; and (3) MPTP-intoxicated monkeys that received oral l-dopa on a chronic basis. In agreement with previous studies, the number of striatal TH-positive (TH+) neurons in l-dopa-free parkinsonian monkeys was significantly higher (p0.05) than in normal (non-parkinsonian) monkeys. However, this increase was abolished in parkinsonian monkeys that received l-dopa treatment. In fact, the number of striatal TH+ neurons in l-dopa-treated parkinsonian monkeys was not significantly different (p0.05) from values obtained in normal monkeys. These findings suggest that the DA concentration regulates the numerical density of this ectopic neuronal population, a phenomenon that is more likely the result of a shift in the phenotype of preexistent striatal interneurons rather than the recruitment of newborn neurons that would eventually develop a DA phenotype. Our data also reinforce the hypothesis that striatal TH+ neurons act as local DA source and, as such, are part of a compensatory mechanism that could be artificially enhanced to alleviate or delay PD symptoms.

Dopamine deficiency, caused by the degeneration of nigrostriatal dopaminergic neurons, is the cause of the major clinical motor symptoms of Parkinson's disease. These symptoms can be treated successfully with a range of drugs that include levodopa, inhibitors of the enzymatic breakdown of levodopa and dopamine agonists delivered by oral, subcutaneous, transcutaneous, intravenous or intra-duodenal routes. However, Parkinson's disease involves degeneration of non-dopaminergic neurons and the treatment of the resulting predominantly non-motor features remains a challenge. This review describes the important recent advances that underlie the development of novel dopaminergic and non-dopaminergic drugs for Parkinson's disease, and also for the motor complications that arise from the use of existing therapies.

Adenosine A2A receptors (A2AR) have received increasing attention for the treatment of L-DOPA-induced dyskinesias in Parkinson disease. In the present study, A2AR messenger RNA (mRNA) and receptor-specific binding in the brain of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys were studied after treatment with L-DOPA and a selective NR1A/2B NMDA receptor antagonist, CI-1041. Four MPTP monkeys received L-DOPA/benserazide and all developed dyskinesias, whereas among the four MPTP monkeys who additionally received CI-1041, only one developed mild dyskinesias. Four normal monkeys and four MPTP-treated monkeys were also studied. All MPTP monkeys had similar striatal dopamine (DA) denervation. A2AR mRNA levels, measured by in situ hybridization, were increased in the rostral lateral caudate and putamen of saline-treated MPTP monkeys as well as in the caudal lateral and medial putamen when compared with those of controls. A2AR mRNA levels remained elevated in the rostral caudate and putamen of L-DOPA-treated MPTP monkeys when compared with those of controls. A2AR mRNA levels of L-DOPA + CI-1041-treated monkeys were at control levels and decreased in the lateral rostral caudate and caudal putamen when compared with those of L-DOPA-treated and saline-treated MPTP monkeys respectively. No change was measured in the caudal medial putamen and caudate nucleus. A2ARs labeled by autoradiography with [3H]SCH-58261 had lower level in the L-DOPA + CI-1041-treated MPTP monkeys compared with saline- or L-DOPA-treated MPTP and control monkeys in the rostral lateral and medial caudate and the putamen. No effect of lesion or L-DOPA treatment was measured on [3H]SCH-58261-specific binding. These findings suggest that blockade of NMDA receptors could prevent the development of dyskinesias by altering A2ARs. Synapse 60:239–250, 2006. © 2006 Wiley-Liss, Inc.

The pharmaceutical industry spends over US$10 billion to fund some 90% of the 40 000–80 000 randomized controlled clinical trials (RCTs) being conducted across the world at any given time.[1][1],[2][2],[3][3] By its own estimation, the pharmaceutical industry in Canada spent Can$501.8 million on

Defects in dendritic spines are common to several forms of cognitive deficits, including mental retardation and Alzheimer disease. Because mutation of p21-activated kinase (PAK) can lead to mental retardation and because PAK-cofilin signaling is critical in dendritic spine morphogenesis and actin dynamics, we hypothesized that the PAK pathway is involved in synaptic and cognitive deficits in Alzheimer disease. Here, we show that PAK and its activity are markedly reduced in Alzheimer disease and that this is accompanied by reduced and redistributed phosphoPAK, prominent cofilin pathology and downstream loss of the spine actin-regulatory protein drebrin, which cofilin removes from actin. We found that beta-amyloid (Abeta) was directly involved in PAK signaling deficits and drebrin loss in Abeta oligomer-treated hippocampal neurons and in the Appswe transgenic mouse model bearing a double mutation leading to higher Abeta production. In addition, pharmacological PAK inhibition in adult mice was sufficient to cause similar cofilin pathology, drebrin loss and memory impairment, consistent with a potential causal role of PAK defects in cognitive deficits in Alzheimer disease.

Enkephalin is reported to play an important role in the pathophysiology of levodopa (LD) -induced dyskinesias. The present study investigated the effect of chronic treatment with a selective NR1A/2B N-methyl-D-aspartate (NMDA) receptor antagonist, CI-1041, on the expression of preproenkephalin-A (PPE-A) in brains of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) -treated monkeys in relation to the development of LD-induced dyskinesias. Four MPTP-monkeys received LD/benserazide alone; they all developed dyskinesias. Four other MPTP-monkeys received LD/benserazide plus CI-1041; only one of them developed mild dyskinesias at the end of the fourth week of treatment. Four normal monkeys and four saline-treated MPTP monkeys were also included. MPTP-treated monkeys had extensive and similar striatal dopamine denervation. An increase of PPE-A mRNA levels assayed by in situ hybridization was observed in the lateral putamen (rostral and caudal) and caudate nucleus (rostral) of saline-treated MPTP monkeys compared to controls, whereas no change or a small increase was observed in their medial parts. Striatal PPE-A mRNA levels remained elevated in LD-treated MPTP monkeys, whereas cotreatment with CI-1041 brought them back to control values. These findings suggest that chronic blockade of striatal NR1A/2B NMDA receptors with CI-1041 normalizes PPE-A mRNA expression and prevents the development of LD-induced dyskinesias in an animal model of Parkinson disease. © 2005 Movement Disorder Society

Epidemiological studies suggest that increased intake of the omega-3 (n-3) polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) is associated with reduced risk of Alzheimer's disease (AD). DHA levels are lower in serum and brains of AD patients, which could result from low dietary intake and/or PUFA oxidation. Because effects of DHA on Alzheimer pathogenesis, particularly on amyloidosis, are unknown, we used the APPsw (Tg2576) transgenic mouse model to evaluate the impact of dietary DHA on amyloid precursor protein (APP) processing and amyloid burden. Aged animals (17-19 months old) were placed in one of three groups until 22.5 months of age: control (0.09% DHA), low-DHA (0%), or high-DHA (0.6%) chow. beta-Amyloid (Abeta) ELISA of the detergent-insoluble extract of cortical homogenates showed that DHA-enriched diets significantly reduced total Abeta by70% when compared with low-DHA or control chow diets. Dietary DHA also decreased Abeta42 levels below those seen with control chow. Image analysis of brain sections with an antibody against Abeta (amino acids 1-13) revealed that overall plaque burden was significantly reduced by 40.3%, with the largest reductions (40-50%) in the hippocampus and parietal cortex. DHA modulated APP processing by decreasing both alpha- and beta-APP C-terminal fragment products and full-length APP. BACE1 (beta-secretase activity of the beta-site APP-cleaving enzyme), ApoE (apolipoprotein E), and transthyretin gene expression were unchanged with the high-DHA diet. Together, these results suggest that dietary DHA could be protective against beta-amyloid production, accumulation, and potential downstream toxicity.

Brain adenosine A2A receptors have recently attracted considerable attention because of their interaction with the dopaminergic system and as potential targets for Parkinson's disease pharmacotherapy. Post mortem adenosine A2A receptor mRNA and [3H]SCH 58261- specific binding to adenosine A2A receptor were studied in the brain of Parkinson's disease patients using in situ hybridization and receptor binding autoradiography, respectively. Fourteen levodopa-treated Parkinson's disease patients, of which seven developed dyskinesias and seven did not, were compared with nine controls. Nigrostriatal denervation was similar between dyskinetic and non-dyskinetic Parkinson's disease patients, as assessed with catecholamine concentrations and [125I]RTI-121-specific binding to dopamine transporters. A2A receptor mRNA levels (+129%; P < 0.01) and [3H]SCH 58261-specific binding (+32%, P < 0.01) were increased in the putamen (lateral and medial) of dyskinetic patients compared with controls. The increase of adenosine A2A receptor mRNA in dyskinetic Parkinson's disease patients was also significant compared with non-dyskinetic Parkinson's disease patients (+60%; P < 0.05) in the lateral putamen. Moreover, [3H]SCH 58261-specific binding to adenosine A2A receptors was increased in the external globus pallidus (+24%; P < 0.001) of Parkinson's disease patients compared with controls, regardless of the dyskinesigenic response to levodopa. No change of adenosine A2A receptors was observed in the caudate nucleus, whereas adenosine A2A receptor protein and mRNA levels in the internal globus pallidus were not different from background. These findings suggest that increased synthesis of adenosine A2A receptors in striatopallidal pathway neurons is associated with the development of dyskinesias following long-term levodopa therapy in Parkinson's disease.

Brain gene-targeting technology is used to reversibly normalize tyrosine hydroxylase (TH) activity in the striatum of adult rats, using the experimental 6-hydroxydopamine model of Parkinson's disease. The TH expression plasmid is encapsulated inside an 85-nm PEGylated immunoliposome (PIL) that is targeted with either the OX26 murine monoclonal antibody (MAb) to the rat transferrin receptor (TfR) or with the mouse IgG2a isotype control antibody. TfRMAb-PIL, or mIgG2a-PIL, is injected intravenously at a dose of 10 microg of plasmid DNA per rat. TfRMAb-PIL, but not mIgG2a-PIL, enters the brain via the transvascular route. The targeting TfRMAb enables the nanocontainer carrying the gene to undergo both receptor-mediated transcytosis across the blood-brain barrier (BBB) and receptor-mediated endocytosis into neurons behind the BBB by accessing the TfR. With this approach, the striatal TH activity ipsilateral to the intracerebral injection of the neurotoxin was normalized and increased from 738 +/- 179 to 5486 +/- 899 pmol/hr per milligram of protein. The TH enzyme activity measurements were corroborated by TH immunocytochemistry, which showed that the entire striatum was immunoreactive for TH after intravenous gene therapy. The normalization of striatal biochemistry was associated with a reversal of apomorphine-induced rotation behavior. Lesioned animals treated with the apomorphine exhibited 20 +/- 5 and 6 +/- 2 rotations/min, respectively, after intravenous administration of the TH plasmid encapsulated in mIgG2a-PIL and TfRMAb-PIL. These studies demonstrate that it is possible to normalize brain enzyme activity by intravenous administration and nonviral gene transfer.