Characterization of brain transduction capability of a BBB-penetrant AAV vector in mice, rats and macaques reveals differences in expression profiles.

Different screening methods are being developed to generate adeno-associated viral vectors (AAV) with the ability to bypass the blood-brain barrier (BBB) upon intravenous administration. Recently, the AAV9P31 stood out as the most efficient version among a library of peptide-displaying capsids selected in C57BL/6 mice using RNA-driven biopanning. In this work we have characterized in detail its biodistribution in different mouse strains (C57BL/6 and Balb/c), as well as in Sprague Dawley rats and non-human primates (Macaca fascicularis). Using GFP and NanoLuc reporter genes, we confirmed homogeneous infection and transgene expression across the CNS of mice injected intravenously with AAV9P31. A more restricted pattern was observed upon either intracerebroventricular or intraparenchymal injection. Following intravenous delivery, region- and cell-specific differential patterns of transduction were observed in the mouse brain, including a preferential transduction of astrocytes and neurons in the cerebral cortex and striatum, whereas neurons were the only transduced cell type in subcortical locations across the hippocampus, thalamus, hypothalamus, mesencephalon, brainstem and cerebellum. Furthermore, transduced microglial cells were never found in any CNS location. Peripheral organs transduced upon intravenous administration included lung, liver, peritoneum, heart and skeletal muscle. However, a comparable performance of AAV9P31 to bypass the BBB in rats and macaques was not observed, although a more limited neuronal transduction was found in the brainstem of rats upon intravenous delivery. Finally, intracerebroventricular delivery in macaques resulted in neuronal transduction in cortical, subcortical structures and cerebellum following a patchy pattern. In conclusion, the widespread CNS transduction obtained in mice upon intravenous delivery of AAV9P31 represents a powerful tool for modeling a wide variety of neurological disorders as well as an appealing choice for the evaluation of gene therapy-based therapeutics.

Development and characterization of a non-human primate model of disseminated synucleinopathy.

Introduction: The presence of a widespread cortical synucleinopathy is the main neuropathological hallmark underlying clinical entities such as Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB). There currently is a pressing need for the development of non-human primate (NHPs) models of PDD and DLB to further overcome existing limitations in drug discovery. Methods: Here we took advantage of a retrogradely-spreading adeno-associated viral vector serotype 9 coding for the alpha-synuclein A53T mutated gene (AAV9-SynA53T) to induce a widespread synucleinopathy of cortical and subcortical territories innervating the putamen. Four weeks post-AAV deliveries animals were sacrificed and a comprehensive biodistribution study was conducted, comprising the quantification of neurons expressing alpha-synuclein, rostrocaudal distribution and their specific location. Results: Intraputaminal deliveries of AAV9-SynA53T lead to a disseminated synucleinopathy throughout ipsi- and contralateral cerebral cortices, together with transduced neurons located in the ipsilateral caudal intralaminar nuclei and in the substantia nigra pars compacta (leading to thalamostriatal and nigrostriatal projections, respectively). Cortical afferent systems were found to be the main contributors to putaminal afferents (superior frontal and precentral gyri in particular). Discussion: Obtained data extends current models of synucleinopathies in NHPs, providing a reproducible platform enabling the adequate implementation of end-stage preclinical screening of new drugs targeting alpha-synuclein.

Neuromelanin accumulation drives endogenous synucleinopathy in non-human primates.

Although neuromelanin is a dark pigment characteristic of dopaminergic neurons in the human substantia nigra pars compacta, its potential role in the pathogenesis of Parkinson's disease (PD) has often been neglected since most commonly used laboratory animals lack neuromelanin. Here we took advantage of adeno-associated viral vectors encoding the human tyrosinase gene for triggering a time-dependent neuromelanin accumulation within substantia nigra pars compacta dopaminergic neurons in macaques up to similar levels of pigmentation as observed in elderly humans. Furthermore, neuromelanin accumulation induced an endogenous synucleinopathy mimicking intracellular inclusions typically observed in PD together with a progressive degeneration of neuromelanin-expressing dopaminergic neurons. Moreover, Lewy body-like intracellular inclusions were observed in cortical areas of the frontal lobe receiving dopaminergic innervation, supporting a circuit-specific anterograde spread of endogenous synucleinopathy by permissive trans-synaptic templating. In summary, the conducted strategy resulted in the development and characterization of a new macaque model of PD matching the known neuropathology of this disorder with unprecedented accuracy. Most importantly, evidence is provided showing that intracellular aggregation of endogenous α-synuclein is triggered by neuromelanin accumulation, therefore any therapeutic approach intended to decrease neuromelanin levels may provide appealing choices for the successful implementation of novel PD therapeutics.

Adeno-Associated Viral Vectors as Versatile Tools for Neurological Disorders: Focus on Delivery Routes and Therapeutic Perspectives.

It is without doubt that the gene therapy field is currently in the spotlight for the development of new therapeutics targeting unmet medical needs. Thus, considering the gene therapy scenario, neurological diseases in general and neurodegenerative disorders in particular are emerging as the most appealing choices for new therapeutic arrivals intended to slow down, stop, or even revert the natural progressive course that characterizes most of these devastating neurodegenerative processes. Since an extensive coverage of all available literature is not feasible in practical terms, here emphasis was made in providing some advice to beginners in the field with a narrow focus on elucidating the best delivery route available for fulfilling any given AAV-based therapeutic approach. Furthermore, it is worth nothing that the number of ongoing clinical trials is increasing at a breath-taking speed. Accordingly, a landscape view of preclinical and clinical initiatives is also provided here in an attempt to best illustrate what is ongoing in this quickly expanding field.

Adeno-Associated Viral Vectors as Versatile Tools for Parkinson's Research, Both for Disease Modeling Purposes and for Therapeutic Uses.

It is without any doubt that precision medicine therapeutic strategies targeting neurodegenerative disorders are currently witnessing the spectacular rise of newly designed approaches based on the use of viral vectors as Trojan horses for the controlled release of a given genetic payload. Among the different types of viral vectors, adeno-associated viruses (AAVs) rank as the ones most commonly used for the purposes of either disease modeling or for therapeutic strategies. Here, we reviewed the current literature dealing with the use of AAVs within the field of Parkinson's disease with the aim to provide neuroscientists with the advice and background required when facing a choice on which AAV might be best suited for addressing a given experimental challenge. Accordingly, here we will be summarizing some insights on different AAV serotypes, and which would be the most appropriate AAV delivery route. Next, the use of AAVs for modeling synucleinopathies is highlighted, providing potential readers with a landscape view of ongoing pre-clinical and clinical initiatives pushing forward AAV-based therapeutic approaches for Parkinson's disease and related synucleinopathies.

Glucocerebrosidase Gene Therapy Induces Alpha-Synuclein Clearance and Neuroprotection of Midbrain Dopaminergic Neurons in Mice and Macaques.

Mutations in the GBA1 gene coding for glucocerebrosidase (GCase) are the main genetic risk factor for Parkinson's disease (PD). Indeed, identifying reduced GCase activity as a common feature underlying the typical neuropathological signatures of PD-even when considering idiopathic forms of PD-has recently paved the way for designing novel strategies focused on enhancing GCase activity to reduce alpha-synuclein burden and preventing dopaminergic cell death. Here we have performed bilateral injections of a viral vector coding for the mutated form of alpha-synuclein (rAAV9-SynA53T) for disease modeling purposes, both in mice as well as in nonhuman primates (NHPs), further inducing a progressive neuronal death in the substantia nigra pars compacta (SNpc). Next, another vector coding for the GBA1 gene (rAAV9-GBA1) was unilaterally delivered in the SNpc of mice and NHPs one month after the initial insult, together with the contralateral delivery of an empty/null rAAV9 for control purposes. Obtained results showed that GCase enhancement reduced alpha-synuclein burden, leading to improved survival of dopaminergic neurons. Data reported here support using GCase gene therapy as a disease-modifying treatment for PD and related synucleinopathies, including idiopathic forms of these disorders.

Expression of GPR55 and either cannabinoid CB1 or CB2 heteroreceptor complexes in the caudate, putamen, and accumbens nuclei of control, parkinsonian, and dyskinetic non-human primates.

Endocannabinoids are neuromodulators acting on specific cannabinoid CB1 and CB2 G-protein-coupled receptors (GPCRs), representing potential therapeutic targets for neurodegenerative diseases. Cannabinoids also regulate the activity of GPR55, a recently "deorphanized" GPCR that directly interacts with CB1 and with CB2 receptors. Our hypothesis is that these heteromers may be taken as potential targets for Parkinson's disease (PD). This work aims at assessing the expression of heteromers made of GPR55 and CB1/CB2 receptors in the striatum of control and parkinsonian macaques (with and without levodopa-induced dyskinesia). For this purpose, double blind in situ proximity ligation assays, enabling the detection of GPCR heteromers in tissue samples, were performed in striatal sections of control, MPTP-treated and MPTP-treated animals rendered dyskinetic by chronic treatment with levodopa. Image analysis and statistical assessment were performed using dedicated software. We have previously demonstrated the formation of heteromers between GPR55 and CB1 receptor (CB1-GPR55_Hets), which is highly expressed in the central nervous system (CNS), but also with the CB2 receptor (CB2-GPR55_Hets). Compared to the baseline expression of CB1-GPR55_Hets in control animals, our results showed increased expression levels in basal ganglia input nuclei of MPTP-treated animals. These observed increases in CB1-GPR55_Hets returned back to baseline levels upon chronic treatment with levodopa in dyskinetic animals. Obtained data regarding CB2-GPR55_Hets were quite similar, with somehow equivalent amounts in control and dyskinetic animals, and with increased expression levels in MPTP animals. Taken together, the detected increased expression of GPR55-endocannabinoid heteromers appoints these GPCR complexes as potential non-dopaminergic targets for PD therapy.

Expression of cannabinoid CB1 R-GPR55 heteromers in neuronal subtypes of the Macaca fascicularis striatum.

The cannabinoid CB1 receptor (CB1 R) is the most abundant G protein-coupled receptor in the central nervous system, consistent with the important role of endocannabinoids as neuromodulators. Cannabinoids also modulate the function of G protein-coupled receptor 55 (GPR55), which forms heteroreceptor complexes with the CB1 R in the striatum. The aim was to characterize cannabinoid CB1 R-GPR55 heteromers (CB1 R/GPR55Hets) in the basal ganglia input nuclei of nonhuman primates, Macaca fascicularis, both in projection neurons and interneurons, by the in situ proximity ligation assay. Striatal projecting neurons were identified by the retrograde neuroanatomical tracer, biotinylated dextran amine (BDA), injected into external or internal subdivisions of the globus pallidus. Triple immunofluorescent stains were carried out to visualize (1) BDA-labeled neurons, (2) CB1 R/GPR55Hets, and (3) striatal interneurons positive for choline acetyltransferase, parvalbumin, calretinin, or nitric oxide synthase. CB1 R/GPR55Hets were identified within both types of projection neurons as well as all interneurons except those that are cholinergic. Moreover, CB1 R/GPR55Hets were found specifically in the neuronal cell surface, and also in intracellular membranes. Further research efforts will be needed to confirm the intracellular occurrence of heteromers and their potential as therapeutic targets in diseases related to motor control imbalances, particularly within a parkinsonian context (with or without levodopa-induced dyskinesia).

Targeting CB1 and GPR55 Endocannabinoid Receptors as a Potential Neuroprotective Approach for Parkinson's Disease.

Cannabinoid CB1 receptors (CB1R) and the GPR55 receptor are expressed in striatum and are potential targets in the therapy of Parkinson's disease (PD), one of the most prevalent neurodegenerative diseases in developed countries. The aim of this paper was to address the potential of ligands acting on those receptors to prevent the action of a neurotoxic agent, MPP+, that specifically affects neurons of the substantia nigra due to uptake via the dopamine DAT transporter. The SH-SY5Y cell line model was used as it expresses DAT and, therefore, is able to uptake MPP+ that inhibits complex I of the respiratory mitochondrial chain and leads to cell death. Cells were transfected with cDNAs coding for either or both receptors. Receptors in cotransfected cells formed heteromers as indicated by the in situ proximity ligation assays. Cell viability was assayed by oxygen rate consumption and by the bromide-based MTT method. Assays of neuroprotection using two concentrations of MPP+ showed that cells expressing receptor heteromers were more resistant to the toxic effect. After correction by effects on cell proliferation, the CB1R antagonist, SR141716, afforded an almost full neuroprotection in CB1R-expressing cells even when a selective agonist, ACEA, was present. In contrast, SR141716 was not effective in cells expressing CB1/GPR55 heteromeric complexes. In addition, an agonist of GPR55, CID1792197, did not enhance neuroprotection in GPR55-expressing cells. These results show that neurons expressing heteromers are more resistant to cell death but question the real usefulness of CB1R, GPR55, and their heteromers as targets to afford PD-related neuroprotection.

Glucocerebrosidase expression patterns in the non-human primate brain.

Glucocerebrosidase (GCase) is a lysosomal enzyme encoded by the GBA1 gene. Mutations in GBA1 gene lead to Gaucher's disease, the most prevalent lysosomal storage disorder. GBA1 mutations reduce GCase activity, therefore promoting the aggregation of alpha-synuclein, a common neuropathological finding underlying Parkinson's disease (PD) and dementia with Lewy bodies. However, it is also worth noting that a direct link between GBA1 mutations and alpha-synuclein aggregation indicating cause and effect is still lacking, with limited experimental evidence to date. Bearing in mind that a number of strategies increasing GCase expression for the treatment of PD are currently under development, here we sought to analyze the baseline expression of GCase in the brain of Macaca fascicularis, which has often been considered as the gold-standard animal model of PD. Although as with other lysosomal enzymes, GCase is expected to be ubiquitously expressed, here a number of regional variations have been consistently found, together with several specific neurochemical phenotypes expressing very high levels of GCase. In this regard, the most enriched expression of GCase was constantly found in cholinergic neurons from the nucleus basalis of Meynert, dopaminergic cells in the substantia nigra pars compacta, serotoninergic neurons from the raphe nuclei, as well as in noradrenergic neurons located in the locus ceruleus. Moreover, it is also worth noting that moderate levels of expression were also found in a number of areas within the paleocortex and archicortex, such as the entorhinal cortex and the hippocampal formation, respectively.

Adeno-Associated Viral Vectors Serotype 8 for Cell-Specific Delivery of Therapeutic Genes in the Central Nervous System.

Adeno-associated viruses (AAVs) have become highly promising tools for research and clinical applications in the central nervous system (CNS). However, specific delivery of genes to the cell type of interest is essential for the success of gene therapy and therefore a correct selection of the promoter plays a very important role. Here, AAV8 vectors carrying enhanced green fluorescent protein (eGFP) as reporter gene under the transcriptional control of different CNS-specific promoters were used and compared with a strong ubiquitous promoter. Since one of the main limitations of AAV-mediated gene delivery lies in its restricted cloning capacity, we focused our work on small-sized promoters. We tested the transduction efficacy and specificity of each vector after stereotactic injection into the mouse striatum. Three glia-specific AAV vectors were generated using two truncated forms of the human promoter for glial fibrillar acidic protein (GFAP) as well as a truncated form of the murine GFAP promoter. All three vectors resulted in predominantly glial expression; however we also observed eGFP expression in other cell-types such as oligodendrocytes, but never in neurons. In addition, robust and neuron-specific eGFP expression was observed using the minimal promoters for the neural protein BM88 and the neuronal nicotinic receptor ß2 (CHRNB2). In summary, we developed a set of AAV vectors designed for specific expression in cells of the CNS using minimal promoters to drive gene expression when the size of the therapeutic gene matters.

Neurochemical evidence supporting dopamine D1-D2 receptor heteromers in the striatum of the long-tailed macaque: changes following dopaminergic manipulation.

Although it has long been widely accepted that dopamine receptor types D1 and D2 form GPCR heteromers in the striatum, the presence of D1-D2 receptor heteromers has been recently challenged. In an attempt to properly characterize D1-D2 receptor heteromers, here we have used the in situ proximity ligation assay (PLA) in striatal sections comprising the caudate nucleus, the putamen and the core and shell territories of the nucleus accumbens. Experiments were carried out in control macaques as well as in MPTP-treated animals (with and without dyskinesia). Obtained data support the presence of D1-D2 receptor heteromers within all the striatal subdivisions, with the highest abundance in the accumbens shell. Dopamine depletion by MPTP resulted in an increase of D1-D2 density in caudate and putamen which was normalized by levodopa treatment. Two different sizes of heteromers were consistently found, thus suggesting that besides individual heteromers, D1-D2 receptor heteromers are sometimes organized in macromolecular complexes made of a number of D1-D2 heteromers. Furthermore, the PLA technique was combined with different neuronal markers to properly characterize the identities of striatal neurons expressing D1-D2 heteromers. We have found that striatal projection neurons giving rise to either the direct or the indirect basal ganglia pathways expressed D1-D2 heteromers. Interestingly, macromolecular complexes of D1-D2 heteromers were only found within cholinergic interneurons. In summary, here we provide overwhelming proof that D1 and D2 receptors form heteromeric complexes in the macaque striatum, thus representing a very appealing target for a number of brain diseases involving dopamine dysfunction.

Stronger Dopamine D1 Receptor-Mediated Neurotransmission in Dyskinesia.

Radioligand binding assays to rat striatal dopamine D1 receptors showed that brain lateralization of the dopaminergic system were not due to changes in expression but in agonist affinity. D1 receptor-mediated striatal imbalance resulted from a significantly higher agonist affinity in the left striatum. D1 receptors heteromerize with dopamine D3 receptors, which are considered therapeutic targets for dyskinesia in parkinsonian patients. Expression of both D3 and D1-D3 receptor heteromers were increased in samples from 6-hydroxy-dopamine-hemilesioned rats rendered dyskinetic by treatment with 3, 4-dihydroxyphenyl-L-alanine (L-DOPA). Similar findings were obtained using striatal samples from primates. Radioligand binding studies in the presence of a D3 agonist led in dyskinetic, but not in lesioned or L-DOPA-treated rats, to a higher dopamine sensitivity. Upon D3-receptor activation, the affinity of agonists for binding to the right striatal D1 receptor increased. Excess dopamine coming from L-DOPA medication likely activates D3 receptors thus making right and left striatal D1 receptors equally responsive to dopamine. These results show that dyskinesia occurs concurrently with a right/left striatal balance in D1 receptor-mediated neurotransmission.

Detection of cannabinoid receptors CB1 and CB2 within basal ganglia output neurons in macaques: changes following experimental parkinsonism.

Although type 1 cannabinoid receptors (CB1Rs) are expressed abundantly throughout the brain, the presence of type 2 cannabinoid receptors (CB2Rs) in neurons is still somewhat controversial. Taking advantage of newly designed CB1R and CB2R mRNA riboprobes, we demonstrate by PCR and in situ hybridization that transcripts for both cannabinoid receptors are present within labeled pallidothalamic-projecting neurons of control and MPTP-treated macaques, whereas the expression is markedly reduced in dyskinetic animals. Moreover, an in situ proximity ligation assay was used to qualitatively assess the presence of CB1Rs and CB2Rs, as well as CB1R-CB2R heteromers within basal ganglia output neurons in all animal groups (control, parkinsonian and dyskinetic macaques). A marked reduction in the number of CB1Rs, CB2Rs and CB1R-CB2R heteromers was found in dyskinetic animals, mimicking the observed reduction in CB1R and CB2R mRNA expression levels. The fact that chronic levodopa treatment disrupted CB1R-CB2R heteromeric complexes should be taken into consideration when designing new drugs acting on cannabinoid receptor heteromers.

Calbindin content and differential vulnerability of midbrain efferent dopaminergic neurons in macaques.

Calbindin (CB) is a calcium binding protein reported to protect dopaminergic neurons from degeneration. Although a direct link between CB content and differential vulnerability of dopaminergic neurons has long been accepted, factors other than CB have also been suggested, particularly those related to the dopamine transporter. Indeed, several studies have reported that CB levels are not causally related to the differential vulnerability of dopaminergic neurons against neurotoxins. Here we have used dual stains for tyrosine hydroxylase (TH) and CB in 3 control and 3 MPTP-treated monkeys to visualize dopaminergic neurons in the ventral tegmental area (VTA) and in the dorsal and ventral tiers of the substantia nigra pars compacta (SNcd and SNcv) co-expressing TH and CB. In control animals, the highest percentages of co-localization were found in VTA (58.2%), followed by neurons located in the SNcd (34.7%). As expected, SNcv neurons lacked CB expression. In MPTP-treated animals, the percentage of CB-ir/TH-ir neurons in the VTA was similar to control monkeys (62.1%), whereas most of the few surviving neurons in the SNcd were CB-ir/TH-ir (88.6%). Next, we have elucidated the presence of CB within identified nigrostriatal and nigroextrastriatal midbrain dopaminergic projection neurons. For this purpose, two control monkeys received one injection of Fluoro-Gold into the caudate nucleus and one injection of cholera toxin (CTB) into the postcommissural putamen, whereas two more monkeys were injected with CTB into the internal division of the globus pallidus (GPi). As expected, all the nigrocaudate- and nigroputamen-projecting neurons were TH-ir, although surprisingly, all of these nigrostriatal-projecting neurons were negative for CB. Furthermore, all the nigropallidal-projecting neurons co-expressed both TH and CB. In summary, although CB-ir dopaminergic neurons seem to be less prone to MPTP-induced degeneration, our data clearly demonstrated that these neurons are not giving rise to nigrostriatal projections and indeed CB-ir/TH-ir neurons only originate nigroextrastriatal projections.

Phosphodiesterase inhibition in cognitive decline.

Understanding the cellular and molecular processes involved in learning and memory will help in the development of safe and effective cognitive enhancers. The cAMP response element-binding (CREB) may be a universal modulator of processes required for memory formation, and increasing the levels of second messengers like cAMP and cGMP could ultimately lead to CREB activation. Phosphodiesterase (PDE) inhibitors regulate signaling pathways by elevating cAMP and/or cGMP levels, and they have been demonstrated to improve learning and memory in a number of rodent models of impaired cognition. The aim of this review is to summarize the outstanding progress that has been made in the application of PDE inhibitors for memory dysfunction. In addition, we have introduced some recent data we generated demonstrating that tadalafil could be considered as an optimal candidate for drug re-positioning and as a good candidate to enhance cognition.

L-DOPA-treatment in primates disrupts the expression of A(2A) adenosine-CB(1) cannabinoid-D(2) dopamine receptor heteromers in the caudate nucleus.

The molecular basis of priming for L-DOPA-induced dyskinesias in Parkinson's disease (PD), which depends on the indirect pathway of motor control, is not known. In rodents, the indirect pathway contains striatopallidal GABAergic neurons that express heterotrimers composed of A(2A) adenosine, CB(1) cannabinoid and D(2) dopamine receptors that regulate dopaminergic neurotransmission. The present study was designed to investigate the expression of these heteromers in the striatum of a primate model of Parkinson's disease and to determine whether their expression and pharmacological properties are altered upon L-DOPA treatment. By using the recently developed in situ proximity ligation assay and by identification of a biochemical fingerprint, we discovered a regional distribution of A(2A)/CB(1) /D(2) receptor heteromers that predicts differential D(2)-mediated neurotransmission in the caudate-putamen of Macaca fascicularis. Whereas heteromers were abundant in the caudate nucleus of both naïve and MPTP-treated monkeys, L-DOPA treatment blunted the biochemical fingerprint and led to weak heteromer expression. These findings constitute the first evidence of altered receptor heteromer expression in pathological conditions and suggest that drugs targeting A(2A)-CB(1) -D(2) receptor heteromers may be successful to either normalize basal ganglia output or prevent L-DOPA-induced side effects.

Tadalafil crosses the blood-brain barrier and reverses cognitive dysfunction in a mouse model of AD.

Previous studies have demonstrated that cognitive function can be restored in mouse models of Alzheimer's disease (AD) following administration of sildenafil, a specific PDE5 inhibitor (Puzzo et al., 2009; Cuadrado-Tejedor et al.). Another very potent PDE5 inhibitor with a longer half-life and safe in chronic treatments, tadalafil, may represent a better alternative candidate for AD therapy. However, tadalafil was proven unable to achieve similar benefits than those of sildenafil in AD animal models (Puzzo et al., 2009). The lack of efficacy was attributed to inability to cross the blood-brain barrier (BBB). In this paper we first measured the blood and brain levels of tadalafil to prove that the compound crosses BBB and that chronic treatment leads to accumulation in the brain of the J20 transgenic mouse model of AD. We demonstrated the presence of PDE5 mRNA in the brain of the mice and also in the human brain. After a 10 week treatment with either of these PDE5 inhibitors, the performance of the J20 mice in the Morris water maze test improved when compared with the transgenic mice that received vehicle. Biochemical analysis revealed that neither sildenafil nor tadalafil altered the amyloid burden, although both compounds reduced Tau phosphorylation in the mouse hippocampus. This study provides evidence of the potential benefits of a chronic tadalafil treatment in AD therapy. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

Unmasking adenosine 2A receptors (A2ARs) in monkey basal ganglia output neurons using cholera toxin subunit B (CTB).

The A(2A)R has become a therapeutic target in Parkinson disease due to its functional role in the striatum, capable of modulating dopaminergic neurotransmission in the basal ganglia. No conclusive evidence, however, has been provided to demonstrate the existence of A(2A)Rs in the output nuclei of the basal ganglia: the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNr). Using immunohistochemistry and in situ hybridization techniques we have confirmed the presence of A(2A)Rs in both the striatum (medium spiny and cholinergic neurons) and the external segment of the globus pallidus (GPe), in the monkey. The antibody routinely used to label A(2A)Rs failed to detect A(2A)R-positive neurons in the GPi and SNr, however, in situ hybridization showed that A(2A)R mRNA transcripts were indeed present in both these nuclei. Surprisingly, by labeling pallidothalamic and nigrothalamic projection neurons originating in the GPi and SNr with the neuronal retrograde tracer cholera toxin subunit B (CTB), the receptor protein was unmasked and detectable using the antibody. This unmasking of the protein was specific to CTB and not an artifact of the tracer. We have shown unequivocally that the A(2A)R is present in the output nuclei of the primate basal ganglia, however, to be able to detect the receptor immunohistochemically, unmasking the protein with CTB was necessary. The presence of A(2A)Rs in the GPi and SNr suggests that these output nuclei could be targeted therapeutically in Parkinson disease to restore abnormal activity in the basal ganglia.

Pallidothalamic-projecting neurons in Macaca fascicularis co-express GABAergic and glutamatergic markers as seen in control, MPTP-treated and dyskinetic monkeys.

GABAergic neurons within the internal division of the globus pallidus (GPi) are the main source of basal ganglia output reaching the thalamic ventral nuclei in monkeys. Following dopaminergic denervation, pallidothalamic-projecting neurons are known to be hyperactive, whereas a reduction in GPi activity is typically observed in lesioned animals showing levodopa-induced dyskinesia. Besides the mRNAs coding for GABAergic markers (GAD65 and GAD67), we show that all GPi neurons innervating thalamic targets also express transcripts for the isoforms 1 and 2 of the vesicular glutamate transporter (vGlut1 and vGlut2 mRNA). Indeed, dual immunofluorescent detection of GAD67 and vGlut1/2 confirmed the data gathered from in situ hybridization experiments, therefore demonstrating that the detected mRNAs are translated into the related proteins. Furthermore, the dopaminergic lesion resulted in an up-regulation of expression levels for both GAD65 and GAD67 mRNA within identified pallidothalamic-projecting neurons. This was coupled with a down-regulation of GAD65/67 mRNA expression levels in GPi neurons innervating thalamic targets in monkeys showing levodopa-induced dyskinesia. By contrast, the patterns of gene expression for both vGlut1 and vGlut2 mRNAs remained unchanged across GPi projection neurons in control, MPTP-treated and dyskinetic monkeys. In summary, both GABAergic and glutamatergic markers were co-expressed by GPi efferent neurons in primates. Although the status of the dopaminergic system directly modulates the expression levels of GAD65/67 mRNA, the observed expression of vGlut1/2 mRNA is not regulated by either dopaminergic removal or by continuous stimulation with dopaminergic agonists.