D1 dopamine receptor stimulation impairs striatal proteasome activity in Parkinsonism through 26S proteasome disassembly.

Among the mechanisms underlying the development of L-dopa-induced dyskinesia (LID) in Parkinson's disease, complex alterations in dopamine signaling in D1 receptor (D1R)-expressing medium spiny striatal neurons have been unraveled such as, but not limited to, dysregulation of D1R expression, lateral diffusion, intraneuronal trafficking, subcellular localization and desensitization, leading to a pathological anchorage of D1R at the plasma membrane. Such anchorage is partly due to a decreased proteasomal activity that is specific of the L-dopa-exposed dopamine-depleted striatum, results from D1R activation and feeds-back the D1R exaggerated cell surface abundance. The precise mechanisms by which L-dopa affects striatal proteasome activity remained however unknown. We here show, in a series of in vitro ex vivo and in vivo models, that such rapid modulation of striatal proteasome activity intervenes through D1R-mediated disassembly of the 26S proteasome rather than change in transcription or translation of proteasome or proteasome subunits intraneuronal relocalization.

PSD-95 expression controls L-DOPA dyskinesia through dopamine D1 receptor trafficking.

L-DOPA-induced dyskinesia (LID), a detrimental consequence of dopamine replacement therapy for Parkinson's disease, is associated with an alteration in dopamine D1 receptor (D1R) and glutamate receptor interactions. We hypothesized that the synaptic scaffolding protein PSD-95 plays a pivotal role in this process, as it interacts with D1R, regulates its trafficking and function, and is overexpressed in LID. Here, we demonstrate in rat and macaque models that disrupting the interaction between D1R and PSD-95 in the striatum reduces LID development and severity. Single quantum dot imaging revealed that this benefit was achieved primarily by destabilizing D1R localization, via increased lateral diffusion followed by increased internalization and diminished surface expression. These findings indicate that altering D1R trafficking via synapse-associated scaffolding proteins may be useful in the treatment of dyskinesia in Parkinson's patients.

L-DOPA impairs proteasome activity in parkinsonism through D1 dopamine receptor.

Aberrant membrane localization of dopamine D(1) receptor (D1R) is associated with L-DOPA-induced dyskinesia (LID), a major complication of L-DOPA treatment in Parkinson's disease (PD). Since the proteasome plays a central role in modulating neuronal response through regulation of neurotransmitter receptor intraneuronal fate, we hypothesized that the ubiquitine-proteasome proteolytic pathway could be impaired in LID. Those LIDs are actually associated with a striatum-specific decrease in proteasome catalytic activity and accumulation of polyubiquitinated proteins in experimental rodent and monkey parkinsonism. We then demonstrated that such decreased proteasome catalytic activity (1) results from D1R activation and (2) feed-back the D1R abnormal trafficking, i.e., its exaggerated cell surface abundance. We further showed that the genetic invalidation of the E3 ubiquitin-protein ligase parkin PD gene leads to exaggerated abnormal involuntary movements compared with wild-type mice. We thus established in an unprecedented series of experimental models that impairment of the ubiquitine-proteasome system at specific nodes (E3 ligase parkin, polyubiquitination, proteasome catalytic activity) leads to the same phenomenon, i.e., aberrant behavioral response to dopamine replacement therapy in PD, highlighting the intimate interplay between dopamine receptor and proteasome activity in a nondegenerative context.

Lentiviral overexpression of GRK6 alleviates L-dopa-induced dyskinesia in experimental Parkinson's disease.

Parkinson's disease is caused primarily by degeneration of brain dopaminergic neurons in the substantia nigra and the consequent deficit of dopamine in the striatum. Dopamine replacement therapy with the dopamine precursor l-dopa is the mainstay of current treatment. After several years, however, the patients develop l-dopa-induced dyskinesia, or abnormal involuntary movements, thought to be due to excessive signaling via dopamine receptors. G protein-coupled receptor kinases (GRKs) control desensitization of dopamine receptors. We found that dyskinesia is attenuated by lentivirus-mediated overexpression of GRK6 in the striatum in rodent and primate models of Parkinson's disease. Conversely, reduction of GRK6 concentration by microRNA delivered with lentiviral vector exacerbated dyskinesia in parkinsonian rats. GRK6 suppressed dyskinesia in monkeys without compromising the antiparkinsonian effects of l-dopa and even prolonged the antiparkinsonian effect of a lower dose of l-dopa. Our finding that increased availability of GRK6 ameliorates dyskinesia and increases duration of the antiparkinsonian action of l-dopa suggests a promising approach for controlling both dyskinesia and motor fluctuations in Parkinson's disease.

Pharmacological analysis demonstrates dramatic alteration of D1 dopamine receptor neuronal distribution in the rat analog of L-DOPA-induced dyskinesia.

We have associated behavioral, pharmacological, and quantitative immunohistochemical study in a rat analog of l-DOPA-induced dyskinesia to understand whether alterations in dopamine receptor fate in striatal neurons may be involved in mechanisms leading to movement abnormalities. Detailed analysis at the ultrastructural level demonstrates specific alterations of dopamine D(1) receptor (D(1)R) subcellular localization in striatal medium spiny neurons in l-DOPA-treated 6-hydroxydopamine-lesioned rats with abnormal involuntary movements (AIMs). This includes exaggerated D(1)R expression at the plasma membrane. However, D(1)R retains ability of internalization, as a challenge with the potent D(1)R agonist SKF-82958 induces a strong decrease of labeling at membrane in animals with AIMs. Since a functional cross talk between D(1)R and D(3)R has been suggested, we hypothesized that their coactivation by dopamine derived from l-DOPA might anchor D(1)R at the membrane. Accordingly, cotreatment with l-DOPA and the D(3)R antagonist ST 198 restores normal level of membrane-bound D(1)R. Together, these results demonstrate that AIMs are related to abnormal D(1)R localization at the membrane and intraneuronal trafficking dysregulation, and suggest that strategies aiming at disrupting the D(1)R-D(3)R cross talk might reduce l-DOPA-induced dyskinesia by reducing D(1)R availability at the membrane.

Antagonizing L-type Ca2+ channel reduces development of abnormal involuntary movement in the rat model of L-3,4-dihydroxyphenylalanine-induced dyskinesia.

BACKGROUND: Chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment of Parkinson's disease (PD) leads to debilitating involuntary movements, termed L-DOPA-induced dyskinesia. Striatofugal medium spiny neurons (MSN) lose their dendritic spines and cortico-striatal glutamatergic synapses in PD and in experimental models of DA depletion. This loss of connectivity is triggered by a dysregulation of intraspine Cav1.3 L-type Ca2+ channels. Here we address the possible implication of DA denervation-induced spine pruning in the development of L-DOPA-induced dyskinesia. METHODS: The L-type Ca2+ antagonist, isradipine was subcutaneously delivered to rats at the doses of .05, .1, or .2 mg/kg/day, for 4 weeks, starting the day after a unilateral nigrostriatal 6-hydroxydopamine (6-OHDA) lesion. Fourteen days later, L-DOPA treatment was initiated. RESULTS: Isradipine-treated animals displayed a dose-dependent reduction in L-DOPA-induced rotational behavior and abnormal involuntary movements. Dendritic spine counting at electron microscopy level showed that isradipine (.2 mg/kg/day) prevented the 6-OHDA-induced spine loss and normalized preproenkephalin-A messenger RNA expression. Involuntary movements were not reduced when isradipine treatment was started concomitantly with L-DOPA. CONCLUSIONS: These results indicate that isradipine, at a therapeutically relevant dose, might represent a treatment option for preventing L-DOPA-induced dyskinesia in PD.

Cytoplasmic expression of human telomerase catalytic protein (hTERT) in neutrophils: an immunoelectron microscopy study.

Human telomerase comprises a catalytic protein subunit (hTERT) and an RNA subunit (hTR). Telomerase extends chromosome ends in compensation for the attrition of the telomeres during replication. In this work, the authors explore the expression of hTERT and hTR in neutrophils, respectively by immunochemistry techniques and in situ hybridization. hTERT was strongly expressed in neutrophils cytoplasm. The ultrastructural study showed that the gold particles were not associated with specific organelles but scattered in the cytosol. hTR was not expressed. hTERT is expressed in the cytoplasm of neutrophils, but its roles-eventually extratelomeric effects-remain to be elucidated.

Differences in ultrastructural localization of dopaminergic D1 receptors between dorsal striatum and nucleus accumbens in the rat.

Dopaminergic receptors of the D1 type are highly expressed in the dorsal striatum and nucleus accumbens. In the dorsal striatum, they are rarely observed on presynaptic terminals. However, their subcellular localization in the nucleus accumbens core and shell had not been compared to that of dorsal striatum. Here we investigated the subcellular localization of D1 receptors in these three brain regions using immunogold labeling and electron microscopy. We showed that, among all presynaptic terminals forming asymmetric contact with dendritic processes, the percentage of D1R immunoreactive terminals was low in the dorsal striatum (8.2%), but reached in the nucleus accumbens core and shell 25.5 and 29%, respectively. These observations are consistent with electrophysiological studies, which showed that D1 stimulation inhibits the response of target neurons to glutamatergic input via presynaptic mechanisms in the nucleus accumbens but not in the dorsal striatum.

Altered D(1) dopamine receptor trafficking in parkinsonian and dyskinetic non-human primates.

Dyskinesias represent a debilitating complication of levodopa therapy for Parkinson's disease (PD). While we recently demonstrated that levodopa-induced dyskinesia results from increased dopamine D(1) receptor-mediated transmission, we also questioned the possible role of subcellular localization of D(1) and D(2) receptors in mediating these effects as we previously showed that D(1) receptors undergo differential trafficking in striatal neurons of non-dyskinetic PD patients. Taking advantage of a monkey brain bank, we here report changes affecting the cellular and subcellular distribution of D(1) and D(2) dopamine receptors within the striatum of three experimental groups: normal, parkinsonian and dyskinetic L-dopa-treated parkinsonian animals. Our studies at both light and electron microscopy levels show a recruitment of D(1) receptor at the plasma membrane of striatal neurons in the parkinsonian animals and a strong increase of D(1) expression both at the membrane and in cytoplasm of dyskinetic animals, whereas D(2) receptor distribution is only modestly affected in all conditions. Our results rule out the hypothesis of a pathological overinternalization of dopamine receptors in levodopa-induced dyskinesia but raise the possibility for involvement of D(1) receptors in the priming phenomenon through massive and sudden internalization in response to the first ever administration of L-dopa and for an altered homologous desensitization mechanism in dyskinesia leading to an increased availability of D(1) receptors at membrane. Further experiments including parkinsonian monkeys chronically treated with L-dopa that show no dyskinesia and parkinsonian monkeys treated only once with L-dopa are now necessary to confirm our hypothesis.

Enhanced preproenkephalin-B-derived opioid transmission in striatum and subthalamic nucleus converges upon globus pallidus internalis in L-3,4-dihydroxyphenylalanine-induced dyskinesia.

BACKGROUND: A role for enhanced opioid peptide transmission has been suggested in the genesis of levodopa-induced dyskinesia. However, basal ganglia nuclei other than the striatum have not been regarded as potential sources, and the opioid precursors have never been quantified simultaneously with the levels of opioid receptors at the peak of dyskinesia severity. METHODS: The levels of messenger RNA (mRNA) encoding the opioid precursors preproenkephalin-A and preproenkephalin-B in the striatum and the subthalamic nucleus and the levels of mu, delta, and kappa opioid receptors were measured within the basal ganglia of four groups of nonhuman primates killed at the peak of effect: normal, parkinsonian, parkinsonian chronically-treated with levodopa without exhibiting dyskinesia, and parkinsonian chronically-treated with levodopa showing overt dyskinesia. RESULTS: Dyskinesia are associated with reduction in opioid receptor binding and specifically of kappa and mu receptor binding in the globus pallidus internalis (GPi), the main output structure of the basal ganglia. This decrease was correlated with enhancement of the expression of preproenkephalin-B mRNA but not that of preproenkephalin-A in the striatum and the subthalamic nucleus. CONCLUSIONS: Abnormal transmission of preproenkephalin-B-derived opioid coming from the striatum and the subthalamic nucleus converges upon GPi at the peak of dose to induce levodopa-induced dyskinesia.

Receptor recycling mediates plasma membrane recovery of dopamine D1 receptors in dendrites and axons after agonist-induced endocytosis in primary cultures of striatal neurons.

The pharmacological stimulation of G-protein-coupled receptor induces receptor internalization. Receptor's fate after the step of internalization remains poorly characterized despite its incidence on the neuronal responsiveness. In this context, we studied the dopamine (DA) D1 receptor (D1R) trafficking in a model of striatal neuronal culture that endogenously express the D1R. We first characterized by immunohistochemistry the spatial distribution of the compartments involved in the endocytic pathways and then the D1R trafficking in dendrites and axons. In dendrites, immunohistochemical analysis showed that acute stimulation by the D1R agonist SKF 82958 (1 microM) induces an internalization of D1R in early endosomes labeled with Alexa-488-conjugated transferrin. We show that, 20 min after removal of the agonist, the D1R immunolabeling pattern returns to the basal state in dendrites and in axons. Recovery was unaffected by cycloheximide (70 microM) but was prevented by monensin (100 microM) that inhibits endosomal acidification and receptor recycling. These data suggest that dendritic and axonal D1Rs are internalized after agonist stimulation and targeted to the recycling pathway demonstrating that the machinery involved in GPCR endocytosis and recycling is functional both in dendrites and in axons. Temporal characteristics observed for the recovery of D1R density to the basal state and those observed for the resensitization process strongly suggest that D1R recycling supports the receptor resensitization.

Intraneuronal trafficking of G-protein-coupled receptors in vivo.

In vitro studies have widely demonstrated that the abundance and availability of G-protein-coupled receptors (GPCRs) at the cell surface is regulated by the neuronal environment and is the result of complex intraneuronal trafficking. However, this regulation is still poorly understood in vivo. Modulation of receptor availability at the neuronal membrane is a key event in the regulation of neuronal functions (e.g. neurotransmitter release or neuronal excitability in physiological, pathological or therapeutic conditions). We discuss the effects of duration of receptor stimulation (acute versus chronic) on the intraneuronal trafficking of GPCRs in vivo, and we show that this trafficking might differ according to subcellular compartment (soma, dendrites or axon terminals).

Levodopa-induced dyskinesia in MPTP-treated macaques is not dependent on the extent and pattern of nigrostrial lesioning.

The extent of nigrostriatal denervation is presumed to play a role in the genesis of levodopa-induced dyskinesia. Yet some parkinsonian patients who have been treated over a long period do not develop dyskinesia, raising the possibility that the pattern of denervation is as important as the extent of lesioning as a risk factor. Here we study the extent and pattern of nigrostriatal denervation in a homogeneous population of parkinsonian macaque monkeys chronically treated with levodopa. Based on the characteristics of the lesioning, non-dyskinetic animals could not be differentiated from those with dyskinesia. Indeed, the number of tyrosine-hydroxylase (TH)-immunopositive neurons in the substantia nigra pars compacta, striatal dopamine transporter (DAT) binding and TH immunostaining, as well as the overall TH striatal content measured by Western blotting were identical. Moreover, the patterns of lesioning assessed by a detailed analysis of the TH- and DAT-immunopositive striatal fibers were comparable in all functional quadrants and at all rostro-caudal levels considered. These data indicate that neither the extent nor the pattern of nigrostriatal lesioning are sufficient to explain the occurrence of levodopa-induced dyskinesia.

Pathogenesis of levodopa-induced dyskinesia: focus on D1 and D3 dopamine receptors.

Involuntary movements, or dyskinesia, represent a debilitating complication of levodopa therapy for Parkinson's disease. Taking advantage of a monkey brain bank constituted to study the pathophysiology of levodopa-induced dyskinesia, we here report the changes affecting D1, D2 and D3 dopamine receptors within the striatum of four experimental groups of non-human primates: normal, parkinsonian, parkinsonian treated with levodopa without or with dyskinesia. We also report the possible role of arrestin and G protein-coupled receptor kinases.

Aging and subcellular localization of m2 muscarinic autoreceptor in basalocortical neurons in vivo.

By using immunohistochemical approaches at the light and electron microscopic levels, we have shown that aging modifies the subcellular distribution of the m2 muscarinic autoreceptor (m2R) differentially at somato-dendritic postsynaptic sites and at axonal presynaptic sites in cholinergic basalocortical neurons, in vivo. In cholinergic perikarya and dendrites of the nucleus basalis magnocellularis (NBM), aging is associated with a decrease of the density of m2R at the plasma membrane and in the cytoplasm, suggesting a decrease of the total number of m2R in the somato-dendritic field. In contrast, the number of substance P receptors per somato-dendritic surface was not affected. In the frontal cortex (FC), we have shown a decrease of cytoplasmic m2R density also leading to a decrease of the number of m2R per surface of varicosities but with no change of the density of m2R at the membrane. Our results suggest that the decrease of m2R in the somato-dendritic field of the NBM, but not a modification of the number of presynaptic m2 autoreceptors at the plasma membrane in the FC, could contribute to the decrease of the efficacy of cholinergic transmission observed with aging in the rat.

Mutation of TP53 gene is involved in carcinogenesis of hepatic undifferentiated (embryonal) sarcoma of the adult, in contrast with Wnt or telomerase pathways: an immunohistochemical study of three cases with genomic relation in two cases.

BACKGROUND/AIMS: Hepatic undifferentiated (embryonal) sarcoma (HUS) is an exceptional hepatic malignant tumor in adults. Genetic studies were never reported in adult cases. METHODS: In this study concerning three cases of HUS occurring in adult, we studied the three classical ways of carcinogenesis i.e. the TP53 (p53), Wnt (CTNNB1/beta-catenin and AXIN1) and telomerase (hTERT) pathways. We studied the expression of p53, beta-catenin and telomerase catalytic subunit hTERT by immunohistochemistry in the three cases; we determined TP53 gene mutation in two cases and the genome-wide allelotype, AXIN1, and CTNNB1/beta-catenin gene mutation in one case. RESULTS: Immunohistochemistry showed an overexpression of p53 in more than 80% of tumoral cells; furthermore, mutations of TP53 were observed in two cases, involving the sequence-specific DNA binding domain. In contrast, no mutation was found in CTNNB1/beta-catenin and AXIN1 genes. Tumoral cells did not show hTERT staining nor nuclear expression of beta-catenin. In addition, allelotype analysis in one case showed loss of heterozygosity of chromosome 7p, 11p, 17p, 22q, and allelic imbalance of 1p, 8p, 20q. CONCLUSIONS: In this report of HUS in three adult patients, we emphasize the role of TP53 pathway in carcinogenesis of this rare tumor. This point could be of interest for therapeutic strategies.

Involvement of sensorimotor, limbic, and associative basal ganglia domains in L-3,4-dihydroxyphenylalanine-induced dyskinesia.

Dyskinesia represents a debilitating complication of L-3,4-dihydroxyphenylalanine (L-dopa) therapy for Parkinson's disease. Such motor manifestations are attributed to pathological activity in the motor parts of basal ganglia. However, because consistent funneling of information takes place between the sensorimotor, limbic, and associative basal ganglia domains, we hypothesized that nonmotor domains play a role in these manifestations. Here we report the changes in 2-deoxyglucose (2-DG) accumulation in the sensorimotor, limbic, and associative domains of basal ganglia and thalamic nuclei of four groups of nonhuman primates: normal, parkinsonian, parkinsonian chronically treated with L-dopa without exhibiting dyskinesia, and parkinsonian chronically treated with L-dopa and exhibiting overt dyskinesia. Although nondyskinetic animals display a rather normalized metabolic activity, dyskinetic animals are distinguished by significant changes in 2-DG accumulation in limbic- and associative-related structures and not simply in sensorimotor-related ones, suggesting that dyskinesia is linked to a pathological processing of limbic and cognitive information. We propose that these metabolic changes reflect the underlying neural mechanisms of not simply motor dyskinesias but also affective, motivational, and cognitive disorders associated with long-term exposure to L-dopa.

Increased D1 dopamine receptor signaling in levodopa-induced dyskinesia.

Involuntary movements, or dyskinesia, represent a debilitating complication of levodopa therapy for Parkinson's disease. Although changes affecting D(1) and D(2) dopamine receptors have been studied in association with this condition, no causal relationship has yet been established. Taking advantage of a monkey brain bank constituted to study levodopa-induced dyskinesia, we report changes affecting D(1) and D(2) dopamine receptors within the striatum of normal, parkinsonian, nondyskinetic levodopa-treated parkinsonian, and dyskinetic levodopa-treated parkinsonian animals. Whereas D(1) receptor expression itself is not related to dyskinesia, D(1) sensitivity per D(1) receptor measured by D(1) agonist-induced [(35)S]GTPgammaS binding is linearly related to dyskinesia. Moreover, the striata of dyskinetic animals show higher levels of cyclin-dependent kinase 5 (Cdk5) and of the dopamine- and cAMP-regulated phosphoprotein of 32kDa (DARPP-32). Our data suggest that levodopa-induced dyskinesia results from increased dopamine D(1) receptor-mediated transmission at the level of the direct pathway.

Determination of DNA ploidy by fluorescence in situ hybridization (FISH) in hydatidiform moles: evaluation of FISH on isolated nuclei.

In the past 20 years, the diagnosis of hydatidiform moles has become more difficult because of the widespread use of early uterine evacuation. Differentiating hydropic degeneration, partial, and complete moles is important because of their different prognosis. However, clinical diagnosis is less obvious, and the pathologist has to separate the different entities on the basis of very subtle morphologic criteria. In difficult cases, ploidy may be determined by various methods, including fluorescence in situ hybridization (FISH) on routine histological sections from paraffin-embedded specimens. However, FISH analysis is often difficult because of the presence of numerous truncated nuclei. In this context, we have tested the advantages of FISH on isolated nuclei, a well-known variant of the technique that might be more sensitive. We reviewed 24 cases of products of abortion: hydropic degenerations, complete hydatidiform moles, partial moles, and nonmolar triploidies. After histological review, FISH on isolated nuclei proved conclusive in all cases. The results could be easily interpreted thanks to the reduced number of truncated nuclei. The percentage of cells with 2 signals was always >70% in the diploid cases and >60% in the triploid cases. In conclusion, this sensitive technique seems to be a valuable tool for the diagnosis of moles.

"In vivo" intraneuronal trafficking of G protein coupled receptors in the striatum: regulation by dopaminergic and cholinergic environment.

We have studied "in vivo" neurochemically identified striatal neurons to analyze the localisation and the trafficking of dopamine and acetylcholine G protein coupled receptors (GPCR) (D1R, D2R, m2R and m4R) under the influence of neurotransmitter environment. We have identified receptors in tissue sections through immunohistochemical detection at the light and electron microscopic level. We have identified receptors in normal animals and after acute and chronic stimulations. We have quantified receptors through image analysis at the electron microscopic level in relation to various subcellular compartments. Our results demonstrate that, in normal conditions, GPCRs are mostly associated with plasma membrane of the striatal neurons, mostly at extra-synaptic sites. In certain instances (m4R; D2R), receptors have prominent localisation inside the rough endoplasmic reticulum. Our results also show that two distinct receptors for a same neurotransmitter may have distinct subcellular localisation in a same neuronal population (m2R versus m4R) and that the same neurotransmitter receptor (m4R) can have distinct localisation in distinct neuronal populations (cytoplasm versus cell surface). After acute stimulation, cell surface receptors undergo dramatic subcellular changes that involve plasma membrane depletion, internalisation in endosomes and in multivesicular bodies. Such changes are reversible after the end of the stimulation and are blocked by antagonist action. Chronic stimulation also provokes changes in subcellular localisation with specific pattern: plasma membrane depletion, and exaggerated storage of receptors in rough endoplasmic reticulum and eventually Golgi complex (D1R; m2R and m4R). Decreasing chronic receptor stimulation reverses such changes. These results demonstrate that, "in vivo", in the striatum, GPCRs undergo complex intraneuronal trafficking under the influence of neurochemical environment in conditions that dramatically modulate the number of cell surface receptors available for interaction with neurotransmitters or drugs. This confirms that "in vivo", the trafficking and the subcellular compartmentalization of GPCRs may contribute to regulate neuronal sensitivity and neuronal interactions in physiological, experimental and pathological conditions, including in therapeutic conditions.