Loss of parkin causes endoplasmic reticulum calcium dyshomeostasis by upregulation of reticulocalbin 1.

Increasing evidence suggests that astrocytes play an important role in the progression of Parkinson's disease (PD). Previous studies on our parkin knockout mouse demonstrated a higher accumulation of damaged mitochondria in astrocytes than in surrounding dopaminergic (DA) neurons, suggesting that Parkin plays a crucial role regarding their interaction during PD pathogenesis. In the current study, we examined primary mesencephalic astrocytes and neurons in a direct co-culture system and discovered that the parkin deletion causes an impaired differentiation of mesencephalic neurons. This effect required the parkin mutation in astrocytes as well as in neurons. In Valinomycin-treated parkin-deficient astrocytes, ubiquitination of Mitofusin 2 was abolished, whereas there was no significant degradation of the outer mitochondrial membrane protein Tom70. This result may explain the accumulation of damaged mitochondria in parkin-deficient astrocytes. We examined differential gene expression in the substantia nigra region of our parkin-KO mouse by RNA sequencing and identified an upregulation of the endoplasmic reticulum (ER) Ca2+ -binding protein reticulocalbin 1 (RCN1) expression, which was validated using qPCR. Immunostaining of the SN brain region revealed RCN1 expression mainly in astrocytes. Our subcellular fractionation of brain extract has shown that RCN1 is located in the ER and in mitochondria-associated membranes (MAM). Moreover, a loss of Parkin function reduced ATP-stimulated calcium-release in ER mesencephalic astrocytes that could be attenuated by siRNA-mediated RCN1 knockdown. Our results indicate that RCN1 plays an important role in ER-associated calcium dyshomeostasis caused by the loss of Parkin function in mesencephalic astrocytes, thereby highlighting the relevance of astrocyte function in PD pathomechanisms.

Identification and pharmacological modification of resistance mechanisms to protoporphyrin-mediated photodynamic therapy in human cutaneous squamous cell carcinoma cell lines.

BACKGROUND: Photodynamic therapy (PDT) is clinically approved to treat neoplastic skin diseases such as precursors of cutaneous squamous cell carcinoma (cSCC). In PDT, 5-aminolevulinic acid (5-ALA) drives the selective formation of the endogenous photosensitizer protoporphyrin IX (PpIX). Although 5-ALA PDT is clinically highly effective, resistance might occur due to decreased accumulation of PpIX in certain tumors. Such resistance may be caused by any fundamental step of PpIX accumulation: 5-ALA uptake, PpIX synthesis and PpIX efflux. METHODS: We investigated PpIX accumulation and photodynamically induced cell death in PDT refractory SCC-13, PDT susceptible A431, and normal human epidermal keratinocytes (NHEK). Expression of genes associated with cellular PpIX kinetics was investigated on mRNA and protein level. PpIX accumulation and cell death upon illumination were pharmacologically manipulated using drugs targeting 5-ALA uptake, PpIX synthesis or efflux. RESULTS: The experiments indicate that taurine transporter (SLC6A6) is the major pathway for 5-ALA uptake in cSCC cells, while being less important in NHEK. Downregulation of PpIX synthesis enzymes in SCC-13 was counteracted by methotrexate (MTX) treatment, which restored PpIX formation and cell death. PpIX efflux inhibitors targeting ABC transporters led to significantly increased PpIX accumulation in SCC-13, thereby fully overcoming resistance. CONCLUSIONS: The results indicate a conserved threshold for PpIX accumulation with respect to PDT-resistance. Cells showed increased viability after PDT at PpIX concentrations below 1.5 nM. Selective uptake of 5-ALA via taurine transporter SLC6A6 in cutaneous tumor cells is novel but unrelated to resistance. MTX can partially abrogate resistance by PpIX synthesis enzyme induction, while efflux mechanisms via ABC transporters seem the main driving force and promising drug targets.

Clinical Pharmacokinetics and Safety of a 10% Aminolevulinic Acid Hydrochloride Nanoemulsion Gel (BF-200 ALA) in Photodynamic Therapy of Patients Extensively Affected With Actinic Keratosis: Results of 2 Maximal Usage Pharmacokinetic Trials.

The nanoemulsion-based 10% aminolevulinic acid (ALA) hydrochloride gel BF-200 ALA optimizes epidermal penetration of its active ingredient and is approved for topical photodynamic therapy (PDT) for the treatment of actinic keratosis in the United States and Europe. To characterize systemic absorption from dermal application during PDT, ALA and its key active metabolite protoporphyrin IX (PpIX) were analyzed in 2 maximal usage pharmacokinetic trials (MUsT) in patients severely affected with actinic keratosis. The primary objective of both MUsTs was to assess baseline-adjusted plasma concentration-time curves for ALA and PpIX after a single PDT treatment applying either 2 g (1 tube) of BF-200 ALA on the face (MUsT-1) or applying 6 g (3 tubes) of BF-200 ALA on the face/scalp or body periphery (MUsT-2), to 20 or 60 cm2 , respectively. All PDTs were performed using red light at around 635 nm wavelength. Safety and tolerability were documented along with pharmacokinetics. In both MUsTs, ALA plasma concentrations were transiently increased to a maximum concentration at about 2.5 to 3.3 times above endogenous baseline with time to maximum concentration at ≈3 hours after dosing. Plasma levels subsequently returned to baseline within 10 hours after dosing. Overall baseline-adjusted mean area under the baseline-adjusted plasma concentration-time curve from time zero to the last sampling time point at which the concentration was at or above the lower limit of quantification ranged from 142.8 to 146.2, indicating that a similar, minor fraction of topical ALA is systemically absorbed under both dosing regimens. Systemic PpIX exposure after administration of either dose of BF-200 ALA was equally minimal. Application site skin reactions were treatment area size-related, albeit transient and consistent with the known safety profile of BF-200 ALA.

Histological investigations on the dura mater vascular system of mice.

The human dura mater encephali is a well innervated and vascularized membrane. Its vascular system plays a crucial role in disorders and pathological cases like dural hematoma, meningitis, and different headache types. To investigate these diseases mouse models are increasingly being used. However, the literature on the vascular system of the mouse dura mater is sparse and explicit studies concerned exclusively with its vasculature are lacking. Here we present a detailed light and scanning electron microscopic investigation of the supratentorial dura mater of the mouse species, with a focus on the largest part of it, the parietal dura mater. By utilizing different immunohistochemical and classical staining methods, a "cartography" of the vascular system was achieved. Additionally, the different blood vessel types with their mural cells were characterized. In contrast to humans, no arteries were found in the mouse parietal dura mater. Its supply is assured through frontolateral and occipital localized arteriolar branches. These arteriolar vessels exhibit in some specimens arteriolar anastomoses with one another. The venous blood is drained to the superior sagittal and transverse sinus through satellite venules accompanying the arterioles or through solitary venules. In all samples, large ruptured venules were identified in the frontolateral dural area. Scanning electron microscopy revealed that these vessels were ruptured on the dorsal side (skull bones-oriented side) of the dura. Our results contribute to the anatomical data on the mouse species and may set up a basis for fundamental investigation of disorders, for which the role of dural blood vessels is not yet clarified.

Helional-induced activation of human olfactory receptor 2J3 promotes apoptosis and inhibits proliferation in a non-small-cell lung cancer cell line.

Studies within the last decade have localized the functional expression of olfactory receptors (ORs) to cells outside of the olfactory epithelium. In human hepatocarcinoma and prostate cancer cells, the activation of ORs by odors modulates elementary physiological processes and leads to an inhibitory effect on proliferation. Cells of the respiratory tract are in direct contact with the surrounding air, in which a myriad of volatile molecules, especially odors, are present. Non-small-cell lung cancer (NSCLC) has a high prevalence, a high mortality rate and is difficult to treat. NSCLC cells are nearly resistant to common chemotherapeutic approaches, and surgical resection provides the only possible chance of a cure for most patients. New approaches for the treatment of NSCLC are the focus of many current studies. Thus, it is of interest to characterize the functional expression of ORs in cancer cells of the lung and to investigate the impact of ORs on pathophysiological processes. In the present study, we demonstrate that the expression of OR2J3 and cytosolic Ca2+ increase via the activation of the agonist helional in the NSCLC cell line A549. We further investigated the underlying pathway. Helional triggers phoshoinositol-3 kinase (PI3K), signaling the release of intracellular Ca2+ and phosphorylation of ERK. We observed that OR2J3 activation induces apoptosis and inhibits cell proliferation and migration in long-term stimulus experiments with helional. Our study provides the first evidence of the functional expression of an OR in NSCLC cells and its putative therapeutic impact.

Olfactory Receptors Modulate Physiological Processes in Human Airway Smooth Muscle Cells.

Pathophysiological mechanisms in human airway smooth muscle cells (HASMCs) significantly contribute to the progression of chronic inflammatory airway diseases with limited therapeutic options, such as severe asthma and COPD. These abnormalities include the contractility and hyperproduction of inflammatory proteins. To develop therapeutic strategies, key pathological mechanisms, and putative clinical targets need to be identified. In the present study, we demonstrated that the human olfactory receptors (ORs) OR1D2 and OR2AG1 are expressed at the RNA and protein levels in HASMCs. Using fluorometric calcium imaging, specific agonists for OR2AG1 and OR1D2 were identified to trigger transient Ca(2+) increases in HASMCs via a cAMP-dependent signal transduction cascade. Furthermore, the activation of OR2AG1 via amyl butyrate inhibited the histamine-induced contraction of HASMCs, whereas the stimulation of OR1D2 with bourgeonal led to an increase in cell contractility. In addition, OR1D2 activation induced the secretion of IL-8 and GM-CSF. Both effects were inhibited by the specific OR1D2 antagonist undecanal. We herein provide the first evidence to show that ORs are functionally expressed in HASMCs and regulate pathophysiological processes. Therefore, ORs might be new therapeutic targets for these diseases, and blocking ORs could be an auspicious strategy for the treatment of early-stage chronic inflammatory lung diseases.

Hypoxia facilitates neurogenic dural plasma protein extravasation in mice: a novel animal model for migraine pathophysiology.

Migraine animal models generally mimic the onset of attacks and acute treatment processes. A guinea pig model used the application of meta-chlorophenylpiperazine (mCPP) to trigger immediate dural plasma protein extravasation (PPE) mediated by 5-HT2B receptors. This model has predictive value for antimigraine drugs but cannot explain the delayed onset of efficacy of 5-HT2B receptor antagonists when clinically used for migraine prophylaxis. We found that mCPP failed to induce dural PPE in mice. Considering the role 5-HT2B receptors play in hypoxia-induced pulmonary vessel muscularization, we were encouraged to keep mice under hypoxic conditions and tested whether this treatment will render them susceptible to mCPP-induced dural PPE. Following four-week of hypoxia, PPE, associated with increased transendothelial transport, was induced by mCPP. The effect was blocked by sumatriptan. Chronic application of 5-HT2B receptor or nitric oxide synthase blockers during hypoxia prevented the development of susceptibility. Here we present a migraine model that distinguishes between a migraine-like state (hypoxic mice) and normal, normoxic mice and mimics processes that are related to chronic activation of 5-HT2B receptors under hypoxia. It seems striking, that chronic endogenous activation of 5-HT2B receptors is crucial for the sensitization since 5-HT2B receptor antagonists have strong, albeit delayed migraine prophylactic efficacy.

BF-1--a novel selective 5-HT2B receptor antagonist blocking neurogenic dural plasma protein extravasation in guinea pigs.

Serotonin 5-HT2B receptor antagonists have been proposed as migraine prophylactic drugs, but previously available 5-HT2B receptor antagonists displayed multiple monoaminergic side effects and had to be withdrawn from the market. Here, we set out to identify a novel antagonist with high affinity and selectivity towards 5-HT2B receptors. To test the affinity of new compounds towards various receptors, we generated a broad series of cells functionally coupling human monoaminergic receptors to luciferase. Using the cell lines we revealed pimethixene (1-methyl-4-(9H-thioxanthen-9-ylidene)piperidine) as highly potent, albeit non-selective 5-HT2B receptor antagonist and optimized its chemical structure to create highly potent and selective 5-HT2B receptor antagonists. We selected the methoxythioxanthene BF-1 for further analysis. In comparison to pimethixene, it lacked high affinities to 5-HT1A, 5-HT2A, 5-HT2C, histamine H1, dopamine D1 and D2 as well as muscarinic M1 and M2 receptors. BF-1 was tested as potential migraine prophylactic drug by blocking meta-chlorophenylpiperazine, (mCPP) or BW723C86 (5-((thiophen-2-yl)methoxy)-α-methyltryptamine) induced neurogenic dural plasma protein extravasation in a guinea pig model that may resemble a migraine attack. BF-1 was significantly more potent in this assay compared to the well know non-selective 5-HT2B antagonists, methysergide ((6aR,9R)-N-[(2S)-1-Hydroxybutan-2-yl]-4,7-dimethyl-6,6a,8,9-tetrahydroindolo[4,3-fg]quinoline-9-carboxamide) or pizotifen (4-(1-methyl-4-piperidylidine)-9,10-dihydro-4H-benzo-[4,5]cyclohepta[1,2]-thiophene). Therefore, we propose BF-1 as a new compound that may be developed for prophylactic migraine treatment without the typical monoaminergic side effects.

Olfaction in three genetic and two MPTP-induced Parkinson's disease mouse models.

Various genetic or toxin-induced mouse models are frequently used for investigation of early PD pathology. Although olfactory impairment is known to precede motor symptoms by years, it is not known whether it is caused by impairments in the brain, the olfactory epithelium, or both. In this study, we investigated the olfactory function in three genetic Parkinson's disease (PD) mouse models and mice treated with MPTP intraperitoneally and intranasally. To investigate olfactory function, we performed electro-olfactogram recordings (EOGs) and an olfactory behavior test (cookie-finding test). We show that neither a parkin knockout mouse strain, nor intraperitoneal MPTP treated animals display any olfactory impairment in EOG recordings and the applied behavior test. We also found no difference in the responses of the olfactory epithelium to odorants in a mouse strain over-expressing doubly mutated α-synuclein, while this mouse strain was not suitable to test olfaction in a cookie-finding test as it displays a mobility impairment. A transgenic mouse expressing mutated α-synuclein in dopaminergic neurons performed equal to control animals in the cookie-finding test. Further we show that intranasal MPTP application can cause functional damage of the olfactory epithelium.

Synaptic adhesion molecules in Cadm family at the neuromuscular junction.

RA175/SynCAM1/Cadm1 (Cadm1), a member of the immunoglobulin superfamily, is a synaptic cell adhesion molecule that has a PDZ-binding motif at the C-terminal region. It promotes the formation of presynaptic terminals and induces functional synapses in the central nervous system. Cadm1-deficient (knockout [KO]) mice show behavioral abnormalities, including excessive aggression and anxiety, but do not show any symptoms of neuromuscular disorder, although neuromuscular junctions (NMJs) have structures similar to synapses. We have examined the expression of members of the Cadm family in the mouse muscle tissues. Cadm4 and Cadm1 were major components of the Cadm family, and Cadm3 was faintly detected, but Cadm2 was not detected by RT-PCR. Cadm4 as well as Cadm1 colocalized with alpha-bungarotoxin at the NMJs and interacted with the multiple PDZ domain protein Mupp1. Cadm4 was expressed in Cadm1-KO mice and might compensate for Cadm1 loss through interactions with Mupp1.

GABAA receptors involved in sleep and anaesthesia: ß1- versus ß3-containing assemblies.

The histaminergic neurons of the posterior hypothalamus (tuberomamillary nucleus-TMN) control wakefulness, and their silencing through activation of GABA(A) receptors (GABA(A)R) induces sleep and is thought to mediate sedation under propofol anaesthesia. We have previously shown that the ß1 subunit preferring fragrant dioxane derivatives (FDD) are highly potent modulators of GABA(A)R in TMN neurons. In recombinant receptors containing the ß3N265M subunit, FDD action is abolished and GABA potency is reduced. Using rat, wild-type and ß3N265M mice, FDD and propofol, we explored the relative contributions of ß1- and ß3-containing GABA(A)R to synaptic transmission from the GABAergic sleep-on ventrolateral preoptic area neurons to TMN. In ß3N265M mice, GABA potency remained unchanged in TMN neurons, but it was decreased in cultured posterior hypothalamic neurons with impaired modulation of GABA(A)R by propofol. Spontaneous and evoked GABAergic synaptic currents (IPSC) showed ß1-type pharmacology, with the same effects achieved by 3 µM propofol and 10 µM PI24513. Propofol and the FDD PI24513 suppressed neuronal firing in the majority of neurons at 5 and 100 µM, and in all cells at 10 and 250 µM, respectively. FDD given systemically in mice induced sedation but not anaesthesia. Propofol-induced currents were abolished (1-6 µM) or significantly reduced (12 µM) in ß3N265M mice, whereas gating and modulation of GABA(A)R by PI24513 as well as modulation by propofol were unchanged. In conclusion, ß1-containing (FDD-sensitive) GABA(A)R represent the major receptor pool in TMN neurons responding to GABA, while ß3-containing (FDD-insensitive) receptors are gated by low micromolar doses of propofol. Thus, sleep and anaesthesia depend on different GABA(A)R types.

Genetic mouse models for Parkinson's disease display severe pathology in glial cell mitochondria.

We recently described mitochondrial pathology in neurons of transgenic mice with genes associated with Parkinson's disease (PD). Now we describe severe mitochondrial damage in glial cells of the mesencephalon in mice carrying a targeted deletion of parkin (PaKO) or overexpressing doubly mutated human alpha-synuclein (asyn). The number of mitochondria with altered morphology in glial cells is cell type-dependent, but always higher than in neurons. Interestingly, mitochondrial damage also occurs in mesencephalic glia of mice carrying mutated asyn controlled by the tyrosine hydroxylase promoter. Such mice do not show glial expression of the transgene, but show expression in neighboring neurons. However, we found strong overexpression of endogenous asyn in mesencephalic astrocytes from these mice. Cortical astrocytes neither display enhanced asyn expression nor mitochondrial damage. Cultivated mesencephalic astrocytes from newborn transgenic mice display various functional defects along with the morphological damage of mitochondria. First, the mitochondrial Ca(2+)-storage capacity is reduced in asyn transgenic mesencephalic astrocytes, but not in astrocytes from PaKO. Second, the expression of the mitochondrial protein PTEN-induced putative kinase is constitutively increased in asyn transgenic mice, while in PaKO it reacts to oxidative stress by overexpressing this protein along with other mitochondria-related proteins. Third, the neurotrophic effects exerted by control astrocytes, stimulating cortical neurons from healthy mice to develop longer processes and larger neuronal areas, are lacking in co-cultures with transgenic mesencephalic astrocytes. In summary, glial mitochondria from transgenic mice display morphological and functional alterations. Such transgenic astrocytes fail to influence neuronal differentiation, reflecting an important role that glia may play in PD pathogenesis.

Spinal cord pathology in alpha-synuclein transgenic mice.

Accumulation of α-synuclein is observed in neurodegenerative diseases like Parkinson's disease and Multiple System Atrophy. In previous studies with transgenic C57BL/6 mice overexpressing α-synuclein carrying the mutations A53T and A30P found in Parkinson's disease or with a parkin-null background, we reported severe mitochondrial impairments in neurons and to a larger extent in glial cells of the mesencephalon. Neuron death was not observed in the brain. Here we show that the mice show severe motor impairments in behavioral tests. In addition, these mice exhibit astrocytic cell death in the spinal cord, accompanied by extensive gliosis and microglial activation. This is shown by cell death staining and immunohistochemistry. Ultrastructural analyses revealed severe mitochondrial impairments not only in astrocytes, but also in oligodendrocytes and, to a small extent, in neurons. Thus, the transgenic mice show a profound pathology in glial cells of the spinal cord.

Parkin directly modulates 26S proteasome activity.

Parkinson's disease (PD) is a common neurodegenerative disease that involves the deterioration of dopaminergic neurons in the substantia nigra pars compacta. Although the etiology of PD remains poorly understood, recent genetic, postmortem, and experimental evidence shows that abnormal protein accumulation and subsequent aggregate formation are prominent features of both sporadic and familial PD. While proteasome dysfunction is observed in PD, diverse mutations in the parkin gene are linked to early-onset autosomal-recessive forms of familial PD. We demonstrate that parkin, an E3 ubiquitin ligase, activates the 26S proteasome in an E3 ligase activity-independent manner. Furthermore, an N-terminal ubiquitin-like domain within parkin is critical for the activation of the 26S proteasome through enhancing the interaction between 19S proteasomal subunits, whereas the PD-linked R42P mutant abolishes this action. The current findings point to a novel role for parkin for 26S proteasome assembly and suggest that parkin mutations contribute to the proteasomal dysfunction in PD.

Fragrant dioxane derivatives identify beta1-subunit-containing GABAA receptors.

Nineteen GABA(A) receptor (GABA(A)R) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of beta1-subunit-containing GABA(A)Rs is unknown. Here we report the discovery of a new structural class of GABA(A)R positive modulators with unique beta1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed alpha1betaxgamma2L (x-for 1,2,3) GABA(A)R FDD were 6 times more potent at beta1- versus beta2- and beta3-containing receptors. Serine at position 265 was essential for the high sensitivity of the beta1-subunit to FDD and the beta1N286W mutation nearly abolished modulation; vice versa the mutation beta3N265S shifted FDD sensitivity toward the beta1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to beta1-negative cerebellar Purkinje neurons. Immunostaining for the beta1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by beta1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of beta1-containing GABA(A)Rs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABA(A)Rs.

Upregulation of cathepsin S in psoriatic keratinocytes.

Cathepsin S (CATS) is a cysteine protease, well known for its role in MHC class II-mediated antigen presentation and extracellular matrix degradation. Disturbance of the expression or metabolism of this protease is a concomitant feature of several diseases. Given this importance we studied the localization and regulation of CATS expression in normal and pathological human/mouse skin. In normal human skin CATS-immunostaining is mainly present in the dermis and is localized in macrophages, Langerhans, T- and endothelial cells, but absent in keratinocytes. In all analyzed pathological skin biopsies, i.e. atopic dermatitis, actinic keratosis and psoriasis, CATS staining is strongly increased in the dermis. But only in psoriasis, CATS-immunostaining is also detectable in keratinocytes. We show that cocultivation with T-cells as well as treatment with cytokines can trigger expression and secretion of CATS, which is involved in MHC II processing in keratinocytes. Our data provide first evidence that CATS expression (i) is selectively induced in psoriatic keratinocytes, (ii) is triggered by T-cells and (iii) might be involved in keratinocytic MHC class II expression, the processing of the MHC class II-associated invariant chain and remodeling of the extracellular matrix. This paper expands our knowledge on the important role of keratinocytes in dermatological disease.

Intra- versus extracellular effects of microglia-derived cysteine proteases in a conditioned medium transfer model.

Activated microglia release inflammatory mediators that display either beneficial or harmful effects on neuronal survival and signaling. In the present study we demonstrate that exposure to lipopolysaccharide leads to an increase in the lysosomal cysteine proteases, cathepsin B, K, S, and X, in culture supernatants of the microglia cell line BV-2. In addition, we observed an up-regulation of cathepsins in the cytoplasmic fraction in response to stimulation with lipopolysaccharide. Conditioned medium from these cells was toxic to the neuroblastoma cell line Neuro2a. Experiments with membrane-permeable and membrane-impermeable cysteine protease inhibitors suggested that blocking extracellular cathepsins had no effect on microglia-mediated neuron death in this medium transfer model. However, intracellular cathepsins seem to trigger the release of neurotoxic factors. In lipopolysaccharide-stimulated BV-2 cells, inhibition of intracellular cathepsins significantly diminished microglial activation characterized by reduced expression of different proinflammatory cytokines, thereby reducing the neurotoxic effects of the medium. This hitherto unknown intracellular effect of cysteine proteases in activated microglia might connect chronic neuroinflammation with neurodegeneration.

Analgesic and antiinflammatory effects of cannabinoid receptor agonists in a rat model of neuropathic pain.

Cannabinoid receptor (CB) agonists are known to attenuate allodynia in a range of pain models, but their long-term effects and their mechanisms of action are controversial. The present study compares the antiallodynic effects of long-term treatment with a mixed CB1/CB2 (WIN55,212-2) and a selective CB2 (GW405833) cannabinoid receptor agonist and correlates these effects with their influences on spinal cord (SC) glial activation. The substances were applied daily in a rat neuropathic pain model. Tactile allodynia was assessed, and the development of gliosis was illustrated with immunohistochemical methods. Both substances reduced mechanical allodynia. Their analgesic effect was accompanied by a significant reduction in reactive gliosis and cathepsins (CAT) X and S expression. A daily injection of either substance for 8 days was sufficient to induce a sustained antiallodynic effect, which persisted up to 6 days after the last injection. The re-appearance of mechanical allodynia after this period was associated with a breakout of a strong gliotic response in the lumbar SC. Our results emphasize the therapeutic efficacy of cannabinoid receptor agonists and their inhibitory effects on the formation of gliosis.

Molecular interaction between parkin and PINK1 in mammalian neuronal cells.

Parkinson's disease (PD) is characterized by the deterioration of dopaminergic neurons in the pars compacta of substantia nigra and the formation of intraneuronal protein inclusions. The etiology of PD is not known, but the recent identification of several mutation genes in familial PD has provided a rich understanding of the molecular mechanisms of PD pathology. Mutations in PTEN-induced putative kinase 1 (PINK1) and parkin are linked to early-onset autosomal recessive forms of familial PD. Here we show molecular and functional interactions between parkin and PINK1. Parkin selectively binds to PINK1 and upregulates PINK1 levels. In addition, PINK1 reduces the solubility of parkin, which induces the formation of microtubule-dependent cytoplasmic aggresomes. Our findings reveal that parkin and PINK1 affect each other's stability, solubility and tendency to form aggresomes, and have important implications regarding the formation of Lewy bodies.

The in vitro receptor profile of rotigotine: a new agent for the treatment of Parkinson's disease.

Rotigotine (Neupro) is a non-ergoline dopamine agonist developed for the once daily treatment of Parkinson's disease (PD) using a transdermal delivery system (patch) which provides patients with the drug continuously over 24 h. To fully understand the pharmacological actions of rotigotine, the present study determined its extended receptor profile. In standard binding assays, rotigotine demonstrated the highest affinity for dopamine receptors, particularly the dopamine D3 receptor (Ki=0.71 nM) with its affinities to other dopamine receptors being (Ki in nM): D4.2 (3.9), D4.7 (5.9), D5 (5.4), D2 (13.5), D4.4 (15), and D1 (83). Significant affinities were also demonstrated at alpha-adrenergic (alpha2B, Ki=27 nM) and serotonin receptors (5-HT1A Ki=30 nM). In newly developed reporter-gene assays for determination of functional activity, rotigotine behaved as a full agonist at dopamine receptors (rank order: D3>D2L>D1=D5>D4.4) with potencies 2,600 and 53 times higher than dopamine at dopamine D3 and D2L receptors, respectively. At alpha-adrenergic sites, rotigotine acted as an antagonist on alpha2B receptors. At serotonergic sites, rotigotine had a weak but significant agonistic activity at 5-HT1A receptors and a minor or nonexistent activity at other serotonin receptors. Thus, in respect to PD, rotigotine can be characterized as a specific dopamine receptor agonist with a preference for the D3 receptor over D2 and D1 receptors. In addition, it exhibits interaction with D4 and D5 receptors, the role of which in relation to PD is not clear yet. Among non-dopaminergic sites, rotigotine shows relevant affinity to only 5-HT1A and alpha2B receptors. Further studies are necessary to investigate the contribution of the different receptor subtypes to the efficacy of rotigotine in Parkinson's disease and possible other indications such as restless legs syndrome.