Cell-to-cell transmitted alpha-synuclein recapitulates experimental Parkinson's disease.

Parkinson's disease is characterized by a progressive accumulation of alpha-Synuclein (αSyn) neuronal inclusions called Lewy bodies in the nervous system. Lewy bodies can arise from the cell-to-cell propagation of αSyn, which can occur via sequential steps of secretion and uptake. Here, by fusing a removable short signal peptide to the N-terminus of αSyn, we developed a novel mouse model with enhanced αSyn secretion and cell-to-cell transmission. Expression of the secreted αSyn in the mouse brain was under the control of a novel hybrid promoter in combination with adeno-associated virus serotype 9 (AAV9). This combination of promoter and viral vector induced a robust expression in neurons but not in the glia of injected mice. Biochemical characterization of the secreted αSyn revealed that, in cultured cells, this protein is released to the extracellular milieu via conventional secretion. The released αSyn is then internalized and processed by acceptor cells via the endosome-lysosome pathway indicating that the secreted αSyn is cell-to-cell transmitted. The secreted αSyn is aggregation-prone and amyloidogenic, and when expressed in the brain of wild-type non-transgenic mice, it induces a Parkinson's disease-like phenotype that includes a robust αSyn pathology in the substantia nigra, neuronal loss, neuroinflammation, and motor deficits, all the key features of experimental animal models of Parkinson's disease. In summary, a novel animal model of Parkinson's disease based on enhanced cell-to-cell transmission of αSyn was developed. The neuron-produced cell-to-cell transmitted αSyn triggers all phenotypic features of experimental Parkinson's disease in mice.

Oxidative stress induced by sustained supraphysiological intrastriatal GDNF delivery is prevented by dose regulation.

Despite its established neuroprotective effect on dopaminergic neurons and encouraging phase I results, intraputaminal GDNF administration failed to demonstrate significant clinical benefits in Parkinson's disease patients. Different human GDNF doses were delivered in the striatum of rats with a progressive 6-hydroxydopamine lesion using a sensitive doxycycline-regulated AAV vector. GDNF treatment was applied either continuously or intermittently (2 weeks on/2 weeks off) during 17 weeks. Stable reduction of motor impairments as well as increased number of dopaminergic neurons and striatal innervation were obtained with a GDNF dose equivalent to 3- and 10-fold the rat endogenous level. In contrast, a 20-fold increased GDNF level only temporarily provided motor benefits and neurons were not spared. Strikingly, oxidized DNA in the substantia nigra increased by 50% with 20-fold, but not 3-fold GDNF treatment. In addition, only low-dose GDNF allowed to preserve dopaminergic neuron cell size. Finally, aberrant dopaminergic fiber sprouting was observed with 20-fold GDNF but not at lower doses. Intermittent 20-fold GDNF treatment allowed to avoid toxicity and spare dopaminergic neurons but did not restore their cell size. Our data suggest that maintaining GDNF concentration under a threshold generating oxidative stress is a pre-requisite to obtain significant symptomatic relief and neuroprotection.

Glial Cell Line-Derived Neurotrophic Factor Gene Delivery in Parkinson's Disease: A Delicate Balance between Neuroprotection, Trophic Effects, and Unwanted Compensatory Mechanisms.

Glial cell line-derived neurotrophic factor (GDNF) and Neurturin (NRTN) bind to a receptor complex consisting of a member of the GDNF family receptor (GFR)-α and the Ret tyrosine kinase. Both factors were shown to protect nigro-striatal dopaminergic neurons and reduce motor symptoms when applied terminally in toxin-induced Parkinson's disease (PD) models. However, clinical trials based on intraputaminal GDNF protein administration or recombinant adeno-associated virus (rAAV)-mediated NRTN gene delivery have been disappointing. In this review, several factors that could have limited the clinical benefits are discussed. Retrograde transport of GDNF/NRTN to the dopaminergic neurons soma is thought to be necessary for NRTN/GFR-α/Ret signaling mediating the pro-survival effect. Therefore, the feasibility of treating advanced patients with neurotrophic factors is questioned by recent data showing that: (i) tyrosine hydroxylase-positive putaminal innervation has almost completely disappeared at 5 years post-diagnosis and (ii) in patients enrolled in the rAAV-NRTN trial more than 5 years post-diagnosis, NRTN was almost not transported to the substantia nigra pars compacta. In addition to its anti-apoptotic and neurotrophic properties, GDNF also interferes with dopamine homeostasis via time and dose-dependent effects such as: stimulation of dopamine neuron excitability, inhibition of dopamine transporter activity, tyrosine hydroxylase phosphorylation, and inhibition of tyrosine hydroxylase transcription. Depending on the delivery parameters, the net result of this intricate network of regulations could be either beneficial or deleterious. In conclusion, further unraveling of the mechanism of action of GDNF gene delivery in relevant animal models is still needed to optimize the clinical benefits of this new therapeutic approach. Recent developments in the design of regulated viral vectors will allow to finely adjust the GDNF dose and period of administration. Finally, new clinical studies in less advanced patients are warranted to evaluate the potential of AAV-mediated neurotrophic factors gene delivery in PD. These will be facilitated by the demonstration of the safety of rAAV administration into the human brain.

Tollip, an early regulator of the acute inflammatory response in the substantia nigra.

BACKGROUND: Tollip is a ubiquitously expressed protein, originally described as a modulator of the IL-1R/TLR-NF-κB signaling pathways. Although this property has been well characterized in peripheral cells, and despite some evidence of its expression in the central nervous system, the role of Tollip in neuroinflammation remains poorly understood. The present study sought to explore the implication of Tollip in inflammation in the substantia nigra pars compacta, the structure affected in Parkinson's disease. METHODS: We first investigated Tollip distribution in the midbrain by immunohistochemistry. Then, we addressed TLR4-mediated response by intra-nigral injections of lipopolysaccharide (LPS), a TLR4 agonist, on inflammatory markers in Tollip knockout (KO) and wild-type (WT) mice. RESULTS: We report an unexpectedly high Tollip immunostaining in dopaminergic neurons of the mice brain. Second, intra-nigral injection of LPS led to increased susceptibility to neuroinflammation in Tollip KO compared to Tollip WT mice. This was demonstrated by a significant increase of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß), interleukin 6 (IL-6), and interferon gamma (IFN-γ) messenger RNA (mRNA) in the midbrain of Tollip KO mice upon LPS injection. Consistently, brain rAAV viral vector transduction with a nuclear factor kappa B (NF-κB)-inducible reporter gene confirmed increased NF-κB activation in Tollip KO mice. Lastly, Tollip KO mice displayed higher inducible NO synthase (iNOS) production, both at the messenger and protein level when compared to LPS-injected WT mice. Tollip deletion also aggravated LPS-induced oxidative and nitrosative damages, as indicated by an increase of 8-oxo-2'-deoxyguanosine and nitrotyrosine immunostaining, respectively. CONCLUSIONS: Altogether, these findings highlight a critical role of Tollip in the early phase of TLR4-mediated neuroinflammation. As brain inflammation is known to contribute to Parkinson's disease, Tollip may be a potential target for neuroprotection.

A regulatable AAV vector mediating GDNF biological effects at clinically-approved sub-antimicrobial doxycycline doses.

Preclinical and clinical data stress the importance of pharmacologically-controlling glial cell line-derived neurotrophic factor (GDNF) intracerebral administration to treat PD. The main challenge is finding a combination of a genetic switch and a drug which, when administered at a clinically-approved dose, reaches the brain in sufficient amounts to induce a therapeutic effect. We describe a highly-sensitive doxycycline-inducible adeno-associated virus (AAV) vector. This vector allowed for the first time a longitudinal analysis of inducible transgene expression in the brain using bioluminescence imaging. To evaluate the dose range of GDNF biological activity, the inducible AAV vector (8.0 × 10(9) viral genomes) was injected in the rat striatum at four delivery sites and increasing doxycycline doses administered orally. ERK/Akt signaling activation as well as tyrosine hydroxylase downregulation, a consequence of long-term GDNF treatment, were induced at plasmatic doxycycline concentrations of 140 and 320 ng/ml respectively, which are known not to increase antibiotic-resistant microorganisms in patients. In these conditions, GDNF covered the majority of the striatum. No behavioral abnormalities or weight loss were observed. Motor asymmetry resulting from unilateral GDNF treatment only appeared with a 2.5-fold higher vector and a 13-fold higher inducer doses. Our data suggest that using the herein-described inducible AAV vector, biological effects of GDNF can be obtained in response to sub-antimicrobial doxycycline doses.

Long-term controlled GDNF over-expression reduces dopamine transporter activity without affecting tyrosine hydroxylase expression in the rat mesostriatal system.

The dopamine (DA) transporter (DAT) is a plasma membrane glycoprotein expressed in dopaminergic (DA-) cells that takes back DA into presynaptic neurons after its release. DAT dysfunction has been involved in different neuro-psychiatric disorders including Parkinson's disease (PD). On the other hand, numerous studies support that the glial cell line-derived neurotrophic factor (GDNF) has a protective effect on DA-cells. However, studies in rodents show that prolonged GDNF over-expression may cause a tyrosine hydroxylase (TH, the limiting enzyme in DA synthesis) decline. The evidence of TH down-regulation suggests that another player in DA handling, DAT, may also be regulated by prolonged GDNF over-expression, and the possibility that this effect is induced at GDNF expression levels lower than those inducing TH down-regulation. This issue was investigated here using intrastriatal injections of a tetracycline-inducible adeno-associated viral vector expressing human GDNF cDNA (AAV-tetON-GDNF) in rats, and doxycycline (DOX; 0.01, 0.03, 0.5 and 3mg/ml) in the drinking water during 5weeks. We found that 3mg/ml DOX promotes an increase in striatal GDNF expression of 12× basal GDNF levels and both DA uptake decrease and TH down-regulation in its native and Ser40 phosphorylated forms. However, 0.5mg/ml DOX promotes a GDNF expression increase of 3× basal GDNF levels with DA uptake decrease but not TH down-regulation. The use of western-blot under non-reducing conditions, co-immunoprecipitation and in situ proximity ligation assay revealed that the DA uptake decrease is associated with the formation of DAT dimers and an increase in DAT-α-synuclein interactions, without changes in total DAT levels or its compartmental distribution. In conclusion, at appropriate GDNF transduction levels, DA uptake is regulated through DAT protein-protein interactions without interfering with DA synthesis.

Cannabinoid receptor activation in the juvenile rat brain results in rapid biomechanical alterations: Neurovascular mechanism as a putative confounding factor.

We have recently reported cannabinoid-induced rapid changes in the structure of individual neurons. In order to investigate the presence of similar effects at the regional level, measures of brain tissue biomechanics are required. However, cannabinoids are known to alter cerebral blood flow (CBF), putatively resulting in presently unexplored changes in cerebral tissue biomechanics. Here we used magnetic resonance elastography (MRE) and flow-sensitive alternating inversion recovery (FAIR) imaging to measure in vivo alterations of mechanical properties and CBF, respectively, in the rat hippocampus, a brain region with a high density of type-1 cannabinoid receptors (CB1R). Systemic injection of the cannabinoid agonist CP55,940 (0.7 mg/kg) induced a significant stiffness decrease of 10.5 ± 1.2% at 15 minutes. FAIR imaging indicated a comparable decrease (11.3 ± 1.9%) in CBF. Both effects were specific to CB1R activation, as shown by pretreatment with the CB1R-specific antagonist AM251. Strikingly, similar rapid parallel changes of brain elasticity and CBF were also observed after systemic treatment with the hypotensive drug nicardipine. Our results reveal important drug-induced parallel changes in CBF and brain mechanical characteristics, and show that blood flow-dependent tissue softening has to be considered as an important putative confounding factor when cerebral viscoelastic changes are investigated.

Cannabinoid-induced actomyosin contractility shapes neuronal morphology and growth.

Endocannabinoids are recently recognized regulators of brain development, but molecular effectors downstream of type-1 cannabinoid receptor (CB1R)-activation remain incompletely understood. We report atypical coupling of neuronal CB1Rs, after activation by endo- or exocannabinoids such as the marijuana component ∆(9)-tetrahydrocannabinol, to heterotrimeric G12/G13 proteins that triggers rapid and reversible non-muscle myosin II (NM II) dependent contraction of the actomyosin cytoskeleton, through a Rho-GTPase and Rho-associated kinase (ROCK). This induces rapid neuronal remodeling, such as retraction of neurites and axonal growth cones, elevated neuronal rigidity, and reshaping of somatodendritic morphology. Chronic pharmacological inhibition of NM II prevents cannabinoid-induced reduction of dendritic development in vitro and leads, similarly to blockade of endocannabinoid action, to excessive growth of corticofugal axons into the sub-ventricular zone in vivo. Our results suggest that CB1R can rapidly transform the neuronal cytoskeleton through actomyosin contractility, resulting in cellular remodeling events ultimately able to affect the brain architecture and wiring.

Involvement of histamine receptors in the atypical antipsychotic profile of clozapine: a reassessment in vitro and in vivo.

RATIONALE: The basis of the unique clinical profile of the antipsychotic clozapine is not yet elucidated. Brain histamine receptors may play a role in schizophrenia and its treatment, but their involvement in the profile of clozapine remained unknown. OBJECTIVES: We explored the properties of clozapine and its two metabolites, N-desmethylclozapine (NDMC) and clozapine N-oxide, at the four human histaminergic receptors. We compared their active concentrations with their blood concentrations in patients treated by clozapine. We investigated the changes in receptor densities induced in rat brain by repeated administration of a therapeutic dose of clozapine. RESULTS: Clozapine and NDMC behaved as very potent, and partial, H(1)-receptor inverse agonists, weak, and full, H(2)-receptor inverse agonists, moderate, and protean, H(3)-receptor agonists, and moderate, and partial, H(4)-receptor agonists. Taking into account their micromolar mean blood concentrations found in 75 treated patients, and assuming that they are enriched in human brain as they are in rat brain, a full occupation of H(1)-, H(3)-, and H(4)-receptors, and a partial occupation of H(2) receptors, is expected. In agreement, repeated administration of clozapine at a therapeutic dose (20 mg/kg/day for 20 days) induced an up-regulation of H(1)- and H(2)-receptors in rat brain. CONCLUSIONS: Clozapine and its active metabolite NDMC interact with the four human histamine receptors at clinically relevant concentrations. This interaction may substantiate, at least in part, the atypical antipsychotic profile of clozapine, as well as its central and peripheral side effects such as sedation and weight gain.

Histamine H3 and dopamine D2 receptor-mediated [35S]GTPgamma[S] binding in rat striatum: evidence for additive effects but lack of interactions.

The interactions in the rat striatum between H(3) receptors (H(3)Rs) and D(2) receptors (D(2)Rs) were investigated with the [(35)S]GTPgamma[S] binding assay. The H(3)R agonist (R)alpha-methylhistamine increased [(35)S]GTPgamma[S] binding to striatal membranes with an EC(50)=14+/-5 nM and a maximal effect of +19+/-1%. This effect was inhibited by the H(3)R antagonist ciproxifan with a K(i)=1.0+/-0.3 nM. The D(2)R agonist quinpirole increased [(35)S]GTPgamma[S] binding to the same membranes with an EC(50)=1.5+/-0.5 microM and a maximal effect of +28+/-2%. Its effect was blocked by haloperidol with a K(i)=0.3+/-0.1 nM. The maximal effects of the H(3)R and D(2)R agonists were additive (+46+/-3%). However, D(2)R ligands did not modify the effects of H(3)R ligands and vice versa. Ciproxifan behaved as an H(3)R inverse agonist and decreased [(35)S]GTPgamma[S] binding. Haloperidol had no effect and did not change the inverse agonist effect of ciproxifan. Administrations for 10 days of ciproxifan (1.5mg/kg/day) or haloperidol (0.5mg/kg/day) did not change the effects of quinpirole and (R)alpha-methylhistamine, respectively. These data suggest that striatal H(3)Rs and D(2)Rs do not interact through their coupling to G-proteins. However, a hyperactivity of histaminergic and dopaminergic neurons being observed in schizophrenia, the additive activations of H(3)Rs and D(2)Rs suggest that they cooperate to generate some schizophrenic symptoms. Such a postsynaptic mechanism may underlie the antipsychotic-like effects of H(3)R inverse agonists and supports their therapeutic interest, alone or as adjunctive treatment with neuroleptics.

Compared pharmacology of human histamine H3 and H4 receptors: structure-activity relationships of histamine derivatives.

Various histamine derivatives were investigated at the human H3 receptor (H3R) and H4 receptor (H4R) stably expressed in human embryonic kidney (HEK)-293 cells using [125I]iodoproxyfan and [3H]histamine binding, respectively. In Tris buffer, [3H]histamine binding to membranes of HEK(hH4R) cells was monophasic (K(D) of 3.8+/-0.8 nM). In phosphate buffer, the Hill coefficient was decreased (n(H) = 0.5+/-0.1) and a large fraction of the binding was converted into a low-affinity component (K(D) = 67+/-27 nM). The inhibition of [3H]histamine binding by two agonists, a protean agonist and five antagonists/inverse agonists confirms that the potency of many H3R ligands is retained or only slightly reduced at the H4R. Histamine derivatives substituted with methyl groups in alpha, beta or N(alpha) position of the side chain retained a nanomolar potency at the H3R, but their affinity was dramatically decreased at the H4R. With relative potencies to histamine of 282 and 0.13% at the H3R and H4R, respectively, (+/-)-alpha,beta-dimethylhistamine is a potent and selective H3R agonist. Chiral alpha-branched analogues exhibited a marked stereoselectivity at the H3R and H4R, the enantiomers with a configuration equivalent to L-histidine being preferred at both receptors. The methylsubstitution of the imidazole ring was also studied. The relative potency to histamine of 4-methylhistamine (4-MeHA) at the H4R (67%) was similar to that reported at H2 receptors but, owing to its high affinity at the H4R (Ki = 7.0+/-1.2 nM) and very low potency at H1- and H3-receptors, it can be considered as a potent and selective H4R agonist. On inhibition of forskolin-induced cAMP formation, all the compounds tested, including 4-MeHA, behaved as full agonists at both receptors. However, the maximal inhibition achieved at the H4R (approximately -30%) was much lower than at the H3R (approximately -80%). Thioperamide behaved as an inverse agonist at both receptors and increased cAMP formation with the same maximal effect (approximately +25%). In conclusion, although the pharmacological profiles of the human H3R and H4R overlap, the structure-activity relationships of histamine derivatives at both receptors strongly differ and lead to the identification of selective compounds.