An in vivo Caenorhabditis elegans model for therapeutic research in human prion diseases.

Human prion diseases are fatal neurodegenerative disorders that include sporadic, infectious and genetic forms. Inherited Creutzfeldt-Jakob disease due to the E200K mutation of the prion protein-coding gene is the most common form of genetic prion disease. The phenotype resembles that of sporadic Creutzfeldt-Jakob disease at both the clinical and pathological levels, with a median disease duration of 4 months. To date, there is no available treatment for delaying the occurrence or slowing the progression of human prion diseases. Existing in vivo models do not allow high-throughput approaches that may facilitate the discovery of compounds targeting pathological assemblies of human prion protein or their effects on neuronal survival. Here, we generated a genetic model in the nematode Caenorhabditis elegans, which is devoid of any homologue of the prion protein, by expressing human prion protein with the E200K mutation in the mechanosensitive neuronal system. Expression of E200K prion protein induced a specific behavioural pattern and neurodegeneration of green fluorescent protein-expressing mechanosensitive neurons, in addition to the formation of intraneuronal inclusions associated with the accumulation of a protease-resistant form of the prion protein. We demonstrated that this experimental system is a powerful tool for investigating the efficacy of anti-prion compounds on both prion-induced neurodegeneration and prion protein misfolding, as well as in the context of human prion protein. Within a library of 320 compounds that have been approved for human use and cross the blood-brain barrier, we identified five molecules that were active against the aggregation of the E200K prion protein and the neurodegeneration it induced in transgenic animals. This model breaks a technological limitation in prion therapeutic research and provides a key tool to study the deleterious effects of misfolded prion protein in a well-described neuronal system.

Doxycycline inhibits α-synuclein-associated pathologies in vitro and in vivo.

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are neurodegenerative disorders characterized by the misfolding and aggregation of alpha-synuclein (aSyn). Doxycycline, a tetracyclic antibiotic shows neuroprotective effects, initially proposed to be due to its anti-inflammatory properties. More recently, an additional mechanism by which doxycycline may exert its neuroprotective effects has been proposed as it has been shown that it inhibits amyloid aggregation. Here, we studied the effects of doxycycline on aSyn aggregation in vivo, in vitro and in a cell free system using real-time quaking induced conversion (RT-QuiC). Using H4, SH-SY5Y and HEK293 cells, we found that doxycycline decreases the number and size of aSyn aggregates in cells. In addition, doxycycline inhibits the aggregation and seeding of recombinant aSyn, and attenuates the production of mitochondrial-derived reactive oxygen species. Finally, we found that doxycycline induces a cellular redistribution of aggregates in a C.elegans animal model of PD, an effect that is associated with a recovery of dopaminergic function. In summary, we provide strong evidence that doxycycline treatment may be an effective strategy against synucleinopathies.

Cycline efficacy on the propagation of human prions in primary cultured neurons is strain-specific.

In prion diseases, a major issue in therapeutic research is the variability of the effect between strains. Stimulated by the report of an antiprion effect in a scrapie model and by ongoing international clinical trials using doxycycline, we studied the efficacy of cyclines against the propagation of human prions. First, we successfully propagated various Creutzfeldt-Jakob disease (CJD) isolates (sporadic, variant, and iatrogenic CJD) in neuronal cultures expressing the human prion protein. Then, we found that doxycycline was the most effective compound, with important variations between isolates. Isolates from sporadic CJD, the most common form of prion disease, showed the highest sensitivity.

The Pesticide Chlordecone Promotes Parkinsonism-like Neurodegeneration with Tau Lesions in Midbrain Cultures and C. elegans Worms.

Chlordecone (CLD) is an organochlorine pesticide (OCP) that is currently banned but still contaminates ecosystems in the French Caribbean. Because OCPs are known to increase the risk of Parkinson's disease (PD), we tested whether chronic low-level intoxication with CLD could reproduce certain key characteristics of Parkinsonism-like neurodegeneration. For that, we used culture systems of mouse midbrain dopamine (DA) neurons and glial cells, together with the nematode C. elegans as an in vivo model organism. We established that CLD kills cultured DA neurons in a concentration- and time-dependent manner while exerting no direct proinflammatory effects on glial cells. DA cell loss was not impacted by the degree of maturation of the culture. The use of fluorogenic probes revealed that CLD neurotoxicity was the consequence of oxidative stress-mediated insults and mitochondrial disturbances. In C. elegans worms, CLD exposure caused a progressive loss of DA neurons associated with locomotor deficits secondary to alterations in food perception. L-DOPA, a molecule used for PD treatment, corrected these deficits. Cholinergic and serotoninergic neuronal cells were also affected by CLD in C. elegans, although to a lesser extent than DA neurons. Noticeably, CLD also promoted the phosphorylation of the aggregation-prone protein tau (but not of α-synuclein) both in midbrain cell cultures and in a transgenic C. elegans strain expressing a human form of tau in neurons. In summary, our data suggest that CLD is more likely to promote atypical forms of Parkinsonism characterized by tau pathology than classical synucleinopathy-associated PD.

Effects of a New Natural Catechol-O-methyl Transferase Inhibitor on Two In Vivo Models of Parkinson's Disease.

A tetrahydroisoquinoline identified in Mucuna pruriens ((1R,3S)-6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline-1,3-dicarboxylic acid, compound 4) was synthesized and assessed for its in vitro pharmacological profile and in vivo effects in two animal models of Parkinson's disease. Compound 4 inhibits catechol-O-methyltransferase (COMT) with no affinity for the dopaminergic receptors or the dopamine transporter. It restores dopamine-mediated motor behavior when it is co-administered with L-DOPA to C. elegans worms with 1-methyl-4-phenylpyridinium-damaged dopaminergic neurons. In a 6-hydroxydopamine rat model of Parkinson's disease, its co-administration at 30 mg/kg with L-DOPA enhances the effect of L-DOPA with an intensity similar to that of tolcapone 1 at 30 mg/kg but for a shorter duration. The effect is not dose-dependent. Compound 4 seems not to cross the blood-brain barrier and thus acts as a peripheral COMT inhibitor. COMT inhibition by compound 4 further validates the traditional use of M. pruriens for the treatment of Parkinson's disease, and compound 4 can thus be considered as a promising drug candidate for the development of safe, peripheral COMT inhibitors.

Neuron dysfunction is induced by prion protein with an insertional mutation via a Fyn kinase and reversed by sirtuin activation in Caenorhabditis elegans.

Although prion propagation is well understood, the signaling pathways activated by neurotoxic forms of prion protein (PrP) and those able to mitigate pathological phenotypes remain largely unknown. Here, we identify src-2, a Fyn-related kinase, as a gene required for human PrP with an insertional mutation to be neurotoxic in Caenorhabditis elegans, and the longevity modulator sir-2.1/SIRT1, a sirtuin deacetylase, as a modifier of prion neurotoxicity. The expression of octarepeat-expanded PrP in C. elegans mechanosensory neurons led to a progressive loss of response to touch without causing cell death, whereas wild-type PrP expression did not alter behavior. Transgenic PrP molecules showed expression at the plasma membrane, with protein clusters, partial resistance to proteinase K (PK), and protein insolubility detected for mutant PrP. Loss of function (LOF) of src-2 greatly reduced mutant PrP neurotoxicity without reducing PK-resistant PrP levels. Increased sir-2.1 dosage reversed mutant PrP neurotoxicity, whereas sir-2.1 LOF showed aggravation, and these effects did not alter PK-resistant PrP. Resveratrol, a polyphenol known to act through sirtuins for neuroprotection, reversed mutant PrP neurotoxicity in a sir-2.1-dependent manner. Additionally, resveratrol reversed cell death caused by mutant PrP in cerebellar granule neurons from prnp-null mice. These results suggest that Fyn mediates mutant PrP neurotoxicity in addition to its role in cellular PrP signaling and reveal that sirtuin activation mitigates these neurotoxic effects. Sirtuin activators may thus have therapeutic potential to protect from prion neurotoxicity and its effects on intracellular signaling.

Brain mitochondrial defects amplify intracellular [Ca2+] rise and neurodegeneration but not Ca2+ entry during NMDA receptor activation.

According to the "indirect" excitotoxicity hypothesis, mitochondrial defects increase Ca2+ entry into neurons by rendering NMDA-R hypersensitive to glutamate. We tested this hypothesis by investigating in the rat striatum and cultured striatal cells how partial mitochondrial complex II inhibition produced by 3-nitropropionic acid (3NP) modifies the toxicity of the NMDA-R agonist quinolinate (QA). We showed that nontoxic 3NP treatment, leading to partial inhibition of complex II activity, greatly exacerbated striatal degeneration produced by slightly toxic QA treatment through an "all-or-nothing" process. The potentiation of QA-induced cell death by 3NP was associated with increased calpain activity and massive calpain-mediated cleavage of several postsynaptic proteins, suggesting major neuronal Ca2+ deregulation in the striatum. However, Ca2+ anomalies probably do not result from NMDA-R hypersensitivity. Indeed, brain imaging experiments using [(18)F]fluorodeoxyglucose indirectly showed that 3NP did not increase QA-induced ionic perturbations at the striatal glutamatergic synapses in vivo. Consistent with this, the exacerbation of QA toxicity by 3NP was not related to an increase in the QA-induced entry of 45Ca2+ into striatal neurons. The present results demonstrate that the potentiation of NMDA-R-mediated excitotoxicity by mitochondrial defects involves primarily intracellular Ca2+ deregulation, in the absence of NMDA-R hypersensitivity.

Calpain is a major cell death effector in selective striatal degeneration induced in vivo by 3-nitropropionate: implications for Huntington's disease.

Striatal cell death in Huntington's Disease (HD) may involve mitochondrial defects, NMDA-mediated excitotoxicity, and activation of death effector proteases such as caspases and calpain. However, the precise contribution of mitochondrial defects in the activation of these proteases in HD is unknown. Here, we addressed this question by studying the mechanism of striatal cell death in rat models of HD using the mitochondrial complex II inhibitor 3-nitropropionic acid (3-NP). The neurotoxin was either given by intraperitoneal injections (acute model) or over 5 d by constant systemic infusion using osmotic pumps (chronic model) to produce either transient or sustained mitochondrial deficits. Caspase-9 activation preceded neurodegeneration in both cases. However, caspase-8 and caspase-3 were activated in the acute model, but not in the chronic model, showing that 3-NP does not require activation of these caspases to produce striatal degeneration. In contrast, activation of calpain was specifically detected in the striatum in both models and this was associated with a calpain-dependent cleavage of huntingtin. Finally, in the chronic model, which mimics a steady blockade of complex II activity reminiscent of HD, selective calpain inhibition prevented the abnormal calpain-dependent processing of huntingtin, reduced the size of the striatal lesions, and almost completely abolished the 3-NP-induced DNA fragmentation in striatal cells. The present results demonstrate that calpain is a predominant effector of striatal cell death associated with mitochondrial defects in vivo. This suggests that calpain may play an important role in HD pathogenesis and could be a potential therapeutic target to slow disease progression.

Neuroprotective effect of zVAD against the neurotoxin 3-nitropropionic acid involves inhibition of calpain.

The contribution of calpains and caspases to cell death has been widely studied using pharmacological inhibitors. Among them, the caspase inhibitor N-benzyloxycarbonyl-valyl-alanyl-aspartyl-fluoromethylketone (zVAD) has been used as a specific caspase inhibitor in nearly 1000 published studies. However, several studies showed that zVAD also behaves as a calpain inhibitor in peripheral cells. The effects of zVAD as a calpain inhibitor have never been assessed in neurodegeneration models. We examined here whether zVAD could reduce neurodegeneration in Huntington's disease models using the mitochondrial inhibitor 3-nitropropionic acid (3NP). In these models, 3NP toxicity has been shown to require calpain activation. In rats, intra-cerebro-ventricular infusion of zVAD significantly reduced 3NP-induced striatal degeneration, and decreased the 3NP-induced activation of calpain and calpain-dependent cleavage of fodrin. zVAD (100 microM) also blocked 3NP-induced death of cultured striatal neurons. In vitro, zVAD inhibited purified mu-calpain with high affinity (IC50=10 nM). The present data demonstrate that zVAD protects neurons against 3NP through calpain inhibition. This suggests that, in certain models of neuronal death where zVAD showed protective effects, caspases but also calpains may be involved.

Effects of cannabinoids in the rat model of Huntington's disease generated by an intrastriatal injection of malonate.

Cannabinoids could provide neuroprotection in neurodegenerative disorders. In this study, we examined whether a treatment with Delta9-tetrahydrocannabinol, a non-selective cannabinoid receptor agonist, or with SR141716, a selective antagonist for the cannabinoid CB(1) receptor subtype, could affect the toxicity of the complex II reversible inhibitor malonate injected into the striatum, which replicates the mitochondrial complex II deficiency seen in Huntington's disease patients. As expected, malonate injection produced a significant reduction in cytochrome oxidase activity in the striatum consistent with the expected neurodegeneration caused by this toxin. The administration of Delta9-tetrahydrocannabinol increased malonate-induced striatal lesions compared to vehicle and, surprisingly, SR141716, far from producing effects opposite to those of Delta9-tetrahydrocannabinol, also enhanced malonate effects, and to an even greater extent. In summary, our results are compatible with the idea that manipulating the endocannabinoid system can modify neurodegeneration in Huntington's disease, and suggest that highly selective CB(1) receptor agonists might be necessary to produce neuroprotective effects against indirect excitotoxicity.

Potential involvement of cannabinoid receptors in 3-nitropropionic acid toxicity in vivo.

Several neurotransmitter systems are involved in the pathogenesis of Huntington's disease. Here, we examined the involvement of cannabinoid CB(1) receptors in striatal degeneration in the rat model of this disease generated by administration of 3-nitropropionic acid (3NP). Several days before onset of striatal degeneration, G-protein activation by cannabinoid agonists was significantly decreased whereas density and mRNA levels of CB(1) receptors remained essentially normal. This change was transient, CB(1) receptors recovering full functionality after few days. Later, at onset of striatal degeneration, profound alterations of CB(1) receptors were detected, including marked reductions of their density, mRNA levels and coupling to G proteins. In these rats, the administration of the cannabinoid agonist Delta(9)-tetrahydrocannabinol was neuroprotective, which indicates that the early loss of CB(1) receptor signaling could be instrumental in 3NP toxicity. In conclusion, the present study supports the hypothesis that cannabinoid receptors, possibly the CB(1) receptor subtype, may be involved in HD pathogenesis and could be an interesting therapeutic target to slow disease progression.

Normal aging modulates the neurotoxicity of mutant huntingtin.

Aging likely plays a role in neurodegenerative disorders. In Huntington's disease (HD), a disorder caused by an abnormal expansion of a polyglutamine tract in the protein huntingtin (Htt), the role of aging is unclear. For a given tract length, the probability of disease onset increases with age. There are mainly two hypotheses that could explain adult onset in HD: Either mutant Htt progressively produces cumulative defects over time or "normal" aging renders neurons more vulnerable to mutant Htt toxicity. In the present study, we directly explored whether aging affected the toxicity of mutant Htt in vivo. We studied the impact of aging on the effects produced by overexpression of an N-terminal fragment of mutant Htt, of wild-type Htt or of a beta-Galactosidase (beta-Gal) reporter gene in the rat striatum. Stereotaxic injections of lentiviral vectors were performed simultaneously in young (3 week) and old (15 month) rats. Histological evaluation at different time points after infection demonstrated that the expression of mutant Htt led to pathological changes that were more severe in old rats, including an increase in the number of small Htt-containing aggregates in the neuropil, a greater loss of DARPP-32 immunoreactivity and striatal neurons as assessed by unbiased stereological counts.The present results support the hypothesis that "normal" aging is involved in HD pathogenesis, and suggest that age-related cellular defects might constitute potential therapeutic targets for HD.

3-Nitropropionic acid: a mitochondrial toxin to uncover physiopathological mechanisms underlying striatal degeneration in Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by a mutation in the gene encoding Huntingtin. The mechanisms underlying the preferential degeneration of the striatum, the most striking neuropathological change in HD, are unknown. Of those probably involved, mitochondrial defects might play an important role. The behavioural and anatomical similarities found between HD and models using the mitochondrial toxin 3-nitropropionic acid (3NP) in rats and primates support this hypothesis. Here, we discuss the recently identified mechanisms of 3NP-induced striatal degeneration. Two types of important factor have been identified. The first are the 'executioner' components that have direct roles in cell death, such as c-Jun N-terminal kinase and Ca2+-activated protease calpains. The second are 'environmental' factors, such as glutamate, dopamine and adenosine, which modulate the striatal degeneration induced by 3NP. Interestingly, these recent studies support the hypothesis that 3NP and mutated Huntingtin have certain mechanisms of toxicity in common, suggesting that the use of 3NP might give new insights into the pathogenesis of HD. Research on 3NP provides additional proof that the neurochemical environment of a given neurone can determine its preferential vulnerability in neurodegenerative diseases.

Death of cortical and striatal neurons induced by mitochondrial defect involves differential molecular mechanisms.

An important aspect of Huntington's disease (HD) pathogenesis which may have important therapeutic implications is that the cellular events leading to cell death may be different in cortical and striatal neurons. In the present study, we characterized cellular changes in cortical and striatal neurons treated with the mitochondrial toxin 3-nitropropionic acid (3NP) in culture. Degeneration induced by 3NP was similar in both striatal and cortical neurons as observed using markers of cell viability and DNA fragmentation. However, in striatal neurons, 3NP produced a marked delocalization of Bad, Bax, cytochrome c and Smac while this was not observed in cortical neurons. Death of striatal neurons was preceded by activation of calpain and was blocked by calpain inhibitor I. In cortical neurons, calpain was not activated and calpain inhibitor I was without effect. In both cell types, caspase-9 and -3 were not activated by 3NP and the caspase inhibitor zVAD-fmk did not provide neuroprotective effect. Interestingly, treatment with staurosporine (STS) triggered caspase-9 and -3 in cortical and striatal cells, suggesting that the molecular machinery related to caspase-dependent apoptosis was functional in both cell types even though this machinery was not involved in 3NP toxicity. The present results clearly demonstrate that under mitochondrial inhibition, striatal and cortical neurons die through different pathways. This suggests that mitochondrial defects in HD may trigger the death of cortical and striatal neurons through different molecular events.

Quantitative assessment of transcriptome differences between brain territories.

Transcriptome analysis of mammalian brain structures is a potentially powerful approach in addressing the diversity of cerebral functions. Here, we used a microassay for serial analysis of gene expression (SAGE) to generate quantitative mRNA expression profiles of normal adult mouse striatum, nucleus accumbens, and somatosensory cortex. Comparison of these profiles revealed 135 transcripts heterogeneously distributed in the brain. Among them, a majority (78), although matching a registered sequence, are novel regional markers. To improve the anatomical resolution of our analysis, we performed in situ hybridization and observed unique expression patterns in discrete brain regions for a number of candidates. We assessed the distribution of the new markers in peripheral tissues using quantitative RT-PCR, Northern hybridization, and published SAGE data. In most cases, expression was higher in the brain than in peripheral tissues. Because the markers were selected according to their expression level, without reference to prior knowledge, our studies provide an unbiased, comprehensive molecular signature for various mammalian brain structures that can be used to investigate their plasticity under a variety of circumstances.

In vivo calpain/caspase cross-talk during 3-nitropropionic acid-induced striatal degeneration: implication of a calpain-mediated cleavage of active caspase-3.

The role of caspases and calpains in neurodegeneration remains unclear. In this study, we focused on these proteases in a rat model of Huntington's disease using the mitochondrial toxin 3-nitropropionic acid (3NP). Results showed that 3NP-induced death of striatal neurons was preceded by cytochrome c redistribution, transient caspase-9 processing, and activation of calpain, whereas levels of the active/processed form of caspase-3 remained low and were even reduced as compared with control animals. We evidenced here that this decrease in active caspase-3 levels could be attributed to calpain activation. Several observations supported this conclusion. 1) Pharmacological blockade of calpain in 3NP-treated rats increased the levels of endogenous processed caspase-9 and caspase-3. 2) Cell-free extracts prepared from the striatum of 3NP-treated rats degraded in vitro the p34 and p20 subunits of active recombinant caspase-9 and caspase-3, respectively. 3) This degradation of p34 and p20 could be mimicked by purified mu-calpain and was prevented by calpain inhibitors. 4) mu-Calpain produced a loss of the DEVDase (Asp-Glu-Val-Asp) activity of active caspase-3. 5) Western blot analysis and experiments with 35S-radiolabeled caspase-3 showed that mu-calpain cleaved the p20 subunit of active caspase-3 near its catalytic site. 6) mu-Calpain activity was selectively inhibited (IC50 of 100 mum) by a 12 amino acid peptide corresponding to the C terminus of p20. Our results showed that calpain can down-regulate the caspase-9/caspase-3 cell death pathway during neurodegeneration due to chronic mitochondrial defects in vivo and that this effect may involve, at least in part, direct cleavage of the caspase-3 p20 subunit.

A novel systemically active caspase inhibitor attenuates the toxicities of MPTP, malonate, and 3NP in vivo.

Molecular machinery involved in apoptosis plays a role in neuronal death in neurodegenerative disorders such as Parkinson's disease (PD) and Huntington's disease (HD). Several caspase inhibitors, such as the well-known peptidyl inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone (zVADfmk), can protect neurons from apoptotic death caused by mitochondrial toxins. However, the poor penetrability of zVADfmk into brain and toxicity limits its use therapeutically. In the present study, a novel peptidyl broad-spectrum caspase inhibitor, Q-VD-OPH, which offers improvements in potency, stability, and toxicity over zVADfmk, showed significant protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 3-nitropropionic acid (3NP), and malonate toxicities. Q-VD-OPH significantly reduced dopamine depletion in striatum produced by MPTP administration and prevented MPTP-induced loss of dopaminergic neurons in the substantia nigra. It significantly reduced the size of striatal lesions produced by intrastriatal malonate injections and systemic administration of 3NP. Western blots performed on tissues from the midbrain following administration of MPTP or the striatum in 3NP-treated animals showed increases of the active forms of caspase-9 and caspase-8, as well as the caspase-8-mediated proapoptotic protein Bid, which were inhibited Q-VD-OPH treatment. These findings suggest that systematically active broad-spectrum caspase inhibitors maybe useful in the treatment of neurodegenerative diseases such as PD and HD.