Etienne Régulier

Akt is altered in an animal model of Huntington"s disease and in patients

The insulin‐like growth factor I (IGF‐1)/Akt pathway plays a crucial role in Huntingtons disease by phosphorylating the causative protein, polyQ‐huntingtin, and abolishing its toxic properties [ Humbert et al. (2002)Dev. Cell, 2, 831–837; Rangone et al. (2004)Eur. J. Neurosci., 19, 273–279]. Therefore, dysregulation of this pathway may be essential for disease progression. In the present report, we thus aimed to analyse the status of Akt in brain or in peripheral tissues in Huntingtons disease. Using a genetic model of Huntingtons disease in rat that reproduces neuronal dysfunction and death, we show a progressive alteration of Akt during neuronal dysfunction and prior neurodegeneration. By analysing a limited number of lymphoblasts and lymphocytes, we detected modifications of Akt in Huntingtons disease patients confirming a dysregulation of Akt in the disease process. Finally, we demonstrate that during late stages of the disease, Akt is cleaved into an inactive form by caspase‐3. These observations demonstrate a progressive but marked alteration of this pro‐survival pathway in Huntingtons disease, and further implicate it as a key transduction pathway regulating the toxicity of huntingtin.

Dose-Dependent Neuroprotective Effect of Ciliary Neurotrophic Factor Delivered via Tetracycline-Regulated Lentiviral Vectors in the Quinolinic Acid Rat Model of Huntington' s Disease

The ability to regulate gene expression constitutes a prerequisite for the development of gene therapy strategies aimed at the treatment of neurologic disorders. In the present work, we used tetracycline (Tet)-regulated lentiviral vectors to investigate the dose-dependent neuroprotective effect of human ciliary neurotrophic factor (CNTF) in the quinolinic acid (QA) model of Huntingtons disease (HD). The Tet system was split in two lentiviruses, the first one containing the CNTF or green fluorescent protein (GFP) cDNAs under the control of the Tet-response element (TRE) and a second vector encoding the transactivator (tTA). Preliminary coinfection study demonstrated that 63.8% +/- 2.0% of infected cells contain at least two viral copies. Adult rats were then injected with CNTF- and GFP-expressing viral vectors followed 3 weeks later by an intrastriatal administration of QA. A significant reduction of apomorphine-induced rotations was observed in the CNTF-on group. In contrast, GFP-treated animals or CNTF-off rats displayed an ipsilateral turning behavior in response to apomorphine. A selective sparing of DARPP-32-, choline acetyltransferase (ChAT)-, and NADPH-d-positive neurons was observed in the striatum of CNTF-on rats compared to GFP animals and CNTF-off group. Enzyme-linked immunosorbent assay (ELISA) performed on striatal samples of rats sacrificed at the same time point indicated that this neuroprotective effect was associated with the production of 15.5 +/- 4.7 ng CNTF per milligram of protein whereas the residual CNTF expression in the off state (0.54 +/- 0.02 ng/mg of protein) was not sufficient to protect against QA toxicity. These results establish the proof of principle of neurotrophic factor dosing for neurodegenerative diseases and demonstrate the feasibility of lentiviral-mediated tetracycline-regulated gene transfer in the brain.

Inhibition of Calcineurin by FK506 Protects against Polyglutamine-Huntingtin Toxicity through an Increase of Huntingtin Phosphorylation at S421

Huntington’s disease (HD) is caused by an abnormal expanded polyglutamine (polyQ) repeat in the huntingtin protein. Insulin-like growth factor-1 acting through the prosurvival kinase Akt mediates the phosphorylation of huntingtin at S421 and inhibits the toxicity of polyQ-expanded huntingtin in cell culture, suggesting that compounds enhancing phosphorylation are of therapeutic interest. However, it is not clear whether phosphorylation of S421 is crucial in vivo. Using a rat model of HD based on lentiviral-mediated expression of a polyQ-huntingtin fragment in the striatum, we demonstrate here that phosphorylation of S421 is neuroprotective in vivo. We next demonstrate that calcineurin (CaN), a calcium/calmodulin-regulated Ser/Thr protein phosphatase, dephosphorylates S421 in vitro and in cells. Inhibition of calcineurin activity, either by overexpression of the dominant-interfering form of CaN or by treatment with the specific inhibitor FK506, favors the phosphorylation of S421, restores the alteration in huntingtin S421 phosphorylation in HD neuronal cells, and prevents polyQ-mediated cell death of striatal neurons. Finally, we show that administration of FK506 to mice increases huntingtin S421 phosphorylation in brain. Collectively, these data highlight the importance of CaN in the modulation of S421 phosphorylation and suggest the potential use of CaN inhibition as a therapeutic approach to treat HD.

Interactions between NEEP21, GRIP1 and GluR2 regulate sorting and recycling of the glutamate receptor subunit GluR2.

Trafficking of AMPA‐type glutamate receptors (AMPAR) between endosomes and the postsynaptic plasma membrane of neurons plays a central role in the control of synaptic strength associated with learning and memory. The molecular mechanisms of its regulation remain poorly understood, however. Here we show by biochemical and atomic force microscopy analyses that NEEP21, a neuronal endosomal protein necessary for receptor recycling including AMPAR, is associated with the scaffolding protein GRIP1 and the AMPAR subunit GluR2. Moreover, the interaction between NEEP21 and GRIP1 is regulated by neuronal activity. Expression of a NEEP21 fragment containing the GRIP1‐binding site decreases surface GluR2 levels and delays recycling of internalized GluR2, which accumulates in early endosomes and lysosomes. Infusion of this fragment into pyramidal neurons of hippocampal slices induces inward rectification of AMPAR‐mediated synaptic responses, suggesting decreased GluR2 expression at synapses. These results indicate that NEEP21–GRIP1 binding is crucial for GluR2‐AMPAR sorting through endosomes and their recruitment to the plasma membrane, providing a first molecular mechanism to differentially regulate AMPAR subunit cycling in internal compartments.

Analysis of potential transcriptomic biomarkers for Huntington's disease in peripheral blood.

Highly quantitative biomarkers of neurodegenerative disease remain an important need in the urgent quest for disease-modifying therapies. For Huntingtons disease (HD), a genetic test is available (trait marker), but necessary state markers are still in development. In this report, we describe a large battery of transcriptomic tests explored as state biomarker candidates. In an attempt to exploit the known neuroinflammatory and transcriptional perturbations of disease, we measured relevant mRNAs in peripheral blood cells. The performance of these potential markers was weak overall, with only one mRNA, immediate early response 3 (IER3), showing a modest but significant increase of 32% in HD samples compared with controls. No statistically significant differences were found for any other mRNAs tested, including a panel of 12 RNA biomarkers identified in a previous report [Borovecki F, Lovrecic L, Zhou J, Jeong H, Then F, Rosas HD, Hersch SM, Hogarth P, Bouzou B, Jensen RV, et al. (2005) Proc Natl Acad Sci USA 102:11023–11028]. The present results may nonetheless inform the future design and testing of HD biomarker strategies.

Continuous delivery of human and mouse erythropoietin in mice by genetically engineered polymer encapsulated myoblasts

The transplantation of polymer encapsulated myoblasts genetically engineered to secrete erythropoietin (Epo) may obviate the need for repeated parenteral administration of recombinant Epo as a treatment for chronic renal failure, cancer or AIDS-associated anemia. To explore this possibility, the human and mouse Epo cDNAs under the control of the housekeeping mouse PGK-1 promoter were transfected into mouse C2C12 myoblasts, which can be terminally differentiated upon exposure to low serum-containing media. Pools releasing 150 IU human Epo per 106 cells per day and 390 IU mouse Epo per 106 cells per day were selected. Polyether-sulfone (PES) capsules loaded with approximately 200 000 transfected myoblasts from these pools were implanted on the dorsal flank of DBA/2J, C3H and C57BL/6 mice. With human Epo secreting capsules, only a transient increase in the hematocrit occurred in DBA/2J mice, whereas no significant response was detected in C3H or C57BL/6 mice. On the contrary, all mice implanted with capsules releasing mouse Epo increased their hematocrit over 85% as early as 7 days after implantation and sustained these levels for at least 80 days. All retrieved implants released Epo and contained well preserved myoblasts. Moreover most capsules were surrounded by a neovascularization. Mice transplanted with nonencapsulated C2C12 cells releasing mouse Epo showed only a transitory elevation of their hematocrit reflecting the poor engraftment of injected myoblasts. These results indicate that polymer encapsulation of genetically engineered myoblasts is a promising approach for the long-term delivery of bioactive molecules, allowing the resolution of the shortcomings of free myoblast transfer.

Dysregulation of Gene Expression in Primary Neuron Models of Huntington's Disease Shows That Polyglutamine-Related Effects on the Striatal Transcriptome May Not Be Dependent on Brain Circuitry

Gene expression changes are a hallmark of the neuropathology of Huntingtons disease (HD), but the exact molecular mechanisms of this effect remain uncertain. Here, we report that in vitro models of disease comprised of primary striatal neurons expressing N-terminal fragments of mutant huntingtin (via lentiviral gene delivery) faithfully reproduce the gene expression changes seen in human HD. Neither viral infection nor unrelated (enhanced green fluorescent protein) transgene expression had a major effect on resultant RNA profiles. Expression of a wild-type fragment of huntingtin [htt171-18Q] also caused only a small number of RNA changes. The disease-related signal in htt171-82Q versus htt171-18Q comparisons was far greater, resulting in the differential detection of 20% of all mRNA probe sets. Transcriptomic effects of mutated htt171 are time- and polyglutamine-length dependent and occur in parallel with other manifestations of polyglutamine toxicity over 4–8 weeks. Specific RNA changes in htt171-82Q-expressing striatal cells accurately recapitulated those observed in human HD caudate and included decreases in PENK (proenkephalin), RGS4 (regulator of G-protein signaling 4), dopamine D1 receptor (DRD1), DRD2, CNR1 (cannabinoid CB1 receptor), and DARPP-32 (dopamine- and cAMP-regulated phosphoprotein-32; also known as PPP1R1B) mRNAs. HD-related transcriptomic changes were also observed in primary neurons expressing a longer fragment of mutant huntingtin (htt853-82Q). The gene expression changes observed in cultured striatal neurons are not secondary to abnormalities of neuronal firing or glutamatergic, dopaminergic, or brain-derived neurotrophic factor signaling, thereby demonstrating that HD-induced dysregulation of the striatal transcriptome might be attributed to intrinsic effects of mutant huntingtin.

Striatal and nigral pathology in a lentiviral rat model of Machado-Joseph disease

Machado-Joseph disease (MJD) is a fatal, dominant neurodegenerative disorder. MJD results from polyglutamine repeat expansion in the MJD-1 gene, conferring a toxic gain of function to the ataxin-3 protein. In this study, we aimed at overexpressing ataxin-3 in the rat brain using lentiviral vectors (LV), to generate an in vivo MJD genetic model and, to study the disorder in defined brain regions: substantia nigra, an area affected in MJD, cortex and striatum, regions not previously reported to be affected in MJD. LV encoding mutant or wild-type human ataxin-3 was injected in the brain of adult rats and the animals were tested for behavioral deficits and neuropathological abnormalities. Striatal pathology was confirmed in transgenic mice and human tissue. In substantia nigra, unilateral overexpression of mutant ataxin-3 led to: apomorphine-induced turning behavior; formation of ubiquitinated ataxin-3 aggregates; alpha-synuclein immunoreactivity; and loss of dopaminergic markers (TH and VMAT2). No neuropathological changes were observed upon wild-type ataxin-3 overexpression. Mutant ataxin-3 expression in striatum and cortex, resulted in accumulation of misfolded ataxin-3, and within striatum, loss of neuronal markers. Striatal pathology was confirmed by observation in MJD transgenic mice of ataxin-3 aggregates and substantial reduction of DARPP-32 immunoreactivity and, in human striata, by ataxin-3 inclusions, immunoreactive for ubiquitin and alpha-synuclein. This study demonstrates the use of LV encoding mutant ataxin-3 to produce a model of MJD and brings evidence of striatal pathology, suggesting that this region may contribute to dystonia and chorea observed in some MJD patients and may represent a target for therapies.

Improvement of mouse β-thalassemia upon erythropoietin delivery by encapsulated myoblasts

The goal of the present study was to analyze if sustained delivery of elevated doses of recombinant erythropoietin (Epo), by genetically modified and immunoprotected allogeneic cells, was able to correct the chronic anemia, characteristic of a spontaneous mouse model of β-thalassemia (Hbb thal 1). Mouse C2C12 myoblast cells were transfected with a plasmid containing the mouse Epo cDNA and a mutated dihydrofolate reductase (DHFR) gene for gene amplification upon administration of increasing doses of methotrexate. In order to immunoprotect the transplanted cells, the stably modified cells were loaded into polyethersulfone microporus hollow fibers which were implanted subcutaneously into Hbb thal 1 mice. An increase in hematocrit starting 2 weeks after implantation was associated with elevated blood levels of Epo and an improved red blood cell phenotype. The latter indicated an improvement of cell morphology and membrane defects, in particular a reduced amount of free α hemoglobin chain, the hallmark of globin chain imbalance in β-thalassemia. A reduction of reticulocyte count contrasting with the increase in hematocrit was also observed suggesting an improved erythrocyte survival. We conclude that the phenotype can be durably improved in some β-thalassemic mice upon in vivo delivery of recombinant Epo by polymer encapsulated cells. Sustained elevated delivery of recombinant Epo holds promise for the treatment of β-thalassemia-associated chronic anemia.

Haloperidol protects striatal neurons from dysfunction induced by mutated huntingtin in vivo.

Huntingtons disease (HD) results from an abnormal polyglutamine extension in the N-terminal region of the huntingtin protein. This mutation causes preferential degeneration of striatal projection neurons. We previously demonstrated, in vitro, that dopaminergic D2 receptor stimulation acted synergistically with mutated huntingtin (expHtt) to increase aggregate formation and striatal death. In the present work, we extend these observations to an in vivo system based on lentiviral-mediated expression of expHtt in the rat striatum. The early and chronic treatment with the D2 antagonist haloperidol decanoate protects striatal neurons from expHtt-induced dysfunction, as analyzed by DARPP-32 and NeuN stainings. Haloperidol treatment also reduces aggregates formation, an effect that is maintained over time. These findings indicate that D2 receptors activation contributes to the deleterious effects of expHtt on striatal function and may represent an interesting early target to alter the subsequent course of neuropathology in HD.