Loss of the thyroid hormone-binding protein Crym renders striatal neurons more vulnerable to mutant huntingtin in Huntington's disease.

The mechanisms underlying preferential atrophy of the striatum in Huntington's disease (HD) are unknown. One hypothesis is that a set of gene products preferentially expressed in the striatum could determine the particular vulnerability of this brain region to mutant huntingtin (mHtt). Here, we studied the striatal protein µ-crystallin (Crym). Crym is the NADPH-dependent p38 cytosolic T3-binding protein (p38CTBP), a key regulator of thyroid hormone (TH) T3 (3,5,3'-triiodo-l-thyronine) transportation. It has been also recently identified as the enzyme that reduces the sulfur-containing cyclic ketimines, which are potential neurotransmitters. Here, we confirm the preferential expression of the Crym protein in the rodent and macaque striatum. Crym expression was found to be higher in the macaque caudate than in the putamen. Expression of Crym was reduced in the BACHD and Knock-in 140CAG mouse models of HD before onset of striatal atrophy. We show that overexpression of Crym in striatal medium-size spiny neurons using a lentiviral-based strategy in mice is neuroprotective against the neurotoxicity of an N-terminal fragment of mHtt in vivo. Thus, reduction of Crym expression in HD could render striatal neurons more susceptible to mHtt suggesting that Crym may be a key determinant of the vulnerability of the striatum. In addition our work points to Crym as a potential molecular link between striatal degeneration and the THs deregulation reported in HD patients

Doublecortin-like kinase 3 (DCLK3), a novel striatum-enriched species, is amodulator of mutant huntingtin in vivo: L. GALVAN1, M.-C. GAILLARD2, M. DE CHALDÉE2, G. AUREGAN1, N.DUFOUR1, M. GUILLERMIER1, D. HOUITTE1, F. PETIT1, C. MALGORN1,G. LIOT3, S. HUMBERT3, J. ELALOUF2, N. DÉGLON1, *E. P. BROUILLET1;1CEA, MIRCen, URA CEA-CNRS 2210, Fontenay-aux-Roses, France; 2CEAIBITec-S/SBIGeM, Saclay, France; 3Inst. Curie, UMR 146, CNRS, Orsay, France

International audienceHuntington's disease (HD) is a neurodegenerative disorder caused by an abnormalCAG repeat expansion coding for an expanded polyglutamine tract in the protein"huntingtin" (Htt). Although this mutant Htt (mHtt) is expressed ubiquitouslythroughout the brain, the striatum is found preferentially affected. One hypothesisto explain this particular vulnerability is that striatal neurons express a particularset of proteins that make them highly vulnerable to mHtt. In order to furtherexamine this hypothesis, we carried out a transcriptome analysis of different brainterritories and identified more than 100 molecular markers i.e. transcripts that arehighly enriched in the mouse striatum. We recently focused our interest on asubset of striatal-enriched transcripts of poorly characterized transcripts.We here report the study of one of these markers, the CAMKII family-relatedkinase DCLK3. We found that DCLK3 is mainly expressed in the adult striatum inrodent with low level of expression in the newborn and striatal primary cultures.Reduced mRNA levels of DCLK3 were found in the striatum of transgenic mousemodels of HD. We thus studied the effect of DCLK3 overexpression and knockdownin a mouse model of HD using lentiviral vectors coding for a N-terminalfragment of mHtt. DCLK3 and its related siRNA were delivered using lentiviralvectors. Striatal degeneration produced by mHtt was characterized usingimmunohistochemistry of DARPP32, Cytochrome oxidase and ubiquitin followedby quantitative histological evaluation. Results showed that lenti-siRNA targetingDCLK3 increased mHtt toxicity when compared to the control. On the contrary,overexpression of DCLK3 reduced the striatal lesions produced by mHtt in vivo.DCLK3 also decreased the number and size of ubiquitin-containing nuclearinclusions. Current experiments are examining the mechanisms that could underliethe neuroprotective effect of DCLK3 in striatal neurons. The present study suggests that DCLK3 is a potential modifier of the disease and might beconsidered in HD therapy to slow disease progression

Doublecortin-like kinase 3 (DCLK3), a novel striatum-enriched species, is amodulator of mutant huntingtin in vivo: L. GALVAN1, M.-C. GAILLARD2, M. DE CHALDÉE2, G. AUREGAN1, N.DUFOUR1, M. GUILLERMIER1, D. HOUITTE1, F. PETIT1, C. MALGORN1,G. LIOT3, S. HUMBERT3, J. ELALOUF2, N. DÉGLON1, *E. P. BROUILLET1;1CEA, MIRCen, URA CEA-CNRS 2210, Fontenay-aux-Roses, France; 2CEAIBITec-S/SBIGeM, Saclay, France; 3Inst. Curie, UMR 146, CNRS, Orsay, France

International audienceHuntington's disease (HD) is a neurodegenerative disorder caused by an abnormalCAG repeat expansion coding for an expanded polyglutamine tract in the protein"huntingtin" (Htt). Although this mutant Htt (mHtt) is expressed ubiquitouslythroughout the brain, the striatum is found preferentially affected. One hypothesisto explain this particular vulnerability is that striatal neurons express a particularset of proteins that make them highly vulnerable to mHtt. In order to furtherexamine this hypothesis, we carried out a transcriptome analysis of different brainterritories and identified more than 100 molecular markers i.e. transcripts that arehighly enriched in the mouse striatum. We recently focused our interest on asubset of striatal-enriched transcripts of poorly characterized transcripts.We here report the study of one of these markers, the CAMKII family-relatedkinase DCLK3. We found that DCLK3 is mainly expressed in the adult striatum inrodent with low level of expression in the newborn and striatal primary cultures.Reduced mRNA levels of DCLK3 were found in the striatum of transgenic mousemodels of HD. We thus studied the effect of DCLK3 overexpression and knockdownin a mouse model of HD using lentiviral vectors coding for a N-terminalfragment of mHtt. DCLK3 and its related siRNA were delivered using lentiviralvectors. Striatal degeneration produced by mHtt was characterized usingimmunohistochemistry of DARPP32, Cytochrome oxidase and ubiquitin followedby quantitative histological evaluation. Results showed that lenti-siRNA targetingDCLK3 increased mHtt toxicity when compared to the control. On the contrary,overexpression of DCLK3 reduced the striatal lesions produced by mHtt in vivo.DCLK3 also decreased the number and size of ubiquitin-containing nuclearinclusions. Current experiments are examining the mechanisms that could underliethe neuroprotective effect of DCLK3 in striatal neurons. The present study suggests that DCLK3 is a potential modifier of the disease and might beconsidered in HD therapy to slow disease progression

The newly identified striatal marker Doublecortin-like kinase 3 (Dclk3) may be amolecular determinant of striatal vulnerability in Huntington’s disease

International audienceHuntington's disease (HD) is an inherited neurodegenerative disorder caused by an abnormal polyglutamine expansion in the protein huntingtin (htt). Mutant htt, despite its ubiquitous expression in the brain, leads to preferential neurodegeneration of the striatum through unknown mechanisms. One hypothesis is that gene products selectively expressed in the striatum may be involved in the high vulnerability of striatal neurons to mutant htt. In the present study, we first show that (i) expression of mRNA coding for a newly identified "striatal" gene product, Doublecortin-like kinase 3 (Dclk3) is reduced in the striatum of BAC-HD mice and knock-in Hdh140 CAG mice, and (ii) overexpression of Dclk3 using lentiviral vectors is neuroprotective against mutant htt in primary culture of striatal neurons and in the mouse striatum in vivo. We next aimed at identifying the mechanisms of Dclk3 neuroprotective effects. Dclk3 contains two putative domains: a doublecortin-like domain in the N-terminal part of the protein and a kinase domain in its C-terminus. Thus, we generated mutants and truncated fragments of Dclk3 to investigate the role of the kinase domain. We found that a mutation inactivating the kinase was devoid of protective effects in striatal neurons. Overexpression of the kinase domain alone was sufficient to produce neuroprotection against mutant htt in vivo. Autophosphorylation experiments demonstrated that Dclk3 is actually a functional kinase, further suggesting that Dclk3 likely acts against mutant htt primarily through its kinase activity. We also found that recombinant Dclk3 is co-localized with microtubules with a higher density in the cell perinuclear region, according to a network-like architecture reminiscent of the cytoskeleton. The kinase domain shows a more diffuse localization throughout the cell. Interestingly, biochemical analyses indicated that Dclk3 is cleaved into several breakdown products, separating its doublecortin-like domain from its kinase domain. An abnormal cleavage of recombinant Dclk3 was found in the striatum of transgenic BAC-HD mice as compared to WT mice. Thus, reduced expression of Dclk3 and possibly alteration of its cleavage and cellular localization may lead to a loss of its “pro-survival” activity in HD models, rendering the striatum highly vulnerable to mutant htt. These novel results suggest that Dclk3 may be an important determinant of striatal vulnerability in HD. The identification of the mechanisms underlying the neuroprotective effects of Dclk3 may lead to novel potential therapeutic strategies for HD

Characterization of the striatal kinase Dclk3 and its neuroprotective effects againstmutant huntingtin

International audienceDouble-cortin-like kinases (Dclks) play major roles in development and maintenance of neuronal functions. The neurobiological role of the third member of this family, the striatal-enriched Dclk3 is unknown. We characterized the cytoplasmic and nuclear localization of Dclk3 in the striatum in non-human primates and in man. A yeast-two hybrid screen identified seven Dclk3 interactors, all possessing zinc finger domains. One candidate is known to regulate many transcription activating factors (TAFs), p53 and lysine acetyl transferases. In line with this, expression of Dclk3 kinase domain in human striatal neural stem cells produces transcriptional changes that are mainly linked to chromatin remodeling. Chromatin and transcriptional perturbations have been described in 4/6/202

The striatal kinase DCLK3 produces neuroprotection against mutant huntingtin.

The neurobiological functions of a number of kinases expressed in the brain are unknown. Here, we report new findings on DCLK3 (doublecortin like kinase 3), which is preferentially expressed in neurons in the striatum and dentate gyrus. Its function has never been investigated. DCLK3 expression is markedly reduced in Huntington's disease. Recent data obtained in studies related to cancer suggest DCLK3 could have an anti-apoptotic effect. Thus, we hypothesized that early loss of DCLK3 in Huntington's disease may render striatal neurons more susceptible to mutant huntingtin (mHtt). We discovered that DCLK3 silencing in the striatum of mice exacerbated the toxicity of an N-terminal fragment of mHtt. Conversely, overexpression of DCLK3 reduced neurodegeneration produced by mHtt. DCLK3 also produced beneficial effects on motor symptoms in a knock-in mouse model of Huntington's disease. Using different mutants of DCLK3, we found that the kinase activity of the protein plays a key role in neuroprotection. To investigate the potential mechanisms underlying DCLK3 effects, we studied the transcriptional changes produced by the kinase domain in human striatal neurons in culture. Results show that DCLK3 regulates in a kinase-dependent manner the expression of many genes involved in transcription regulation and nucleosome/chromatin remodelling. Consistent with this, histological evaluation showed DCLK3 is present in the nucleus of striatal neurons and, protein-protein interaction experiments suggested that the kinase domain interacts with zinc finger proteins, including the transcriptional activator adaptor TADA3, a core component of the Spt-ada-Gcn5 acetyltransferase (SAGA) complex which links histone acetylation to the transcription machinery. Our novel findings suggest that the presence of DCLK3 in striatal neurons may play a key role in transcription regulation and chromatin remodelling in these brain cells, and show that reduced expression of the kinase in Huntington's disease could render the striatum highly vulnerable to neurodegeneration