Pathogenesis of Huntington’s Disease: How to Fight Excitotoxicity and Transcriptional Dysregulation

Huntington’s disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat in the exon-1 of the huntingtin (htt) gene. The presence of mutant htt (mhtt) results in multiple physiopathological changes, including protein aggregation, transcriptional deregulation, decreased trophic support, alteration in signaling pathways and excitotoxicity. Indeed, the presence of mhtt induces changes in the activities/levels of different kinases, phosphatases and transcription factors that can impact on cell survival. Many studies have provided evidence that transcription may be a major target of mhtt, as gene dysregulation occurs before the onset of symptoms. The greatest number of downregulated genes in HD has led to test the ability of a large number of compounds to restore gene transcription in mouse models of HD. On the other hand, mhtt engenders multiple cellular dysfunctions including an increase of pathological glutamate-mediated excitotoxicity. For that reason, targeting the excess of glutamate has been the goal for many promising drugs leading to clinical trials. Although advances in developing effective therapies are evident, currently, there is no known cure for HD and existing symptomatic treatments are limited

Novel Epigallocatechin-3-Gallate (EGCG) Derivative as a New Therapeutic Strategy for Reducing Neuropathic Pain after Chronic Constriction Nerve Injury in Mice

Abstract Neuropathic pain is common in peripheral nerve injury and often fails to respond to ordinary medication. Here, we investigated whether the two novel epigallocatechin-3-gallate (EGCG) polyphenolic derivatives, compound 23 and 30, reduce the neuropathic pain in mice chronic constriction nerve injury (CCI). First, we performed a dose-response study to evaluate nociceptive sensation after administration of EGCG and its derivatives 23 and 30, using the Hargreaves test at 7 and 21 days after injury (dpi). We daily administered EGCG, 23 and 30 (10 to 100 mg/Kg; i.p.) during the first week post-CCI. None of the doses of compound 23 caused significant pain diminution, whereas 50mg/kg was optimal for both EGCG and 30 to delay the latency of paw withdrawal. With 50 mg/Kg, we showed that EGCC prevented the thermal hyperalgesia from 7 to 21 dpi and compound 30 from 14 to 56 dpi. To evaluate the molecular mechanisms underpinning why EGCG and compound 30 differentially prevented the thermal hyperalgesia, we studied several biochemical parameters in the dorsal horn of the spinal cord at 14 and 56 dpi. We showed that the effect observed with EGCG and compound 30 was related to the inhibition of fatty acid synthase (FASN), a known target of these polyphenolic compounds. Additionally, we observed that EGCG and compound 30 reduced the expression of CCI-mediated inflammatory proteins and the nuclear localization of nuclear factor-kappa B at 14 dpi, but not at 56 dpi. We also strongly detected a decrease of synaptic plasma membrane levels of N-methyl-D-asparte receptor 2B in CCI-mice treated with compound 30 at 56 dpi. Altogether, compound 30 reduced the chronic thermal hyperalgesia induced by CCI better than the natural compound EGCG. Thus, our findings provide a rationale for the preclinical development of compound 30 as an agent to treat neuropathic pain

Striatal-Enriched Protein Tyrosine Phosphatase Expression and Activity in Huntington's Disease: A STEP in the Resistance to Excitotoxicity

Striatal-enriched protein tyrosine phosphatase (STEP) is highly expressed in striatal projection neurons, the neuronal population most affected in Huntington's disease. Here, we examined STEP expression and phosphorylation, which regulates its activity, in N-terminal exon-1 and full-length mutant huntingtin mouse models. R6/1 mice displayed reduced STEP protein levels in the striatum and cortex, whereas its phosphorylation was increased in the striatum, cortex, and hippocampus. The early increase in striatal STEP phosphorylation levels correlated with a deregulation of the protein kinase A pathway, and decreased calcineurin activity at later stages further contributes to an enhancement of STEP phosphorylation and inactivation. Accordingly, we detected an accumulation of phosphorylated ERK2 and p38, two targets of STEP, in R6/1 mice striatum at advanced stages of the disease. Activation of STEP participates in excitotoxic-induced cell death. Because Huntington's disease mouse models develop resistance to excitotoxicity, we analyzed whether decreased STEP activity was involved in this process. After intrastriatal quinolinic acid (QUIN) injection, we detected higher phosphorylated STEP levels in R6/1 than in wild-type mice, suggesting that STEP inactivation could mediate neuroprotection in R6/1 striatum. In agreement, intrastriatal injection of TAT-STEP increased QUIN-induced cell death. R6/2, Tet/HD94, and Hdh(Q7/Q111) mice striatum also displayed decreased STEP protein and increased phosphorylation levels. In Tet/HD94 mice striatum, mutant huntingtin transgene shutdown reestablished STEP expression. In conclusion, the STEP pathway is severely downregulated in the presence of mutant huntingtin and may participate in compensatory mechanisms activated by striatal neurons that lead to resistance to excitotoxicity

Novel epigallocatechin-3-gallate (EGCG) derivative as a new therapeutic strategy for reducing neuropathic pain after chronic constriction nerve injury in mice

Neuropathic pain is common in peripheral nerve injury and often fails to respond to ordinary medication. Here, we investigated whether the two novel epigallocatechin-3-gallate (EGCG) polyphenolic derivatives, compound 23 and 30, reduce the neuropathic pain in mice chronic constriction nerve injury (CCI). First, we performed a dose-response study to evaluate nociceptive sensation after administration of EGCG and its derivatives 23 and 30, using the Hargreaves test at 7 and 21 days after injury (dpi). We daily administered EGCG, 23 and 30 (10 to 100 mg/Kg; i.p.) during the first week post-CCI. None of the doses of compound 23 caused significant pain diminution, whereas 50mg/kg was optimal for both EGCG and 30 to delay the latency of paw withdrawal. With 50 mg/Kg, we showed that EGCC prevented the thermal hyperalgesia from 7 to 21 dpi and compound 30 from 14 to 56 dpi. To evaluate the molecular mechanisms underpinning why EGCG and compound 30 differentially prevented the thermal hyperalgesia, we studied several biochemical parameters in the dorsal horn of the spinal cord at 14 and 56 dpi. We showed that the effect observed with EGCG and compound 30 was related to the inhibition of fatty acid synthase (FASN), a known target of these polyphenolic compounds. Additionally, we observed that EGCG and compound 30 reduced the expression of CCI-mediated inflammatory proteins and the nuclear localization of nuclear factor-kappa B at 14 dpi, but not at 56 dpi. We also strongly detected a decrease of synaptic plasma membrane levels of N-methyl-D-asparte receptor 2B in CCI-mice treated with compound 30 at 56 dpi. Altogether, compound 30 reduced the chronic thermal hyperalgesia induced by CCI better than the natural compound EGCG. Thus, our findings provide a rationale for the preclinical development of compound 30 as an agent to treat neuropathic pain

Increased 90-kDa ribosomal S6 kinase (Rsk) activity is protective against mutant huntingtin toxicity

Background The 90-kDa ribosomal S6 kinase (Rsk) family is involved in cell survival. Rsk activation is regulated by sequential phosphorylations controlled by extracellular signal-regulated kinase (ERK) 1/2 and 3-phosphoinositide-dependent protein kinase 1 (PDK1). Altered ERK1/2 and PDK1 phosphorylation have been described in Huntington's disease (HD), characterized by the expression of mutant huntingtin (mhtt) and striatal degeneration. However, the role of Rsk in this neurodegenerative disease remains unknown. Here, we analyzed the protein levels, activity and role of Rsk in in vivo and in vitro HD models. Results We observed increased protein levels of Rsk1 and Rsk2 in the striatum of HdhQ111/Q111 and R6/1 mice, STHdhQ111/Q111 cells and striatal cells transfected with full-length mhtt. Analysis of the phosphorylation of Rsk in Hdh mice and STHdh cells showed reduced levels of phospho Ser-380 (dependent on ERK1/2), whereas phosphorylation at Ser-221 (dependent on PDK1) was increased. Moreover, we found that elevated Rsk activity in STHdhQ111/Q111 cells was mainly due to PDK1 activity, as assessed by transfection with Rsk mutant constructs. The increase of Rsk in STHdhQ111/Q111 cells occurred in the cytosol and in the nucleus, which results in enhanced phosphorylation of both cytosolic and nuclear Rsk targets. Finally, pharmacological inhibition of Rsk, knock-down and overexpression experiments indicated that Rsk activity exerts a protective effect against mhtt-induced cell death in STHdhQ7/Q7 cells transfected with mhtt. Conclusion The increase of Rsk levels and activity would act as a compensatory mechanism with capacity to prevent mhtt-mediated cell death. We propose Rsk as a good target for neuroprotective therapies in HD

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Protects Striatal Cells and Improves Motor Function in Huntington’s Disease Models: Role of PAC1 Receptor

International audienceHuntington’s disease (HD) is a hereditary neurodegenerative disorder caused by the expression of mutant huntingtin (mHtt). One of the main features of HD is the degeneration of the striatum that leads to motor discoordination. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that acts through three receptors named PAC1R, VPAC1R, and VPAC2R. In the present study, we first investigated the effect of PACAP on STHdhQ7/Q7 and STHdhQ111/Q111 cells that express wild-type Htt with 7 and mHtt with 111 glutamines, respectively. Then we explored the capacity of PACAP to rescue motor symptoms in the R6/1, a murine model of HD. We found that PACAP treatment (10 –7 M) for 24 h protects STHdhQ111/Q111 cells from mHtt-induced apoptosis. This effect is associated with an increase in PAC1R transcription, phosphorylation of ERK and Akt, and an increase of intracellular c-fos, egr1, CBP, and BDNF protein content. Moreover, the use of pharmacological inhibitors revealed that activation of ERK and Akt mediates these antiapoptotic and neurotrophic effects of PACAP. To find out PAC1R implication, we treated STHdh cells with vasoactive intestinal peptide (VIP), which exhibits equal affinity for VPAC1R and VPAC2R, but lower affinity for PAC1R, in contrast to PACAP which has same affinity for the three receptors. VIP reduced cleaved caspase-3 protein level, without promoting the expression of c-fos, egr1, CBP, and the neurotrophin BDNF. We next measured the protein level of PACAP receptors in the striatum and cortex of R6/1 mice. We observed a specific reduction of PAC1R at the onset of motor symptoms. Importantly, the intranasal administration of PACAP to R6/1 animals restored the motor function and increased the striatal levels of PAC1R, CBP, and BDNF. In conclusion, PACAP exerts antiapoptotic and neurotrophic effects in striatal neurons mainly through PAC1R. This effect in HD striatum allows the recovery of motor function and point out PAC1R as a therapeutic target for treatment of HD

Increased 90-kDa ribosomal S6 kinase (Rsk) activity is protective against mutant huntingtin toxicity

Background: The 90-kDa ribosomal S6 kinase (Rsk) family is involved in cell survival. Rsk activation is regulated by sequential phosphorylations controlled by extracellular signal-regulated kinase (ERK) 1/2 and 3 phosphoinositidedependent protein kinase 1 (PDK1). Altered ERK1/2 and PDK1 phosphorylation have been described in Huntington’s disease (HD), characterized by the expression of mutant huntingtin (mhtt) and striatal degeneration. However, the role of Rsk in this neurodegenerative disease remains unknown. Here, we analyzed the protein levels, activity and role of Rsk in in vivo and in vitro HD models. Results: We observed increased protein levels of Rsk1 and Rsk2 in the striatum of HdhQ111/Q111 and R6/1 mice, STHdhQ111/Q111 cells and striatal cells transfected with full-length mhtt. Analysis of the phosphorylation of Rsk in Hdh mice and STHdh cells showed reduced levels of phospho Ser-380 (dependent on ERK1/2), whereas phosphorylation at Ser-221 (dependent on PDK1) was increased. Moreover, we found that elevated Rsk activity in STHdhQ111/Q111 cells was mainly due to PDK1 activity, as assessed by transfection with Rsk mutant constructs. The increase of Rsk in STHdhQ111/Q111 cells occurred in the cytosol and in the nucleus, which results in enhanced phosphorylation of both cytosolic and nuclear Rsk targets. Finally, pharmacological inhibition of Rsk, knock-down and overexpression experiments indicated that Rsk activity exerts a protective effect against mhtt-induced cell death in STHdhQ7/Q7 cells transfected with mhtt. Conclusion: The increase of Rsk levels and activity would act as a compensatory mechanism with capacity to prevent mhtt-mediated cell death. We propose Rsk as a good target for neuroprotective therapies in H

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Protects Striatal Cells and Improves Motor Function in Huntington's Disease Models: Role of PAC1 Receptor.

Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by the expression of mutant huntingtin (mHtt). One of the main features of HD is the degeneration of the striatum that leads to motor discoordination. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that acts through three receptors named PAC1R, VPAC1R, and VPAC2R. In the present study, we first investigated the effect of PACAP on STHdhQ7/Q7 and STHdhQ111/Q111 cells that express wild-type Htt with 7 and mHtt with 111 glutamines, respectively. Then we explored the capacity of PACAP to rescue motor symptoms in the R6/1, a murine model of HD. We found that PACAP treatment (10-7 M) for 24 h protects STHdhQ111/Q111 cells from mHtt-induced apoptosis. This effect is associated with an increase in PAC1R transcription, phosphorylation of ERK and Akt, and an increase of intracellular c-fos, egr1, CBP, and BDNF protein content. Moreover, the use of pharmacological inhibitors revealed that activation of ERK and Akt mediates these antiapoptotic and neurotrophic effects of PACAP. To find out PAC1R implication, we treated STHdh cells with vasoactive intestinal peptide (VIP), which exhibits equal affinity for VPAC1R and VPAC2R, but lower affinity for PAC1R, in contrast to PACAP which has same affinity for the three receptors. VIP reduced cleaved caspase-3 protein level, without promoting the expression of c-fos, egr1, CBP, and the neurotrophin BDNF. We next measured the protein level of PACAP receptors in the striatum and cortex of R6/1 mice. We observed a specific reduction of PAC1R at the onset of motor symptoms. Importantly, the intranasal administration of PACAP to R6/1 animals restored the motor function and increased the striatal levels of PAC1R, CBP, and BDNF. In conclusion, PACAP exerts antiapoptotic and neurotrophic effects in striatal neurons mainly through PAC1R. This effect in HD striatum allows the recovery of motor function and point out PAC1R as a therapeutic target for treatment of HD