Huntingtin regulates post-Golgi trafficking of secreted proteins. Here, we studied the mechanism by which mutant huntingtin impairs this process. Colocalization studies and Western blot analysis of isolated Golgi membranes showed a reduction of huntingtin in the Golgi apparatus of cells expressing mutant huntingtin. These findings correlated with a decrease in the levels of optineurin and Rab8 in the Golgi apparatus that can be reverted by overexpression of full-length wild-type huntingtin. In addition, immunoprecipitation studies showed reduced interaction between mutant huntingtin and optineurin/Rab8. Cells expressing mutant huntingtin produced both an accumulation of clathrin adaptor complex 1 at the Golgi and an increase of clathrin-coated vesicles in the vicinity of Golgi cisternae as revealed by electron microscopy. Furthermore, inverse fluorescence recovery after photobleaching analysis for lysosomal-associated membrane protein-1 and mannose-6-phosphate receptor showed that the optineurin/Rab8-dependent post-Golgi trafficking to lysosomes was impaired in cells expressing mutant huntingtin or reducing huntingtin levels by small interfering RNA. Accordingly, these cells showed a lower content of cathepsin D in lysosomes, which led to an overall reduction of lysosomal activity. Together, our results indicate that mutant huntingtin perturbs post-Golgi trafficking to lysosomal compartments by delocalizing the optineurin/Rab8 complex, which, in turn, affects the lysosomal function.

Huntington's disease (HD) patients and mouse models show learning and memory impairment even before the onset of motor symptoms. However, the molecular events involved in this cognitive decline are still poorly understood. Here, using three different paradigms, the novel object recognition test, the T-maze spontaneous alternation task and the Morris water maze, we detected severe cognitive deficits in the R6/1 mouse model of HD before the onset of motor symptoms. When we examined the putative molecular pathways involved in these alterations, we observed hippocampal cAMP-dependent protein kinase (PKA) hyper-activation in naïve R6/1 mice compared with wild-type (WT) mice, whereas extracellular signal-regulated kinase 1/2 and calcineurin activities were not modified. Increased PKA activity resulted in hyper-phosphorylation of its substrates N-methyl-d-aspartate receptor subunit 1, Ras-guanine nucleotide releasing factor-1 and striatal-enriched protein tyrosine phosphatase, but not cAMP-responsive element binding protein or the microtubule-associated protein tau. In correlation with the over-activation of the PKA pathway, we found a down-regulation of the protein levels of some phosphodiesterase (PDE) 4 family members. Similar molecular changes were found in the hippocampus of R6/2 mice and HD patients. Furthermore, chronic treatment of WT mice with the PDE4 inhibitor rolipram up-regulated PKA activity, and induced learning and memory deficits similar to those seen in R6 mice, but had no effect on R6/1 mice cognitive impairment. Importantly, hippocampal PKA inhibition by infusion of Rp-cAMPS restored long-term memory in R6/2 mice. Thus, our results suggest that occlusion of PKA-dependent processes is one of the molecular mechanisms underlying cognitive decline in R6 animals.

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/Q111mice 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.

Cognitive impairment is one of the major symptoms in most neurodegenerative disorders such as Alzheimer’s (AD), Parkinson (PD), and Huntington diseases (HD), affecting millions of people worldwide. Unfortunately, there is no treatment to cure or prevent the progression of those diseases. Cognitive impairment has been related to neuronal cell death and/or synaptic plasticity alteration in important brain regions, such as the cerebral cortex, substantia nigra, striatum, and hippocampus. Therefore, compounds that can act to protect the neuronal loss and/or to reestablish the synaptic activity are needed to prevent cognitive decline in neurodegenerative diseases. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two highly related multifunctional neuropeptides widely distributed in the central nervous system (CNS). PACAP and VIP exert their action through two common receptors, VPAC1 and VPAC2, while PACAP has an additional specific receptor, PAC1. In this review article, we first presented evidence showing the therapeutic potential of PACAP and VIP to fight the cognitive decline observed in models of AD, PD, and HD. We also reviewed the main transduction pathways activated by PACAP and VIP receptors to reduce cognitive dysfunction. Furthermore, we identified the therapeutic targets of PACAP and VIP, and finally, we evaluated different novel synthetic PACAP and VIP analogs as promising pharmacological tools.