Xavier Xifró

Mutant huntingtin impairs post-Golgi trafficking to lysosomes by delocalizing optineurin/Rab8 complex from the Golgi apparatus.

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.

THE EXPANDING CLINICAL PROFILE OF ANTI-AMPA RECEPTOR ENCEPHALITIS

Antibodies to the glutamate receptor 1 (GluR1) and GluR2 subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR-ab) were recently described in 10 patients from a series of 109 with limbic encephalitis (LE).1 Besides the hippocampus, GluR1/2 subunits of AMPAR are also widely expressed in the cerebral cortex, basal ganglia, and cerebellum,2 suggesting that the clinical presentation could also extend beyond the clinical profile of LE. In this study, we analyzed the presence of AMPAR-ab in a consecutive series of patients whose serum or CSF was sent to our laboratory for analysis of antineuronal antibodies in the last 2 years. ### Methods. We review all patients with final diagnosis of limbic encephalitis3 or with an immunohistochemical pattern of NMDAR-ab or AMPAR-ab in the serum or CSF (figure e-1 on the Neurology ® Web site at www.neurology.org). AMPAR-ab immunoreactivity on brain sections was confirmed on HEK293 cells transfected with plasmids containing rodent GluR1/2 subunits as described (figure).1 The study was approved by the Ethical Committee of the Hospital Clinic. Figure Immunoreactivity of antibodies against AMPA receptors (A) Section of paraformaldehyde-perfused rat brain incubated with the CSF of a patient with AMPAR-ab. There is a robust reactivity with the neuropil of hippocampus. Bar = 25 μm. (B) HEK293 cells transfected with GluR1/2 AMPAR subunits show intense reactivity with the positive CSF (green) …

Increased PKA signaling disrupts recognition memory and spatial memory: role in Huntington's disease

Huntingtons 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.

Altered cholesterol homeostasis contributes to enhanced excitotoxicity in Huntington’s disease

J. Neurochem. (2010) 115, 153–167.

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 Huntingtons 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 Huntingtons 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 HdhQ7/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.

Calcineurin is involved in the early activation of NMDA‐mediated cell death in mutant huntingtin knock‐in striatal cells

Excitotoxicity has been proposed as one of the mechanisms involved in the specific loss of striatal neurons that occurs in Huntington’s disease. Here, we studied the role of calcineurin in the vulnerability of striatal neurons expressing mutant huntingtin to excitotoxicity. To this end, we induced excitotoxicity by adding NMDA to a striatal precursor cell line expressing full‐length wild‐type (STHdhQ7/Q7) or mutant (STHdhQ111/Q111) huntingtin. We observed that cell death appeared earlier in STHdhQ111/Q111 cells than in STHdhQ7/Q7 cells. Interestingly, these former cells expressed higher levels of calcineurin A that resulted in a greater increase of its activity after NMDA receptor stimulation. Moreover, transfection of full‐length mutant huntingtin in different striatal‐derived cells (STHdhQ7/Q7, M213 and primary cultures) increased calcineurin A protein levels. To determine whether high levels of calcineurin A might account for the earlier activation of cell death in mutant huntingtin knock‐in cells, wild‐type cells were transfected with calcineurin A. Calcineurin A‐transfected STHdhQ7/Q7 cells displayed a significant increase in cell death compared with that recorded in green fluorescent protein‐transfected cells after NMDA treatment. Notably, addition of the calcineurin inhibitor FK‐506 produced a more robust reduction in cell death in mutant huntingtin knock‐in cells than it did in wild‐type cells. These results suggest that high levels of calcineurin A could account for the increased vulnerability of striatal cells expressing mutant huntingtin to excitotoxicity.

Contribution of caspase-mediated apoptosis to the cell death caused by oxygen-glucose deprivation in cortical cell cultures.

Several evidences suggest that cell death after cerebral ischemia involves both necrosis and apoptosis. However, it is still unknown which is the relative contribution of both types of cell death. Exposing rat cortical cultures to oxygen-glucose deprivation (OGD), we show the simultaneous presence of necrotic and apoptotic cells. The relative contribution of necrosis and apoptosis was dependent on the duration of the OGD. OGD-mediated apoptotic cell death is caspase-dependent because the addition of a pan-caspase inhibitor specifically blocked the apoptotic component of the OGD-mediated cell death. Moreover, we observed the activation of caspase-3, -7, and -9 after OGD in neurons and microglial cells. No activation of these caspases was observed in GFAP positive cells. Our results also show that calpain is related to OGD-mediated proteolysis of caspase-3 and -9 but not of caspase-7. These data suggest that different pathways could be involved in OGD-mediated caspase activation.

PH domain leucine-rich repeat protein phosphatase 1 contributes to maintain the activation of the PI3K/Akt pro-survival pathway in Huntington's disease striatum

Dysregulation of gene expression is one of the mechanisms involved in the pathophysiology of Huntingtons disease (HD). Here, we examined whether mutant huntingtin regulates the levels of PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), a phosphatase that specifically dephosphorylates Akt at Ser473. Our results show decreased PHLPP1 protein levels in knock-in models (HdhQ111/Q111 mouse striatum and STHdhQ111/Q111 cells), in the striatum of N-terminal exon-1 mutant huntingtin transgenic mouse models (R6/1; R6/1 : BDNF +/−, R6/2 and Tet/HD94) and in the putamen of HD patients. Quantitative PCR analysis revealed a reduction in PHLPP1 mRNA levels in the striatum of R6/1 compared with wild-type mice. Coincident with reduced PHLPP1 protein levels, we observed increased phosphorylated Akt (Ser473) levels specifically in the striatum. The analysis of the conditional mouse model Tet/HD94 disclosed that after mutant huntingtin shutdown PHLPP1 levels returned to wild-type levels whereas phospho-Akt levels were partially reduced. In conclusion, our results show that mutant huntingtin downregulates PHLPP1 expression. In the striatum, these reduced levels of PHLPP1 can contribute to maintain high levels of activated Akt that may delay cell death and allow the recovery of neuronal viability after mutant huntingtin silencing.

Reduced calcineurin protein levels and activity in exon-1 mouse models of Huntington's disease: Role in excitotoxicity

Calcineurin is a serine/threonine phosphatase involved in the regulation of glutamate receptors signaling. Here, we analyzed whether the regulation of calcineurin protein levels and activity modulates the susceptibility of striatal neurons to excitotoxicity in R6/1 and R6/1:BDNF+/- mouse models of Huntingtons disease. We show that calcineurin inhibition in wild-type mice drastically reduced quinolinic acid-induced striatal cell death. Moreover, calcineurin A and B were differentially regulated during disease progression with a specific reduction of calcineurin A protein levels and calcineurin activity at the onset of the disease in R6/1:BDNF+/- mice. Analysis of the conditional mouse model Tet/HD94 showed that mutant huntingtin specifically controls calcineurin A protein levels. Finally, calcineurin activation induced by intrastriatal quinolinic acid injection in R6/1 mouse was lower than in wild-type mice. Therefore, reduction of calcineurin activity by alteration of calcineurin A expression participates in the pathophysiology of Huntingtons disease and contributes to the excitotoxic resistance observed in exon-1 mouse models.

Estradiol facilitates neurite maintenance by a Src/Ras/ERK signalling pathway

Different reports suggest the estrogens are involved in neuritic outgrowth, maintenance of dendritic morphology and spine formation in the CNS. However, the molecular mechanisms regulated by estrogens on neuronal integrity are not fully understood. We have addressed the relationship between 17beta-estradiol-dependent ERK pathway stimulation and the maintenance of neuritic morphology in cerebellar granule cell cultures (CGC). We report that 17beta-estradiol clearly activates ERK phosphorylation in CGC cultured in low potassium via ERalpha localized in the plasma membrane and without the activation of the insulin-like growth factor-I receptor. 17beta-estradiol activates the ERK pathway through Ras-dependent Src kinase activity. A concomitant activation of the cAMP-response element-binding protein (CREB) is observed. Moreover, we demonstrate that 17beta-estradiol-mediated ERK activation is involved in the maintenance of neuritic arborisation and neuronal morphology in proapoptotic conditions.