Imbalance of p75(NTR)/TrkB protein expression in Huntington's disease: implication for neuroprotective therapies.

Neuroprotective therapies based on brain-derived neurotrophic factor (BDNF) administration have been proposed for Huntington's disease (HD) treatment. However, our group has recently reported reduced levels of TrkB in HD mouse models and HD human brain suggesting that besides a decrease on BDNF levels a reduction of TrkB expression could also contribute to diminished neurotrophic support in HD. BDNF can also bind to p75 neurotrophin receptor (p75(NTR)) modulating TrkB signaling. Therefore, in this study we have analyzed the levels of p75(NTR) in several HD models, as well as in HD human brain. Our data demonstrates a p75(NTR)/TrkB imbalance in the striatum of two different HD mouse models, Hdh(Q111/111) homozygous knockin mice and R6/1 mice that was also manifested in the putamen of HD patients. The imbalance between TrkB and p75(NTR) levels in a HD cellular model did not affect BDNF-mediated TrkB activation of prosurvival pathways but induced activation of apoptotic cascades as demonstrated by increased JNK phosphorylation. Moreover, BDNF failed to protect mutant huntingtin striatal cells transfected with p75(NTR) against NMDA-mediated excitotoxicity, which was associated with decreased Akt phosphorylation. Interestingly, lack of Akt activation following BDNF and NMDA treatment correlated with increased PP1 levels. Accordingly, pharmacological inhibition of PP1 by okadaic acid (OA) prevented mutant huntingtin striatal cell death induced by NMDA and BDNF. Altogether, our findings demonstrate that the p75(NTR)/TrkB imbalance induced by mutant huntingtin in striatal cells associated with the aberrant activity of PP1 disturbs BDNF neuroprotection likely contributing to increasing striatal vulnerability in HD. On the basis of this data we hypothesize that normalization of p75(NTR) and/or TrkB expression or their signaling will improve BDNF neuroprotective therapies in HD.

The dopaminergic stabilizer, (-)-OSU6162, rescues striatal neurons with normal and expanded polyglutamine chains in huntingtin protein from exposure to free radicals and mitochondrial toxins.

Huntington's disease (HD) is a neurodegenerative disease characterized by progressive motor, cognitive and psychiatric deficits, associated with predominant loss of striatal neurons and caused by a polyglutamine expansion in the huntingtin protein. There is so far neither cure nor approved disease-slowing therapy for HD, though recent clinical studies have shown a beneficial long-term effect of pridopidine in patients with HD. The nature of this effect, purely symptomatic or, in addition, neuroprotective, is difficult to elucidate in clinical trials. Pridopidine and (-)-OSU6162 are members of a new family of compounds referred to as dopaminergic stabilizers, which normalize abnormal dopamine neurotransmission. We investigated the effects of (-)-OSU6162 on huntingtin knocked-in striatal neurons in culture. Control neurons had normal full-length huntingtin with 7 glutamines while "mutant" neurons had large expansions (Q=111). We studied the dose-effect curves of (-)-OSU6162 on mitochondrial activity, LDH levels, necrosis and apoptosis in untreated Q7 and Q111 cells. In addition, we investigated the effects of (-)-OSU6162 on Q7 and Q111 neurons challenged with different neurotoxins such as sodium glutamate, H(2)O(2), rotenone and 3-nitropropionic acid (3NP). As we found prevention of toxicity of some of these neurotoxins, we investigated the putative neuroprotective mechanisms of action of (-)-OSU6162 measuring the effects of this dopaminergic stabilizer on expression and release of BDNF, the ratios of Bcl2/Bax proteins and of p-ERK/ERK, the levels of chaperones and GSH, and the effects of (-)-OSU6162 on dopamine uptake and release. We found that (-)-OSU6162, 3-150 µM, produces a dose dependent increase of mitochondrial activity and a reduction of cell death. (-)-OSU6162 does not change glutamate toxicity, but it partially prevents that of H(2)O(2), rotenone and 3-nitropropionic acid. (-)-OSU6162 increases the intracellular levels of BDNF and Bcl2/Bax and decreases those of p-ERK/ERK and CHIP in Q111 cells. (-)-OSU6162 increased (3)H-dopamine uptake and amphetamine-induced (3)H-dopamine release in E13 mouse mid brain neurons. Our studies demonstrate that (-)-OSU6162 improves survival and mitochondrial function in striatal Q111 neurons and the resistance of these cells to several striatal neurotoxins, suggesting that (-)-OSU6162 and related compounds should be tested for neuroprotection in animal models and, eventually, in patients with HD.

Long-term memory deficits in Huntington's disease are associated with reduced CBP histone acetylase activity.

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by an expanded CAG/polyglutamine repeat in the coding region of the huntingtin (htt) gene. Although HD is classically considered a motor disorder, there is now considerable evidence that early cognitive deficits appear in patients before the onset of motor disturbances. Here we demonstrate early impairment of long-term spatial and recognition memory in heterozygous HD knock-in mutant mice (Hdh(Q7/Q111)), a genetically accurate HD mouse model. Cognitive deficits are associated with reduced hippocampal expression of CREB-binding protein (CBP) and diminished levels of histone H3 acetylation. In agreement with reduced CBP, the expression of CREB/CBP target genes related to memory, such c-fos, Arc and Nr4a2, was significantly reduced in the hippocampus of Hdh(Q7/Q111) mice compared with wild-type mice. Finally, and consistent with a role of CBP in cognitive impairment in Hdh(Q7/Q111) mice, administration of the histone deacetylase inhibitor trichostatin A rescues recognition memory deficits and transcription of selective CREB/CBP target genes in Hdh(Q7/Q111) mice. These findings demonstrate an important role for CBP in cognitive dysfunction in HD and suggest the use of histone deacetylase inhibitors as a novel therapeutic strategy for the treatment of memory deficits in this disease.

Neuroprotective effects of white tea against oxidative stress-induced toxicity in striatal cells.

Tea is one of the most widely consumed beverages in the world and represents an important source of antioxidants mainly catechins that confer beneficial effects in reducing the risk of cardiovascular diseases, age-related disorders or cancer. In the central nervous system, oxidative stress caused by increased production of reactive oxygen and nitrogen species represents an important mechanism for neuronal dysfunction and cell loss in different neurodegenerative disorders. The neuroprotective effects of green-tea-derived polyphenols have extensively been demonstrated in different models of neurotoxicity. However, few data have been reported on the antioxidant activity of white tea extracts in the nervous system. In the present study, we demonstrate that white tea extracts protect striatal cell lines against oxidative stress-mediated cell death. The effects of white tea on protection of striatal cell cultures are likely associated with the antioxidant properties of white tea components since neuronal cell loss induced by nonoxidative insults such as D1 dopamine receptor activation cannot be prevented by pre-treatment with white tea. Altogether our results suggest that regular consumption of white tea may contribute to reduce oxidative stress associated with brain injury and be clinically useful for treating age-related and neurodegenerative disorders.

Prescripciones ineficaces e inapropiadas en ancianos. ¿Sirven los criterios de Beers? / In effective and inappropriate prescriptions in the elderly. Are the Beers criteria useful?

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Presenilin modulates EGFR signaling and cell transformation by regulating the ubiquitin ligase Fbw7.

The epidermal growth factor receptor (EGFR) and Notch signaling pathways have antagonistic roles during epidermal differentiation and carcinogenesis. The molecular mechanisms regulating the crosstalk between EGFR and Notch during epidermal transformation are largely unknown. We found enhanced EGFR-dependent signaling, proliferation and oncogenic transformation caused by loss of presenilins (PS), the catalytic components of gamma-secretase that generates the Notch1 intracellular domain (NICD). The underlying mechanism for abnormal EGFR signaling in PS-deficient cells involves gamma-secretase-independent transcriptional upregulation of the E3 ubiquitin ligase Fbw7. Fbw7alpha, which targets NICD for degradation, regulates positively EGFR by affecting a proteasome-dependent ubiquitination step essential for constitutive degradation and stability of EGFR. To investigate the pathological relevance of this findings in vivo, we generated a novel epidermal conditional PS-deficient (ePS cDKO) mouse by deleting both PS in keratinocytes of the basal layer of the epidermis. The ePS cDKO mice develop epidermal hyperplasia associated with enhanced expression of both EGFR and Fbw7 and reduced NICD levels in keratinocytes. These findings establish a novel role for PS on epidermal growth and transformation by reciprocally regulating the EGFR and Notch signaling pathways through Fbw7.

Cellular and molecular mechanisms involved in the selective vulnerability of striatal projection neurons in Huntington's disease.

Neurodegenerative disorders affecting the central nervous system, such as Alzheimer's disease, Parkinson's disease, Huntington's chorea (HD) and amyotrophic lateral sclerosis are characterized by the loss of selected neuronal populations. Another striking feature shared by these diseases is the deposition of proteinaceous inclusion bodies in the brain, which may be intracytoplasmatic or intranuclear, or even extracellular. However, the density and prevalence of aggregates are not always directly related to neurodegeneration. Although some of these diseases are the result of mutations in known proteins, with HD a clear example, the expression and location of the affected protein do not explain the selective neurodegeneration. Therefore, other intrinsic mechanisms, characteristic of each neuronal population, might be involved in the neurodegenerative process. In this review we focus on several proposed mechanisms such as excitotoxicity, mitochondrial dysfunction and altered expression of trophic factors, which could account for the pathogenesis of HD.

Interactions among adenosine deaminase, adenosine A(1) receptors and dopamine D(1) receptors in stably cotransfected fibroblast cells and neurons.

The role of adenosine deaminase in the interactions between adenosine A(1) and dopamine D(1) receptors was studied in a mouse fibroblast cell line stably cotransfected with human D(1) receptor and A(1) receptor cDNAs (A(1)D(1) cells). Confocal laser microscopy analysis showed a high degree of adenosine deaminase immunoreactivity on the membrane of the A(1)D(1) cells but not of the D(1) cells (only cotransfected with human D(1) receptor cDNAs). In double immunolabelling experiments in A(1)D(1) cells and cortical neurons a marked overlap in the distribution of the A(1) receptor and adenosine deaminase immunoreactivities and of the D(1) receptor and adenosine deaminase immunoreactivities was found. Quantitative analysis of A(1)D(1) cells showed that adenosine deaminase immunoreactivity to a large extent colocalizes with A(1) and D(1) receptor immunoreactivity, respectively. The A(1) receptor agonist caused in A(1)D(1) cells and in cortical neurons coaggregation of A(1) receptors and adenosine deaminase, and of D(1) receptors and adenosine deaminase. The A(1) receptor agonist-induced aggregation was blocked by R-deoxycoformycin, an irreversible adenosine deaminase inhibitor. The competitive binding experiments with the D(1) receptor antagonist [(3)H]SCH-23390 showed that the D(1) receptors had a better fit for two binding sites for dopamine, and treatment with the A(1) receptor agonist produced a disappearance of the high-affinity site for dopamine at the D(1) receptor. R-Deoxycoformycin treatment, which has previously been shown to block the interaction between adenosine deaminase and A(1) receptors, and which is crucial for the high-affinity state of the A(1) receptor, also blocked the A(1) receptor agonist-induced loss of high-affinity D(1) receptor binding. The conclusion of the present studies is that the high-affinity state of the A(1) receptor is essential for the A(1) receptor-mediated antagonistic modulation of D(1) receptors and for the A(1) receptor-induced coaggregates of A(1) and adenosine deaminase, and of D(1) and adenosine deaminase. Thus, the confocal experiments indicate that both A(1) and D(1) receptors form agonist-regulated clusters with adenosine deaminase, where the presence of a structurally intact adenosine deaminase bound to A(1) receptors is important for the A(1)-D(1) receptor-receptor interaction at the level of the D(1) receptor recognition.

Involvement of caveolin in ligand-induced recruitment and internalization of A(1) adenosine receptor and adenosine deaminase in an epithelial cell line.

Chronic exposure of A(1) adenosine receptors (A(1)R) to A(1)R agonists leads to activation, phosphorylation, desensitization, and internalization to intracellular compartments of the receptor. Desensitization and internalization of A(1)R is modulated by adenosine deaminase (ADA), an enzyme that regulates the extracellular concentration of adenosine. ADA interacts with A(1)R on the cell surface of the smooth muscle cell line DDT1 MF-2, and both proteins are internalized following agonist stimulation of the receptor. The mechanism involved in A(1)R and ADA internalization upon agonist exposure is poorly understood in epithelial cells. In this report, we show that A(1)R and ADA interact in LLC-PK(1) epithelial cells. Exposure of LLC-PK(1) cells to A(1)R agonists induces aggregation of A(1)R and ADA on the cell surface and their translocation to intracellular compartments. Biochemical and cell biology assays were used to characterize the intracellular vesicles containing both proteins after agonist treatment. A(1)R and ADA colocalized together with the rafts marker protein caveolin. Filipin, a sterol-binding agent that disrupts rafts (small microdomains of the plasma membrane), was able to inhibit A(1)R internalization. In contrast, acid treatment of the cells, which disrupts internalization via clathrin-coated vesicles, did not inhibit agonist-stimulated A(1)R internalization. We demonstrated that A(1)R agonist N(6)-(R)-phenylisopropyl adenosine promotes the translocation of A(1)R into low-density gradient fractions containing caveolin. Furthermore, a direct interaction of the C-terminal domain of A(1)R with caveolin-1 was demonstrated by pull down experiments. These results indicate that A(1)R and ADA form a stable complex in the cell surface of LLC-PK(1) cells and that agonist-induced internalization of the A(1) adenosine receptor and ADA is mediated by clathrin-independent endocytosis.

Purification and characterization of invertase from Lactobacillus reuteri CRL 1100.

The invertase of Lactobacillus reuteri CRL 1100 is a glycoprotein composed by a single subunit with a molecular weight of 58 kDa. The enzyme was stable below 45 degrees C over a wide pH range (4.5-7.0) with maximum activity at pH 6.0 and 37 degrees C. The invertase activity was significantly inhibited by bivalent metal ions (Ca(++), Cu(++), Cd(++), and Hg(++)), beta-mercaptoethanol, and dithiothreitol and partially improved by ethylenediaminetetraacetic acid. The enzyme was purified 32 times over the crude extract by gel filtration and ion-exchange chromatography with a recovery of 17%. The K(m) and V(max) values for sucrose were 6.66 mM and 0.028 micromol/min, respectively. An invertase is purified and characterized for the first time in Lactobacillus, and it proved to be a beta-fructofuranosidase.

Evidence for adenosine/dopamine receptor interactions: indications for heteromerization.

Evidence has been obtained for adenosine/dopamine interactions in the central nervous system. There exists an anatomical basis for the existence of functional interactions between adenosine A(1)R and dopamine D(1)R and between adenosine A(2A) and dopamine D(2) receptors in the same neurons. Selective A(1)R agonists affect negatively the high affinity binding of D(1) receptors. Activation of A(2A) receptors leads to a decrease in receptor affinity for dopamine agonists acting on D(2) receptors, specially of the high-affinity state. These interactions have been reproduced in cell lines and found to be of functional significance. Adenosine/dopamine interactions at the behavioral level probably reflect those found at the level of dopamine receptor binding and transduction. All these findings suggest receptor subtype-specific interactions between adenosine and dopamine receptors that may be achieved by molecular interactions (e.g., receptor heterodimerization). At the molecular level adenosine receptors can serve as a model for homomeric and heteromeric protein-protein interactions. A1R forms homodimers in membranes and also form high-order molecular structures containing also heterotrimeric G-proteins and adenosine deaminase. The occurrence of clustering also clearly suggests that G-protein- coupled receptors form high-order molecular structures, in which multimers of the receptors and probably other interacting proteins form functional complexes. In view of the occurrence of homodimers of adenosine and of dopamine receptors it is speculated that heterodimers between these receptors belonging to two different families of G-protein-coupled receptors can be formed. Evidence that A1/D1 can form heterodimers in cotransfected cells and in primary cultures of neurons has in fact been obtained. In the central nervous system direct and indirect receptor-receptor interactions via adaptor proteins participate in neurotransmission and neuromodulation and, for example, in the establishment of high neural functions such as learning and memory.

Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes.

The possible molecular basis for the previously described antagonistic interactions between adenosine A(1) receptors (A(1)R) and dopamine D(1) receptors (D(1)R) in the brain have been studied in mouse fibroblast Ltk(-) cells cotransfected with human A(1)R and D(1)R cDNAs or with human A(1)R and dopamine D(2) receptor (long-form) (D(2)R) cDNAs and in cortical neurons in culture. A(1)R and D(1)R, but not A(1)R and D(2)R, were found to coimmunoprecipitate in cotransfected fibroblasts. This selective A(1)R/D(1)R heteromerization disappeared after pretreatment with the D(1)R agonist, but not after combined pretreatment with D(1)R and A(1)R agonists. A high degree of A(1)R and D(1)R colocalization, demonstrated in double immunofluorescence experiments with confocal laser microscopy, was found in both cotransfected fibroblast cells and cortical neurons in culture. On the other hand, a low degree of A(1)R and D(2)R colocalization was observed in cotransfected fibroblasts. Pretreatment with the A(1)R agonist caused coclustering (coaggregation) of A(1)R and D(1)R, which was blocked by combined pretreatment with the D(1)R and A(1)R agonists in both fibroblast cells and in cortical neurons in culture. Combined pretreatment with D(1)R and A(1)R agonists, but not with either one alone, substantially reduced the D(1)R agonist-induced accumulation of cAMP. The A(1)R/D(1)R heteromerization may be one molecular basis for the demonstrated antagonistic modulation of A(1)R of D(1)R receptor signaling in the brain. The persistence of A(1)R/D(1)R heteromerization seems to be essential for the blockade of A(1)R agonist-induced A(1)R/D(1)R coclustering and for the desensitization of the D(1)R agonist-induced cAMP accumulation seen on combined pretreatment with D(1)R and A(1)R agonists, which indicates a potential role of A(1)R/D(1)R heteromers also in desensitization mechanisms and receptor trafficking.