Raquel Marin

Lipid Alterations in Lipid Rafts from Alzheimer's Disease Human Brain Cortex

Lipid rafts are membrane microdomains intimately associated with cell signaling. These biochemical microstructures are characterized by their high contents of sphingolipids, cholesterol and saturated fatty acids and a reduced content of polyunsaturated fatty acids (PUFA). Here, we have purified lipid rafts of human frontal brain cortex from normal and Alzheimers disease (AD) and characterized their biochemical lipid composition. The results revealed that lipid rafts from AD brains exhibit aberrant lipid profiles compared to healthy brains. In particular, lipid rafts from AD brains displayed abnormally low levels of n-3 long chain polyunsaturated fatty acids (LCPUFA, mainly 22:6n-3, docosahexaenoic acid) and monoenes (mainly 18:1n-9, oleic acid), as well as reduced unsaturation and peroxidability indexes. Also, multiple relationships between phospholipids and fatty acids were altered in AD lipid rafts. Importantly, no changes were observed in the mole percentage of lipid classes and fatty acids in rafts from normal brains throughout the lifespan (24-85 years). These indications point to the existence of homeostatic mechanisms preserving lipid raft status in normal frontal cortex. The disruption of such mechanisms in AD brains leads to a considerable increase in lipid raft order and viscosity, which may explain the alterations in lipid raft signaling observed in AD.

Plasma membrane oestrogen receptor mediates neuroprotection against β‐amyloid toxicity through activation of Raf‐1/MEK/ERK cascade in septal‐derived cholinergic SN56 cells

Rapid oestrogen neuroprotection against β‐amyloid peptide (Aβ)‐induced toxicity, a main feature of Alzheimers disease, may be partially initiated at the plasma membrane. However, the mechanism by which this oestrogen effect occurs is unknown. In a septal murine cell line (SN56), we observed that short exposures to either 17β‐oestradiol (E2) or membrane impermeant E2 bound to horseradish peroxidase (E‐HRP) induced a biphasic stimulation of extracellular‐signal regulated protein kinase (ERK1/2) phosphorylation, with peak inductions detected around 4–8 min in the early phase and a second maximum around 8 h after treatment. ERK1/2 phosphorylation was abolished by ERK1/2 kinase (MEK) inhibitors PD98059 and U0126. Interestingly, PD98059 was also shown to block rapid E2‐related prevention of death in cells exposed to Aβ fragment 1–40 (Aβ1−40) for 24 h. In contrast, no neuroprotective effects were obtained when MEK inhibitor was used to selectively abolish the late phosphorylation phase. Furthermore, both ERK1/2 activation and E2‐associated protection were blocked by an inhibitor of Raf‐1 kinase. Raf‐1 may be involved in these effects because oestrogen caused the rapid serine 338 (Ser338) phosphorylation of this protein. In addition, the oestrogen receptor (ER) antagonist ICI 182 780 was also observed to block ERK1/2 phosphorylation. We propose a novel mechanism in SN56 cells by which rapid effects of oestrogen leading to neuroprotection are signalled through Raf‐1/MEK/ERK1/2 pathway, possibly by activation of a membrane‐related ER.

Estrogen Activates Classical and Alternative Mechanisms to Orchestrate Neuroprotection

Evidence for a protective role of estradiol in neurodegenerative diseases has steadily increased over the past decade, though the mechanisms of action and the participation of true estrogen receptors (ERs) have proven a complex score. The protective effects of estrogens take place partly through pathways involving canonical ER activation, which is constitutively expressed in many brain regions and is able to initiate gene transcription after specifically binding to estradiol. In addition, non-genomic (or alternative) signalling pathways, involving extranuclear ERs, respond to physiological concentration of estrogens to elicit neuroprotection. Often, rapid activation of intracellular signallers such as mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K) underlie alternative estrogen-induced neuroprotection upon activation of specific binding sites at the plasma membrane. Although the molecular characteristics of these unconventional ERs are still largely unknown, the generally held view maintains that plasma membrane ER (mER) originates from, or is related to, classical nuclear ERs. The present article will review some of the most recent evidence revealing the relevance of alternative mechanisms in estrogen-dependent neuroprotection. Special emphasis will be paid to cellular models of amyloid-beta toxicity where classical and alternative pathways activated by estrogens seem to coexist to orchestrate neuroprotection.

VDAC and ERα interaction in caveolae from human cortex is altered in Alzheimer's disease

Voltage-dependent anion channel (VDAC) is a mitochondrial porin also found in the neuronal membrane (pl-VDAC), where its function may be related to redox homeostasis and apoptosis. Murine models have evidenced pl-VDAC into caveolae in a complex with estrogen receptor alpha (mERalpha), which participates in neuroprotection against amyloid beta (Abeta), and whose integration into this hydrophobic domain remains unclear. Here, we have demonstrated in caveolae of human cortex and hippocampus the presence of pl-VDAC and mERalpha, in a complex with scaffolding caveolin-1 which likely provides mERalpha stability at the plasma membrane. In Alzheimers disease (AD) brains, VDAC was accumulated in caveolae, and it was observed in dystrophic neurites of senile plaques, whereas ERalpha was expressed in astrocytes surrounding the plaques. Together with previous data in murine neurons demonstrating the participation of pl-VDAC in Abeta-induced neurotoxicity, these data suggest that the channel may be involved in membrane dysfunctioning observed in AD neuropathology.

Voltage-dependent anion channel (VDAC) participates in amyloid beta-induced toxicity and interacts with plasma membrane estrogen receptor α in septal and hippocampal neurons

Voltage-dependent anion channel (VDAC) is a porin known by its role in metabolite transport across mitochondria and participation in apoptotic processes. Although traditionally accepted to be located within mitochondrial outer membrane, some data has also reported its presence at the plasma membrane level where it seems to participate in regulation of normal redox homeostasis and apoptosis. Here, exposure of septal SN56 and hippocampal HT22 cells to specific anti-VDAC antibodies prior to amyloid beta (Aβ) peptide was observed to prevent neurotoxicity. In these cell lines, we identified a VDAC form associated with the plasma membrane that seems to be particularly abundant in caveolae. The two membrane-related isoforms of estrogen receptor α (mERα) (80 and 67 kDa), known in SN56 cells to participate in estrogen-induced neuroprotection against Aβ injury, were also observed to be present in caveolae. Interestingly, we demonstrated for the first time that both VDAC and mERα interact at the plasma membrane of these neurons as well as in microsomal fractions of the corresponding murine septal and hippocampal tissues. These proteins were also shown to associate with caveolin-1, thereby corroborating their presence in caveolar microdomains. Taken together, these results suggest that VDAC-mERα association at the plasma membrane level may participate in the modulation of Aβ-induced cell death.

Estradiol prevents amyloid-β peptide-induced cell death in a cholinergic cell line via modulation of a classical estrogen receptor

The pathology of Alzheimers disease includes amyloid-beta peptide aggregation that contributes to degeneration of cholinergic neurons. Even though the underlying molecular mechanisms remain unclear, recent in vitro evidence supports a protective role for estrogens against several neurotoxic agents. Here we report that, in a murine cholinergic cell line (SN56), the massive cell death induced by 1-40 fragment of amyloid-beta peptide was prevented by 17beta-estradiol through a mechanism that may involve estrogen receptor activation. The protective effect of estradiol was observed in a dose-dependent manner, and was completely blocked by the pure antiestrogen ICI 182,780. In contrast, the inactive isomer 17alpha-estradiol consistently showed weaker neuroprotection than the native hormone that was unaffected by ICI 182,780 treatment. In addition, equivalent concentrations of 17beta-estradiol enhanced luciferase activity in cells transfected with a luciferase reporter gene driven by tandem estrogen response elements. Estrogen-induced luciferase activity was blocked by ICI 182,780, indicating estrogen receptor-dependent transcriptional activity. We also observed by reverse transcription-polymerase chain reaction, Western blot and immunocytochemistry that increasing concentrations of 17beta-estradiol enhanced the expression of estrogen receptor alpha mRNA and protein during amyloid-beta-induced toxicity. Under these conditions, it was found by confocal microscopy that the localization of estrogen receptor alpha in the absence of hormone was mainly extranuclear. However, the receptor was consistently observed also at the nuclear region after estrogen exposure. Overall, these data suggest that estrogen may exert neuroprotective effects against amyloid-beta-induced toxicity by activation of estrogen receptor-mediated pathways. In addition, intracellular estrogen receptors are up-regulated by their cognate hormone even during exposure to neurotoxic agents.

Changes in Mating Behavior, Erectile Function, and Nitric Oxide Levels in Penile Corpora Cavernosa in Streptozotocin-Diabetic Rats

Abstract This study assessed whether the in vivo production of nitric oxide (NO) in the penis is impaired in experimental diabetes and whether this phenomenon can be explained by abnormal levels of NO synthase isoenzymes and/or plasma androgens. Adult male Sprague-Dawley rats were injected with streptozotocin (STZ) (40 mg/kg, i.p.) or vehicle. One half of the STZ-treated animals received daily insulin replacement. Twelve weeks later, the animals were tested for mating behavior and erectile reflexes. They were then anesthetized with urethane (1 g/kg), and the NO levels in their corpora cavernosa were monitored electrochemically with porphyrin microsensors before and after electrostimulation of the cavernous nerve. The intracavernous pressure (ICP) was measured simultaneously. The diabetic animals had substantial impairment in the mating and erectile reflexes tests, decreased basal and stimulated NO levels in the corpora, and a reduced ICP response to cavernous nerve stimulation. Insulin replacement fully reversed the effects of diabetes on the mating reflexes, the basal NO signals, and the ICP responses to electrical field stimulation and partially restored the stimulated NO release. Neither diabetes nor diabetes with insulin treatment had significant effects on serum testosterone levels or NOS isoform (nNOS, eNOS, and iNOS) protein content in penile homogenates, indicating that the changes found in erectile function were independent of such variables. These results also suggest that the diabetes-induced reduction in corporeal NO levels could be mainly due to the lack of some essential cofactors for NOS activity rather than to changes in the amount of enzyme proteins.

Role of estrogen receptor α in membrane-initiated signaling in neural cells: Interaction with IGF-1 receptor

The mechanisms of action of estradiol in the nervous system involve nuclear-initiated steroid signaling and membrane-initiated steroid signaling. Estrogen receptors (ERs) are involved in both mechanisms. ERalpha interacts with the signaling of IGF-1 receptor in neural cells: ERalpha transcriptional activity is regulated by IGF-1 receptor signaling and estradiol regulates IGF-1 receptor signaling. The interaction between ERalpha and the IGF-1 receptor in the brain may occur at the plasma membrane of neurons and glial cells. Caveolin-1 may provide the scaffolding for the interaction of different membrane-associated molecules, including voltage-dependent anion channel, ERalpha and IGF-I receptor.

Altered lipid composition in cortical lipid rafts occurs at early stages of sporadic Alzheimer's disease and facilitates APP/BACE1 interactions

The presence of lipid alterations in lipid rafts from the frontal cortex in late stages of Alzheimers disease (AD) has been recently demonstrated. Here, we have isolated and analyzed the lipid composition of lipid rafts from different brain areas from control and AD subjects at initial neuropathologic stages. We have observed that frontal cortex lipid rafts are profoundly altered in AD brains from the earliest stages of AD, namely AD I/II. These changes in the lipid matrix of lipid rafts affected both lipid classes and fatty acids and were also detected in the entorhinal cortex, but not in the cerebellum from the same subjects. Paralleling these changes, lipid rafts from AD frontal and entorhinal cortices displayed higher anisotropy for environment-sensitive probes, indicating that lipid changes in AD lipid rafts increased membrane order and viscosity in these domains. The pathophysiological consequences of these alterations in the development and progression of AD were strengthened by the significant, and specific, accumulation of β-secretase within the lipid rafts of AD subjects even at the earliest stages. Our results provide a mechanistic connection between lipid alterations in these microdomains and amyloidogenic processing of amyloid precursor protein.

An oestrogen membrane receptor participates in estradiol actions for the prevention of amyloid-β peptide1-40-induced toxicity in septal-derived cholinergic SN56 cells

Although oestrogen [17β‐estradiol (E2)]‐related neuroprotection has been demonstrated in different models, the involvement of non‐classical oestrogen receptors (ERs) remains unexplored. Using the SN56 cholinergic cell line, we present evidence indicating that an ER associated with the plasma membrane participates in oestrogen‐dependent inhibition of cell death induced by amyloid‐β peptide (Aβ) toxicity. Similarly to E2 alone, a 15‐min exposure to estradiol‐horseradish peroxidase (E‐HRP) significantly reduced Aβ‐induced cell death. This effect was decreased by the ER antagonist ICI 182,780 as well as by MC‐20 antibody directed to a region neighbouring the ligand‐binding domain of ERα. Using confocal microscopy on unpermeabilized SN56 cells exposed to MC‐20 antibody, we identified a protein at the plasma membrane level. Western blot analysis of purified SN56 cell membrane fractions using MC‐20 antibody revealed the presence of one band with the same electrophoretic mobility as intracellular ERα. Using conjugated forms of the steroid, E‐HRP and E2 conjugated to bovine serum albumin‐FITC, we demonstrated by confocal microscopy that SN56 cells contain surface binding sites for E2. Binding of both conjugates was blocked by pre‐incubation with E2 and decreased by either ICI 182,780 or MC‐20 antibody in a concentration‐dependent manner. Thus, a membrane‐related ER that shares some structural homologies with ERα may participate in oestrogen‐mediated neuroprotection.