Phytosterols reverse antiretroviral-induced hearing loss, with potential implications for cochlear aging.

Cholesterol contributes to neuronal membrane integrity, supports membrane protein clustering and function, and facilitates proper signal transduction. Extensive evidence has shown that cholesterol imbalances in the central nervous system occur in aging and in the development of neurodegenerative diseases. In this work, we characterize cholesterol homeostasis in the inner ear of young and aged mice as a new unexplored possibility for the prevention and treatment of hearing loss. Our results show that cholesterol levels in the inner ear are reduced during aging, an effect that is associated with an increased expression of the cholesterol 24-hydroxylase (CYP46A1), the main enzyme responsible for cholesterol turnover in the brain. In addition, we show that pharmacological activation of CYP46A1 with the antiretroviral drug efavirenz reduces the cholesterol content in outer hair cells (OHCs), leading to a decrease in prestin immunolabeling and resulting in an increase in the distortion product otoacoustic emissions (DPOAEs) thresholds. Moreover, dietary supplementation with phytosterols, plant sterols with structure and function similar to cholesterol, was able to rescue the effect of efavirenz administration on the auditory function. Altogether, our findings point towards the importance of cholesterol homeostasis in the inner ear as an innovative therapeutic strategy in preventing and/or delaying hearing loss.

SIRT-1 Activity Sustains Cholesterol Synthesis in the Brain.

SIRT-1 is a potent energy regulator that has been implicated in the aging of different tissues, and cholesterol synthesis demands high amounts of cellular adenosine triphosphate. An efficient synaptic transmission depends on processes that are highly influenced by cholesterol levels, like endocytosis, exocytosis and membrane lateral diffusion of neurotransmitter receptors. We set out to investigate whether SIRT-1 activity affects brain cholesterol metabolism. We found that pharmacological inhibition of SIRT-1 with EX-527 reduces the mRNA amounts of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR), Cytochrome P450 46A1 (CYP46A1) and Apolipoprotein E (APO-E) in rat primary cortical cultures. The decreased expression of these genes was paralleled by a significant reduction of the cholesterol levels in this type of neuronal culture. Interestingly, a cholesterol decrease of similar extent was observed in mouse astroglial cultures after EX-527 treatment. In agreement, mice administered with EX-527 for 5 days showed a down-regulation of cholesterol synthesis in the cortex, with significant reductions in the mRNA amounts of the transcription factor Sterol Regulatory Element Binding Protein 2 (SREBP-2) and the enzyme HMGCR, two key regulators of the cholesterol synthesis. These transcriptional changes were paralleled by reduced cholesterol levels at cortical synapses. SIRT-1 inhibition also reduced the amount of cholesterol in the hippocampus but without affecting the HMGCR expression levels. Altogether, these results uncover a role for SIRT-1 in the regulation of cholesterol metabolism, and demonstrate that SIRT-1 is required to sustain adequate levels of cholesterol synthesis in the adult brain.

24S-hydroxycholesterol: Cellular effects and variations in brain diseases.

The adult brain exhibits a characteristic cholesterol homeostasis, with low synthesis rate and active catabolism. Brain cholesterol turnover is possible thanks to the action of the enzyme cytochrome P450 46A1 (CYP46A1) or 24-cholesterol hydroxylase, that transforms cholesterol into 24S-hydroxycholesterol (24S-HC). But before crossing the blood-brain barrier (BBB), this oxysterol, that is the most abundant in the brain, can act locally, affecting the functioning of neurons, astrocytes, oligodendrocytes, and vascular cells. The first part of this review addresses different aspects of 24S-HC production and elimination from the brain. The second part concentrates in the effects of 24S-HC at the cellular level, describing how this oxysterol affects cell viability, amyloid ß production, neurotransmission, and transcriptional activity. Finally, the role of 24S-HC in Alzheimer, Huntington and Parkinson diseases, multiple sclerosis and amyotrophic lateral sclerosis, as well as the possibility of using this oxysterol as predictive and/or evolution biomarker in different brain disorders is discussed.

Pleiotropic effects of statins on brain cells.

Starting with cholesterol homeostasis, the first part of the review addresses various aspects of cholesterol metabolism in neuronal and glial cells and the mutual crosstalk between the two cell types, particularly the transport of cholesterol from its site of synthesis to its target loci in neuronal cells, discussing the multiple mechanistic aspects and transporter systems involved. Statins are next analyzed from the point of view of their chemical structure and its impingement on their pharmacological properties and permeability through cell membranes and the blood-brain barrier in particular. The following section then discusses the transcriptional effects of statins and the changes they induce in brain cell genes associated with a variety of processes, including cell growth, signaling and trafficking, uptake and synthesis of cholesterol. We review the effects of statins at the cellular level, analyzing their impact on the cholesterol composition of the nerve and glial cell plasmalemma, neurotransmitter receptor mobilization, myelination, dendritic arborization of neurons, synaptic vesicle release, and cell viability. Finally, the role of statins in disease is exemplified by Alzheimer and Parkinson diseases and some forms of epilepsy, both in animal models and in the human form of these pathologies.

Analysis of cholesterol in mouse brain by HPLC with UV detection.

We describe a sensitive high performance liquid chromatography (HPLC)-based method for the determination of cholesterol in brain tissue. The method does not require the derivatization of the analyte and uses separation and quantification by reversed-phase HPLC coupled to UV detection. Lipids were methanol/chloroform extracted following the method of Bligh and Dyer, and separated using isopropanol/acetonitrile/water (60/30/10, v/v/v) as mobile phase. We observed lineal detection in a wide range of concentrations, from 62.5 to 2000 ng/µL, and were able to detect a significant increase in the brain cholesterol levels between postnatal days 2 and 10 in C57BL6 mice. Based on our validation parameters, we consider this analytical method a useful tool to assess free cholesterol in rodent brain samples and cell cultures.

Sab is differentially expressed in the brain and affects neuronal activity.

Sab (SH3 binding protein 5 or SH3BP5) is a mitochondrial scaffold protein involved in signaling associated with mitochondrial dysfunction and apoptosis; furthermore, Sab is a crucial signaling platform for neurodegenerative disease. To determine how this signaling nexus could have a significant effect on disease, we examined the regional abundance of Sab in the brain and sub-neuronal distribution, and we monitored the effect of Sab-mediated signaling on neuronal activity. We found that Sab is widely expressed in the adult mouse brain with increased abundance in hippocampus, ventral midbrain, and cerebellum. Sab was found in purified synaptosomes and in cultures of hippocampal neurons and astrocytes. Confocal and electron microscopy of mouse hippocampal sections confirmed the mitochondrial localization of Sab in the soma, dendrites, and axons. Given the localization and sub-neuronal distribution of Sab, we postulated that Sab-mediated signaling could affect neuronal function, so we measured the impact of inhibiting Sab-mediated events on the spontaneous activity in cultured hippocampal neurons. Treatment with a Sab-inhibitory peptide (Tat-SabKIM1), but not a scrambled control peptide, decreased the firing frequency and spike amplitudes. Our results demonstrate that brain-specific Sab-mediated signaling plays a role in neuronal activity through the manipulation of mitochondrial physiology by interacting kinases.

Presenilin transmembrane domain 8 conserved AXXXAXXXG motifs are required for the activity of the γ-secretase complex.

Understanding the molecular mechanisms controlling the physiological and pathological activity of γ-secretase represents a challenging task in Alzheimer disease research. The assembly and proteolytic activity of this enzyme require the correct interaction of the 19 transmembrane domains (TMDs) present in its four subunits, including presenilin (PS1 or PS2), the γ-secretase catalytic core. GXXXG and GXXXG-like motifs are critical for TMDs interactions as well as for protein folding and assembly. The GXXXG motifs on γ-secretase subunits (e.g. APH-1) or on γ-secretase substrates (e.g. APP) are known to be involved in γ-secretase assembly and in Aß peptide production, respectively. We identified on PS1 and PS2 TMD8 two highly conserved AXXXAXXXG motifs. The presence of a mutation causing an inherited form of Alzheimer disease (familial Alzheimer disease) in the PS1 motif suggested their involvement in the physiopathological configuration of the γ-secretase complex. In this study, we targeted the role of these motifs on TMD8 of PSs, focusing on their role in PS assembly and catalytic activity. Each motif was mutated, and the impact on complex assembly, activity, and substrate docking was monitored. Different amino acid substitutions on the same motif resulted in opposite effects on γ-secretase activity, without affecting the assembly or significantly impairing the maturation of the complex. Our data suggest that AXXXAXXXG motifs in PS TMD8 are key determinants for the conformation of the mature γ-secretase complex, participating in the switch between the physiological and pathological functional conformations of the γ-secretase.

Cholesterol loss during glutamate-mediated excitotoxicity.

The deregulation of brain cholesterol metabolism is typical in acute neuronal injury (such as stroke, brain trauma and epileptic seizures) and chronic neurodegenerative diseases (Alzheimer's disease). Since both conditions are characterized by excessive stimulation of glutamate receptors, we have here investigated to which extent excitatory neurotransmission plays a role in brain cholesterol homeostasis. We show that a short (30 min) stimulation of glutamatergic neurotransmission induces a small but significant loss of membrane cholesterol, which is paralleled by release to the extracellular milieu of the metabolite 24S-hydroxycholesterol. Consistent with a cause-effect relationship, knockdown of the enzyme cholesterol 24-hydroxylase (CYP46A1) prevented glutamate-mediated cholesterol loss. Functionally, the loss of cholesterol modulates the magnitude of the depolarization-evoked calcium response. Mechanistically, glutamate-induced cholesterol loss requires high levels of intracellular Ca(2+), a functional stromal interaction molecule 2 (STIM2) and mobilization of CYP46A1 towards the plasma membrane. This study underscores the key role of excitatory neurotransmission in the control of membrane lipid composition, and consequently in neuronal membrane organization and function.

Regulation of tyrosine kinase B activity by the Cyp46/cholesterol loss pathway in mature hippocampal neurons: relevance for neuronal survival under stress and in aging.

It is well established that memory formation and retention involve the coordinated flow of information from the post-synaptic site of particular neuronal populations to the nucleus, where short and long-lasting modifications of gene expression occur. With age, mnemonic, motor and sensorial alterations occur, and it is believed that extra failures in the mechanisms used for memory formation and storage are the cause of neurodegenerative pathologies like Alzheimer's disease. A prime candidate responsible for damage and loss of function during aging is the accumulation of reactive oxygen species, derived from normal oxidative metabolism. However, dysfunction in the aged brain is not paralleled by an increase in neuronal death, indicative that the brain is better suited to fight against the death signals generated from reactive oxygen species than against loss-of-function stimuli. A main aim of this laboratory is to understand how neurons perform and survive in the constitutive stress background represented by aging. In this report, we summarize our recent findings in relation to survival.

Oxidative stress activates the pro-survival TrkA pathway through membrane cholesterol loss.

Neuronal activity is a highly demanding energetic process, resulting in the gradual accumulation of reactive oxygen species (ROS). Despite comparatively weak anti-oxidant defence systems, neurons outlive the pressure of ROS by activating most robust anti-stress mechanisms. We recently showed that one such mechanism is the activation of the TrkB receptor pathway, in turn determined by the loss of membrane cholesterol. It is not known however what causes the loss of membrane cholesterol. We here show that in differentiated PC12 cells induction of ROS is paralleled by a moderate loss of membrane cholesterol and the activation of the pro-survival TrkA receptor. Pharmacological reduction of cholesterol in non-stressed cells triggers TrkA activation while cholesterol replenishment inhibits receptor activation induced by stress. Moreover, addition of a ROS inhibitor prevented cholesterol loss and receptor activation under stress. These results highlight cholesterol loss as a compensatory protective mechanism against acute stress.

Cellular stress from excitatory neurotransmission contributes to cholesterol loss in hippocampal neurons aging in vitro.

After approximately 3 weeks in vitro, hippocampal neurons present many of the typical hallmarks accompanying neuronal aging in vivo, including accumulation of reactive oxygen species (ROS), lipofuscin granules, heterochromatic foci, and activation of the Jun N-terminal protein kinase (pJNK) and p53/p21 pathways. In addition, hippocampal neurons in vitro undergo a gradual loss of cholesterol, which is important for the activation of the prosurvival tyrosine kinase receptor TrkB. Here, we used the hippocampal in vitro system to investigate the possible cause of age-accompanying cholesterol loss. We report that cholesterol loss during in vitro aging is paralleled by upregulation and translocation to the neuronal surface of cholesterol-24-hydroxylase (Cyp46), the enzyme responsible for cholesterol removal from neurons. Chronic reduction of electrical activity diminished cholesterol loss in aged neurons and precluded the upregulation of cholesterol-24-hydroxylase. In agreement with a cause-effect relationship, stimulation of excitatory neurotransmission in young neurons led to cholesterol loss. Mechanistically, N-methyl-D-aspartate (NMDA)-mediated excitatory neurotransmission leads to cholesterol loss through generation of reactive oxygen species derived from the activation of the stress-responsive enzyme NADPH oxidase. Supporting the relevance of the in vitro data, reduced cholesterol was also detected in synaptic membranes from old mice brains. Furthermore, excitatory neurotransmission via the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase pathway induced cholesterol loss in purified brain synaptosomes. The current studies highlight excitatory neurotransmission as 1 of the mechanisms involved in cholesterol loss during aging.

Altered serotonergic function of dorsal raphe nucleus in perinatally protein-deprived rats: effects of fluoxetine administration.

We have previously described that perinatally undernourished rats showed increased locus coeruleus activity, a phenomenon reversed by repeated desipramine or fluoxetine administration. Since there is reciprocal modulation between the locus coeruleus and the dorsal raphe nucleus, and because these structures are associated with the pathophysiology of different states of anxiety, we evaluated the activity of serotonergic dorsal raphe neurons from early malnourished animals compared with controls, using in vivo extracellular single-unit recordings. The number of spontaneously active cells/track was significantly higher in protein-deprived animals, although the firing rate and the sensitivity of 5-HT(1A) receptors did not differ from those of controls. Five days of fluoxetine administration (5 mg/kg/day i.p.) was able to reverse the increased number of active serotonergic cells without affecting their firing rate. Furthermore, subsensitivity of 5-HT(1A) autoreceptors developed in the same way after repeated fluoxetine administration in both control and protein-deprived animals. These results suggest that the increased noradrenergic transmission observed in protein-deprived animals may induce an activation of serotonergic neurons in the dorsal raphe nucleus, and that this effect is normalized following fluoxetine treatment, which normalizes locus coeruleus activity.

Locus coeruleus activity in perinatally protein-deprived rats: effects of fluoxetine administration.

We have previously described an increased locus coeruleus activity in perinatally protein-deprived rats. Since locus coeruleus dysfunction has been involved in different types of anxiety disorders and considering the modulating action of serotonergic transmission on locus coeruleus activity, we assessed the effect of fluoxetine, a selective serotonin reuptake inhibitor (SSRI), on locus coeruleus activity as measured by the firing rate and the number of spontaneously active cells/track. Repeated fluoxetine administration reduced locus coeruleus activity in both control and protein-deprived rats, although the reduction was greater in protein-deprived rats. Dose-response curves for the inhibitory effect of clonidine showed subsensitivity of alpha2-adrenergic autoreceptors in protein-deprived rats, a phenomenon reversed by fluoxetine treatment. Dose-response curves for the inhibitory effect of 2,5-dimethoxy-4-iodoamphetamine (DOI) were similar in both groups of rats. Following fluoxetine administration, subsensitivity to this effect developed in control but not in protein-deprived rats. Extracellular noradrenaline level in the prefrontal cortex, as measured by microdialysis procedure, was higher in protein-deprived rats compared to controls, and this difference was reduced after fluoxetine administration. A challenge with yohimbine increased the extracellular noradrenaline level in control but not in protein-deprived rats, suggesting subsensitivity of alpha2-adrenergic autoreceptors in early protein malnourished animals. These results stress the complexity of plastic changes induced by early protein malnutrition and sustain the hypothesis that perinatally protein-deprived rats may represent a useful animal model for screening antipanic agents.