Pharmacological inhibition of mTORC1 reduces neural death and damage volume after MCAO by modulating microglial reactivity.

Ischemic stroke is a sudden and acute disease characterized by neuronal death, increment of reactive gliosis (reactive microglia and astrocytes), and a severe inflammatory process. Neuroinflammation is an early event after cerebral ischemia, with microglia playing a leading role. Reactive microglia involve functional and morphological changes that drive a wide variety of phenotypes. In this context, deciphering the molecular mechanisms underlying such reactive microglial is essential to devise strategies to protect neurons and maintain certain brain functions affected by early neuroinflammation after ischemia. Here, we studied the role of mammalian target of rapamycin (mTOR) activity in the microglial response using a murine model of cerebral ischemia in the acute phase. We also determined the therapeutic relevance of the pharmacological administration of rapamycin, a mTOR inhibitor, before and after ischemic injury. Our data show that rapamycin, administered before or after brain ischemia induction, reduced the volume of brain damage and neuronal loss by attenuating the microglial response. Therefore, our findings indicate that the pharmacological inhibition of mTORC1 in the acute phase of ischemia may provide an alternative strategy to reduce neuronal damage through attenuation of the associated neuroinflammation.

Clearance of ß-amyloid mediated by autophagy is enhanced by MTORC1 inhibition but not AMPK activation in APP/PSEN1 astrocytes.

Proteostasis mechanisms mediated by macroautophagy/autophagy are altered in neurodegenerative diseases such as Alzheimer disease (AD) and their recovery/enhancement has been proposed as a therapeutic approach. From the two central nodes in the anabolism-catabolism balance, it is generally accepted that mechanistic target of rapamycin kinase complex 1 (MTORC1)_ activation leads to the inhibition of autophagy, whereas adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) has the opposite role. In AD, amyloid beta (Aß) production disturbs the optimal neuronal/glial proteostasis. As astrocytes are essential for brain homeostasis, the purpose of this work was to analyze if the upregulation of autophagy in this cell type, either by MTORC1 inhibition or AMPK activation, could modulate the generation/degradation of ß-amyloid. By using primary astrocytes from amyloid beta precursor protein (APP)/Presenilin 1 (PSEN1) mouse model of AD, we confirmed that MTORC1 inhibition reduced Aß secretion through moderate autophagy induction. Surprisingly, pharmacologically increased activity of AMPK did not enhance autophagy but had different effects on Aß secretion. Conversely, AMPK inhibition did not affect autophagy but reduced Aß secretion. These puzzling data were confirmed through the overexpression of different mutant AMPK isoforms: while only the constitutively active AMPK increased autophagy, all versions augmented Aß secretion. We conclude that AMPK has a significantly different role in primary astrocytes than in other reported cells, similar to our previous findings in neurons. Our data support that perhaps only a basal AMPK activity is needed to maintain autophagy whereas the increased activity, either physiologically or pharmacologically, has no direct effect on autophagy-dependent amyloidosis. These results shed light on the controversy about the therapeutic effect of AMPK activation on autophagy induction.

Altered Clock Gene Expression in Female APP/PS1 Mice and Aquaporin-Dependent Amyloid Accumulation in the Retina.

Alzheimer's disease (AD), the most prevalent form of dementia, is a neurodegenerative disorder characterized by different pathological symptomatology, including disrupted circadian rhythm. The regulation of circadian rhythm depends on the light information that is projected from the retina to the suprachiasmatic nucleus in the hypothalamus. Studies of AD patients and AD transgenic mice have revealed AD retinal pathology, including amyloid-ß (Aß) accumulation that can directly interfere with the regulation of the circadian cycle. Although the cause of AD pathology is poorly understood, one of the main risk factors for AD is female gender. Here, we found that female APP/PS1 mice at 6- and 12-months old display severe circadian rhythm disturbances and retinal pathological hallmarks, including Aß deposits in retinal layers. Since brain Aß transport is facilitated by aquaporin (AQP)4, the expression of AQPs were also explored in APP/PS1 retina to investigate a potential correlation between retinal Aß deposits and AQPs expression. Important reductions in AQP1, AQP4, and AQP5 were detected in the retinal tissue of these transgenic mice, mainly at 6-months of age. Taken together, our findings suggest that abnormal transport of Aß, mediated by impaired AQPs expression, contributes to the retinal degeneration in the early stages of AD.

Amyloid-ß impairs mitochondrial dynamics and autophagy in Alzheimer's disease experimental models.

The most accepted hypothesis in Alzheimer's disease (AD) is the amyloid cascade which establishes that Aß accumulation may induce the disease development. This accumulation may occur years before the clinical symptoms but it has not been elucidated if this accumulation is the cause or the consequence of AD. It is however, clear that Aß accumulation exerts toxic effects in the cerebral cells. It is important then to investigate all possible associated events that may help to design new therapeutic strategies to defeat or ameliorate the symptoms in AD. Alterations in the mitochondrial physiology have been found in AD but it is not still clear if they could be an early event in the disease progression associated to amyloidosis or other conditions. Using APP/PS1 mice, our results support published evidence and show imbalances in the mitochondrial dynamics in the cerebral cortex and hippocampus of these mice representing very early events in the disease progression. We demonstrate in cellular models that these imbalances are consequence of Aß accumulation that ultimately induce increased mitophagy, a mechanism which selectively removes damaged mitochondria by autophagy. Along with increased mitophagy, we also found that Aß independently increases autophagy in APP/PS1 mice. Therefore, mitochondrial dysfunction could be an early feature in AD, associated with amyloid overload.

R-Ras1 and R-Ras2 Expression in Anatomical Regions and Cell Types of the Central Nervous System.

Since the optic nerve is one of the most myelinated tracts in the central nervous system (CNS), many myelin diseases affect the visual system. In this sense, our laboratory has recently reported that the GTPases R-Ras1 and R-Ras2 are essential for oligodendrocyte survival and maturation. Hypomyelination produced by the absence of one or both proteins triggers axonal degeneration and loss of visual and motor function. However, little is known about R-Ras specificity and other possible roles that they could play in the CNS. In this work, we describe how a lack of R-Ras1 and/or R-Ras2 could not be compensated by increased expression of the closely related R-Ras3 or classical Ras. We further studied R-Ras1 and R-Ras2 expression within different CNS anatomical regions, finding that both were more abundant in less-myelinated regions, suggesting their expression in non-oligodendroglial cells. Finally, using confocal immunostaining colocalization, we report for the first time that R-Ras2 is specifically expressed in neurons. Neither microglia nor astrocytes expressed R-Ras1 or R-Ras2. These results open a new avenue for the study of neuronal R-Ras2's contribution to the process of myelination.

Pathways Involved in Remyelination after Cerebral Ischemia.

Brain ischemia, also known as ischemic stroke, occurs when there is a lack of blood supply into the brain. When an ischemic insult appears, both neurons and glial cells can react in several ways that will determine the severity and prognosis. This high heterogeneity of responses has been a major obstacle in developing effective treatments or preventive methods for stroke. Although white matter pathophysiology has not been deeply assessed in stroke, its remodelling can greatly influence the clinical outcome and the disability degree. Oligodendrocytes, the unique cell type implied in CNS myelination, are sensible to ischemic damage. Loss of myelin sheaths can compromise axon survival, so new Oligodendrocyte Precursor Cells are required to restore brain function. Stroke can, therefore, enhance oligodendrogenesis to regenerate those new oligodendrocytes that will ensheath the damaged axons. Given that myelination is a highly complex process that requires coordination of multiple pathways such as Sonic Hedgehog, RTKs or Wnt/ß-catenin, we will analyse new research highlighting their importance after brain ischemia. In addition, oligodendrocytes are not isolated cells inside the brain, but rather form part of a dynamic environment of interactions between neurons and glial cells. For this reason, we will put some context into how microglia and astrocytes react against stroke and influence oligodendrogenesis to highlight the relevance of remyelination in the ischemic brain. This will help to guide future studies to develop treatments focused on potentiating the ability of the brain to repair the damage.

Diets with Higher ω-6/ω-3 Ratios Show Differences in Ceramides and Fatty Acid Levels Accompanied by Increased Amyloid-Beta in the Brains of Male APP/PS1 Transgenic Mice.

Senile plaque formation as a consequence of amyloid-ß peptide (Aß) aggregation constitutes one of the main hallmarks of Alzheimer's disease (AD). This pathology is characterized by synaptic alterations and cognitive impairment. In order to either prevent or revert it, different therapeutic approaches have been proposed, and some of them are focused on diet modification. Modification of the ω-6/ω-3 fatty acids (FA) ratio in diets has been proven to affect Aß production and senile plaque formation in the hippocampus and cortex of female transgenic (TG) mice. In these diets, linoleic acid is the main contribution of ω-6 FA, whereas alpha-linoleic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA) are the contributors of ω-3 FA. In the present work, we have explored the effect of ω-6/ω-3 ratio modifications in the diets of male double-transgenic APPswe/PS1ΔE9 (AD model) and wild-type mice (WT). Amyloid burden in the hippocampus increased in parallel with the increase in dietary ω-6/ω-3 ratio in TG male mice. In addition, there was a modification in the brain lipid profile proportional to the ω-6/ω-3 ratio of the diet. In particular, the higher the ω-6/ω-3 ratio, the lower the ceramides and higher the FAs, particularly docosatetraenoic acid. Modifications to the cortex lipid profile was mostly similar between TG and WT mice, except for gangliosides (higher levels in TG mice) and some ceramide species (lower levels in TG mice).

WIP, YAP/TAZ and Actin Connections Orchestrate Development and Transformation in the Central Nervous System.

YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) are transcription co-regulators that make up the terminal components of the Hippo signaling pathway, which plays a role in organ size control and derived tissue homeostasis through regulation of the proliferation, differentiation and apoptosis of a wide variety of differentiated and stem cells. Hippo/YAP signaling contributes to normal development of the nervous system, as it participates in self-renewal of neural stem cells, proliferation of neural progenitor cells and differentiation, activation and myelination of glial cells. Not surprisingly, alterations in this pathway underlie the development of severe neurological diseases. In glioblastomas, YAP and TAZ levels directly correlate with the amount of the actin-binding molecule WIP (WASP interacting protein), which regulates stemness and invasiveness. In neurons, WIP modulates cytoskeleton dynamics through actin polymerization/depolymerization and acts as a negative regulator of neuritogenesis, dendrite branching and dendritic spine formation. Our working hypothesis is that WIP regulates the YAP/TAZ pools using a Hippo-independent pathway. Thus, in this review we will present some of the data that links WIP, YAP and TAZ, with a focus on their function in cells from the central and peripheral nervous systems. It is hoped that a better understanding of the mechanisms involved in brain and nervous development and the pathologies that arise due to their alteration will reveal novel therapeutic targets for neurologic diseases.

Trehalose Reduces the Secreted Beta-Amyloid Levels in Primary Neurons Independently of Autophagy Induction.

The disaccharide trehalose was described as possessing relevant neuroprotective properties as an mTORC1-independent inducer of autophagy, with the ability to protect cellular membranes and denaturation, resulting from desiccation, and preventing the cellular accumulation of protein aggregates. These properties make trehalose an interesting therapeutic candidate against proteinopathies such as Alzheimer's disease (AD), which is characterized by deposits of aggregated amyloid-beta (Aß) and hyperphosphorylated tau. In this study, we observed that trehalose was able to induce autophagy in neurons only in the short-term, whereas long-term treatment with trehalose provoked a relevant anti-amyloidogenic effect in neurons from an AD mouse model that was not mediated by autophagy. Trehalose treatment reduced secreted Aß levels in a manner unrelated to its intracellular accumulation or its elimination through endocytosis or enzymatic degradation. Moreover, the levels of Aß precursor protein (APP) and beta-secretase (BACE1) remained unaltered, as well as the proper acidic condition of the endo-lysosome system. Instead, our results support that the neuroprotective effect of trehalose was mediated by a reduced colocalization of APP and BACE1 in the cell, and, therefore, a lower amyloidogenic processing of APP. This observation illustrates that the determination of the mechanism, or mechanisms, that associate APP and BACE is a relevant therapeutic target to investigate.

AMPK activation does not enhance autophagy in neurons in contrast to MTORC1 inhibition: different impact on ß-amyloid clearance.

The physiological AKT-MTORC1 and AMPK signaling pathways are considered key nodes in the regulation of anabolism-catabolism, and particularly of macroautophagy/autophagy. Indeed, it is reported that these are altered processes in neurodegenerative proteinopathies such as Alzheimer disease (AD), mainly characterized by deposits of ß-amyloid (Aß) and hyperphosphorylated MAPT. These accumulations disrupt the optimal neuronal proteostasis, and hence, the recovery/enhancement of autophagy has been proposed as a therapeutic approach against these proteinopathies. The purpose of the present study was to characterize the modulation of autophagy by MTORC1 and AMPK signaling pathways in the highly specialized neurons, as well as their repercussions on Aß production. Using a double transgenic mice model of AD, we demonstrated that MTORC1 inhibition, either in vivo or ex vivo (primary neuronal cultures), was able to reduce amyloid secretion through moderate autophagy induction in neurons. The pharmacological prevention of autophagy in neurons augmented the Aß secretion and reversed the effect of rapamycin, confirming the anti-amyloidogenic effects of autophagy in neurons. Inhibition of AMPK with compound C generated the expected decrease in autophagy induction, though surprisingly did not increase the Aß secretion. In contrast, increased activity of AMPK with metformin, AICAR, 2DG, or by gene overexpression did not enhance autophagy but had different effects on Aß secretion: whereas metformin and 2DG diminished the secreted Aß levels, AICAR and PRKAA1/AMPK gene overexpression increased them. We conclude that AMPK has a significantly different role in primary neurons than in other reported cells, lacking a direct effect on autophagy-dependent amyloidosis.Abbreviations: 2DG: 2-deoxy-D-glucose; Aß: ß-amyloid; ACACA: acetyl-CoA carboxylase alpha; ACTB: actin beta; AD: Alzheimer disease; AICAR: 5-aminoimidazole-4-carboxamide-1-ß-riboside; AKT: AKT kinases group (AKT1 [AKT serine/threonine kinase 1], AKT2 and AKT3); AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; APP: amyloid beta precursor protein; APP/PSEN1: B6.Cg-Tg (APPSwe, PSEN1dE9) 85Dbo/J; ATG: autophagy related; ATP: adenosine triphosphate; BafA1: bafilomycin A1; CA: constitutively active; CGN: cerebellar granule neuron; CoC/compound C: dorsommorphin dihydrochloride; ELISA: enzyme-linked immunosorbent assay; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; Gmax: GlutaMAX™; IN1: PIK3C3/VPS34-IN1; KI: kinase-inactive; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3; MAPT/TAU: microtubule associated protein tau; Metf: metformin; MRT: MRT68921; MTORC1: mechanistic target of rapamycin kinase complex 1; NBR1: NBR1 autophagy cargo receptor; PRKAA: 5'-AMP-activated protein kinase catalytic subunit alpha; PtdIns3K: phosphatidylinositol 3-kinase; Rapa: rapamycin; RPS6KB1/S6K: ribosomal protein S6 (RPS6) kinase polypeptide 1; SCR: scramble; SQSTM1/p62: sequestosome 1; ULK1/2: unc-51 like autophagy activating kinase 1/2; WT: wild type.

Absence of R-Ras1 and R-Ras2 causes mitochondrial alterations that trigger axonal degeneration in a hypomyelinating disease model.

Fast synaptic transmission in vertebrates is critically dependent on myelin for insulation and metabolic support. Myelin is produced by oligodendrocytes (OLs) that maintain multilayered membrane compartments that wrap around axonal fibers. Alterations in myelination can therefore lead to severe pathologies such as multiple sclerosis. Given that hypomyelination disorders have complex etiologies, reproducing clinical symptoms of myelin diseases from a neurological perspective in animal models has been difficult. We recently reported that R-Ras1-/- and/or R-Ras2-/- mice, which lack GTPases essential for OL survival and differentiation processes, present different degrees of hypomyelination in the central nervous system with a compounded hypomyelination in double knockout (DKO) mice. Here, we discovered that the loss of R-Ras1 and/or R-Ras2 function is associated with aberrant myelinated axons with increased numbers of mitochondria, and a disrupted mitochondrial respiration that leads to increased reactive oxygen species levels. Consequently, aberrant myelinated axons are thinner with cytoskeletal phosphorylation patterns typical of axonal degeneration processes, characteristic of myelin diseases. Although we observed different levels of hypomyelination in a single mutant mouse, the combined loss of function in DKO mice lead to a compromised axonal integrity, triggering the loss of visual function. Our findings demonstrate that the loss of R-Ras function reproduces several characteristics of hypomyelinating diseases, and we therefore propose that R-Ras1-/- and R-Ras2-/- neurological models are valuable approaches for the study of these myelin pathologies.

WIP Modulates Oxidative Stress through NRF2/KEAP1 in Glioblastoma Cells.

Due to their high metabolic rate, tumor cells produce exacerbated levels of reactive oxygen species that need to be under control. Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) is a scaffold protein with multiple yet poorly understood functions that participates in tumor progression and promotes cancer cell survival. However, its participation in the control of oxidative stress has not been addressed yet. We show that WIP depletion increases the levels of reactive oxygen species and reduces the levels of transcription factor NRF2, the master regulator of redox homeostasis. We found that WIP stabilizes NRF2 by restraining the activity of its main NRF2 repressor, the E3 ligase adapter KEAP1, because the overexpression of a NRF2ΔETGE mutant that is resistant to targeted proteasome degradation by KEAP1 or the knock-down of KEAP1 maintains NRF2 levels in the absence of WIP. Mechanistically, we show that the increased KEAP1 activity in WIP-depleted cells is not due to the protection of KEAP1 from autophagic degradation, but is dependent on the organization of the Actin cytoskeleton, probably through binding between KEAP1 and F-Actin. Our study provides a new role of WIP in maintaining the oxidant tolerance of cancer cells that may have therapeutic implications.

R-Ras GTPases Signaling Role in Myelin Neurodegenerative Diseases.

Myelination is required for fast and efficient synaptic transmission in vertebrates. In the central nervous system, oligodendrocytes are responsible for creating myelin sheaths that isolate and protect axons, even throughout adulthood. However, when myelin is lost, the failure of remyelination mechanisms can cause neurodegenerative myelin-associated pathologies. From oligodendrocyte progenitor cells to mature myelinating oligodendrocytes, myelination is a highly complex process that involves many elements of cellular signaling, yet many of the mechanisms that coordinate it, remain unknown. In this review, we will focus on the three major pathways involved in myelination (PI3K/Akt/mTOR, ERK1/2-MAPK, and Wnt/ß-catenin) and recent advances describing the crosstalk elements which help to regulate them. In addition, we will review the tight relation between Ras GTPases and myelination processes and discuss its potential as novel elements of crosstalk between the pathways. A better understanding of the crosstalk elements orchestrating myelination mechanisms is essential to identify new potential targets to mitigate neurodegeneration.

Nanoliposomes as a Therapeutic Tool for Alzheimer's Disease.

The accumulation of extracellular amyloid-beta (Aß), denoted as senile plaques, and intracellular neurofibrillary tangles (formed by hyperphosphorylated Tau protein) in the brain are two major neuropathological hallmarks of Alzheimer's disease (AD). The current and most accepted hypothesis proposes that the oligomerization of Aß peptides triggers the polymerization and accumulation of amyloid, which leads to the senile plaques. Several strategies have been reported to target Aß oligomerization/polymerization. Since it is thought that Aß levels in the brain and peripheral blood maintain equilibrium, it has been hypothesized that enhancing peripheral clearance (by shifting this equilibrium towards the blood) might reduce Aß levels in the brain, known as the sink effect. This process has been reported to be effective, showing a reduction in Aß burden in the brain as a consequence of the peripheral reduction of Aß levels. Nanoparticles (NPs) may have difficulty crossing the blood-brain barrier (BBB), initially due to their size. It is not clear whether particles in the range of 50-100 nm should be able to cross the BBB without being specifically modified for it. Despite the size limitation of crossing the BBB, several NP derivatives may be proposed as therapeutic tools. The purpose of this review is to summarize some therapeutic approaches based on nanoliposomes using two complementary examples: First, unilamellar nanoliposomes containing Aß generic ligands, such as sphingolipids, gangliosides or curcumin, or some sphingolipid bound to the binding domain of ApoE; and second, nanoliposomes containing monoclonal antibodies against Aß. Following similar rationale NPs of poly(lactide-co-glycolide)-poly (ethylene glycol) conjugated with curcumin-derivate (PLGA-PEG-B6/Cur) were reported to improve the spatial learning and memory capability of APP/PS1 mice, compared with native curcumin treatment. Also, some new nanostructures such as exosomes have been proposed as a putative therapeutic and prevention strategies of AD. Although the unquestionable interest of this issue is beyond the scope of this review article. The potential mechanisms and significance of nanoliposome therapies for AD, which are still are in clinical trials, will be discussed.

Crosstalk between WIP and Rho family GTPases.

Through actin-binding proteins such as the neural Wiskott-Aldrich syndrome protein (N-WASP) and WASP-interacting protein (WIP), the Rho family GTPases RhoA, Rac1 and Cdc42 are major modulators of the cytoskeleton. (N-)WASP and WIP control Rho GTPase activity in various cell types, either by direct WIP/(N-)WASP/Cdc42 or potential WIP/RhoA binding, or through secondary links that regulate GTPase distribution and/or transcription levels. WIP helps to regulate filopodium generation and participates in the Rac1-mediated ruffle formation that determines cell motility. In neurons, lack of WIP increases dendritic spine size and filamentous actin content in a RhoA-dependent manner. In contrast, WIP deficiency in an adenocarcinoma cell line significantly reduces RhoA levels. These data support a role for WIP in the GTPase-mediated regulation of numerous actin-related cell functions; we discuss the possibility that this WIP effect is linked to cell proliferative status.

Energy-Sensing Pathways in Ischemia: The Counterbalance Between AMPK and mTORC.

Stroke is an important cause of death and disability, and it is the second leading cause of death worldwide. In humans, middle cerebral artery occlusion (MCAO) is the most common cause of ischemic stroke. The damage occurs due to the lack of nutrients and oxygen contributed by the blood flow. The present review aims to analyze to what extent the lack of each of the elements of the system leads to damage and which mechanisms are unaffected by this deficiency. We believe that the specific analysis of the effect of lack of each component could lead to the emergence of new therapeutic targets for this important brain pathology.

Botulinum Neurotoxin Light Chains Expressed by Defective Herpes Simplex Virus Type-1 Vectors Cleave SNARE Proteins and Inhibit CGRP Release in Rat Sensory Neurons.

A set of herpes simplex virus type 1 (HSV-1) amplicon vectors expressing the light chains (LC) of botulinum neurotoxins (BoNT) A, B, C, D, E and F was constructed. Their properties have been assessed in primary cultures of rat embryonic dorsal root ganglia (DRG) neurons, and in organotypic cultures of explanted DRG from adult rats. Following infection of primary cultures of rat embryonic DRG neurons, the different BoNT LC induced efficient cleavage of their corresponding target Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor (SNARE) protein (VAMP, SNAP25, syntaxin). A similar effect was observed following infection by BoNT-A LC of organotypic cultures of adult rat DRG. To quantify and compare the functional activities of the different BoNT LC, the inhibition of calcitonin gene-related protein (CGRP) secretion was assessed in DRG neurons following infection by the different vectors. All BoNT-LC were able to inhibit CGRP secretion although to different levels. Vectors expressing BoNT-F LC displayed the highest inhibitory activity, while those expressing BoNT-D and -E LC induced a significantly lower CGRP release inhibition. Cleavage of SNARE proteins and inhibition of CGRP release could be detected in neuron cultures infected at less than one transducing unit (TU) per neuron, showing the extreme efficacy of these vectors. To our knowledge this is the first study investigating the impact of vector-expressed transgenic BoNT LC in sensory neurons.