RESUMEN
Charcot-Marie-Tooth type 4B1 (CMT4B1) is a severe autosomal recessive demyelinating neuropathy with childhood onset, caused by loss-of-function mutations in the myotubularin-related 2 (MTMR2) gene. MTMR2 is a ubiquitously expressed catalytically active 3-phosphatase, which in vitro dephosphorylates the 3-phosphoinositides PtdIns3P and PtdIns(3,5)P2, with a preference for PtdIns(3,5)P2 A hallmark of CMT4B1 neuropathy are redundant loops of myelin in the nerve termed myelin outfoldings, which can be considered the consequence of altered growth of myelinated fibers during postnatal development. How MTMR2 loss and the resulting imbalance of 3'-phosphoinositides cause CMT4B1 is unknown. Here we show that MTMR2 by regulating PtdIns(3,5)P2 levels coordinates mTORC1-dependent myelin synthesis and RhoA/myosin II-dependent cytoskeletal dynamics to promote myelin membrane expansion and longitudinal myelin growth. Consistent with this, pharmacological inhibition of PtdIns(3,5)P2 synthesis or mTORC1/RhoA signaling ameliorates CMT4B1 phenotypes. Our data reveal a crucial role for MTMR2-regulated lipid turnover to titrate mTORC1 and RhoA signaling thereby controlling myelin growth.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/metabolismo , Vaina de Mielina/metabolismo , Fosfatos de Fosfatidilinositol/biosíntesis , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Transducción de Señal , Animales , Enfermedad de Charcot-Marie-Tooth/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Noqueados , Vaina de Mielina/genética , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Fosfatos de Fosfatidilinositol/genética , Proteínas Tirosina Fosfatasas no Receptoras/genética , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Alpha-synuclein oligomers (α-synOs) are emerging as crucial factors in the pathogenesis of synucleinopathies. Although the connection between neuroinflammation and α-syn still remains elusive, increasing evidence suggests that extracellular moieties activate glial cells leading to neuronal damage. Using an acute mouse model, we explored whether α-synOs induce memory impairment in association to neuroinflammation, addressing Toll-like receptors 2 and 4 (TLR2 and TLR4) involvement. We found that α-synOs abolished mouse memory establishment in association to hippocampal glial activation. On brain slices α-synOs inhibited long-term potentiation. Indomethacin and Ibuprofen prevented the α-synOs-mediated detrimental actions. Furthermore, while the TLR2 functional inhibitor antibody prevented the memory deficit, oligomers induced memory deficits in the TLR4 knockout mice. In conclusion, solely α-synOs induce memory impairment likely inhibiting synaptic plasticity. α-synOs lead to hippocampal gliosis that is involved in memory impairment. Moreover, while the oligomer-mediated detrimental actions are TLR2 dependent, the involvement of TLR4 was ruled out.
Asunto(s)
Hipocampo/efectos de los fármacos , Memoria/efectos de los fármacos , Neuroglía/efectos de los fármacos , Receptor Toll-Like 2/metabolismo , alfa-Sinucleína/farmacología , Animales , Hipocampo/metabolismo , Potenciación a Largo Plazo/efectos de los fármacos , Ratones , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Reconocimiento en Psicología/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinapsis/metabolismoRESUMEN
BACKGROUND: Amyloid-ß oligomers (AßO) are species mainly involved in the synaptic and cognitive dysfunction in Alzheimer's disease. Although their action has been described mainly at neuronal level, it is now clear that glial cells govern synaptic activity in their resting state, contributing to new learning and memory establishment. In contrast, when activated, they may lead to synaptic and cognitive dysfunction. Using a reliable acute AßO-mediated mouse model of AD, we explored whether the memory alteration AßOs induce relies on the activation of glial cells, and if Toll-like receptor 4 (TLR4), pivotal in the initiation of an immune response, is involved. METHODS: C57 naïve mice were given a single intracerebroventricular injection of synthetic AßO-containing solution (1µM), which induces substantial impairment in the establishment of recognition memory. Then, first we assessed glial cell activation at different times post-injection by western blot, immunohistochemistry and ELISA in the hippocampus. After that we explored the efficacy of pre-treatment with anti-inflammatory drugs (indomethacin and an IL-1ß receptor antagonist) to prevent impairment in the novel object recognition task, and compared AßO's effects in TLR4 knockout mice. RESULTS: A single AßO injection rapidly activated glial cells and increased pro-inflammatory cytokine expression. Both anti-inflammatory drugs prevented the AßO-mediated impairment in memory establishment. A selective TLR4 receptor antagonist abolished AßO's action on memory, and in TLR4 knockout mice it had no effect on either memory or glial activation. CONCLUSIONS: These data provide new information on AßO's mechanism of action, indicating that besides direct action at the synapses, they also act through the immune system, with TLR4 playing a major role. This suggests that in a potential therapeutic setting inflammation must be considered as well.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Memoria/efectos de los fármacos , Microglía/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Antiinflamatorios/farmacología , Disfunción Cognitiva/metabolismo , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Masculino , Ratones Endogámicos C57BL , Neuronas/metabolismo , Sinapsis/metabolismoRESUMEN
The multiplicity of systems affected in Alzheimer's disease (AD) brains calls for multi-target therapies. Although mesenchymal stem cells (MSC) are promising candidates, their clinical application is limited because of risks related to their direct implantation in the host. This could be overcome by exploiting their paracrine action. We herein demonstrate that in vivo systemic administration of secretome collected from MSC exposed in vitro to AD mouse brain homogenates (MSC-CS), fully replicates the cell-mediated neuroreparative effects in APP/PS1 AD mice. We found a complete but transient memory recovery by 7 days, which vanished by 14 days, after a single MSC-CS intravenous administration in 12-month or 22-24-month-old mice. Treatment significantly reduced plaque load, microglia activation, and expression of cytokines in astrocytes in younger, but not aged, mice at 7 days. To optimize efficacy, we established a sustained treatment protocol in aged mice through intranasal route. Once-weekly intranasal administration of MSC-CS induced persistent memory recovery, with dramatic reduction of plaques surrounded by a lower density of ß-amyloid oligomers. Gliosis and the phagocytic marker CD68 were decreased. We found a higher neuronal density in cortex and hippocampus, associated with a reduction in hippocampal shrinkage and a longer lifespan indicating healthier conditions of MSC-CS-treated compared to vehicle-treated APP/PS1 mice. Our data prove that MSC-CS displays a great multi-level therapeutic potential, and lay the foundation for identifying the therapeutic secretome bioreactors leading to the development of an efficacious multi-reparative cocktail drug, towards abrogating the need for MSC implantation and risks related to their direct use.
Asunto(s)
Enfermedad de Alzheimer/patología , Encéfalo/patología , Trasplante de Células Madre Mesenquimatosas/métodos , Placa Amiloide/patología , Administración Intranasal , Enfermedad de Alzheimer/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Biomarcadores , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Gliosis/metabolismo , Masculino , Ratones , Ratones Transgénicos , Neuronas/metabolismoRESUMEN
Inherited peripheral neuropathies (IPNs) represent a broad group of disorders including Charcot-Marie-Tooth (CMT) neuropathies characterized by defects primarily arising in myelin, axons, or both. The molecular mechanisms by which mutations in nearly 100 identified IPN/CMT genes lead to neuropathies are poorly understood. Here we show that the Ras-related GTPase Rab35 controls myelin growth via complex formation with the myotubularin-related phosphatidylinositol (PI) 3-phosphatases MTMR13 and MTMR2, encoded by genes responsible for CMT-types 4B2 and B1 in humans, and found that it downregulates lipid-mediated mTORC1 activation, a pathway known to crucially regulate myelin biogenesis. Targeted disruption of Rab35 leads to hyperactivation of mTORC1 signaling caused by elevated levels of PI 3-phosphates and to focal hypermyelination in vivo. Pharmacological inhibition of phosphatidylinositol 3,5-bisphosphate synthesis or mTORC1 signaling ameliorates this phenotype. These findings reveal a crucial role for Rab35-regulated lipid turnover by myotubularins to repress mTORC1 activity and to control myelin growth.
Asunto(s)
Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Vaina de Mielina/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Astrocitos , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/patología , Regulación hacia Abajo , Técnicas de Sustitución del Gen , Células HEK293 , Células HeLa , Humanos , Metabolismo de los Lípidos/genética , Ratones Transgénicos , Mutación , Vaina de Mielina/patología , Cultivo Primario de Células , Proteínas Tirosina Fosfatasas no Receptoras/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas no Receptoras/genética , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteínas de Unión al GTP rab/genéticaRESUMEN
ß-Amyloid oligomers (AßOs) and neuroinflammation are 2 main culprits to counteract in Alzheimer's disease (AD). Doxycycline (DOXY) is a second generation antibiotic of the tetracycline class that are promising drugs tested in many clinical trials for a number of different pathologies. DOXY is endowed with antiamyloidogenic properties and better crosses the blood-brain barrier, but its efficacy has never been tested in AD mice. We herein show that 15- to 16-month-old APP/PS1dE9 (APP/PS1) AD mice receiving DOXY under different treatment regimens recovered their memory without plaque reduction. An acute DOXY treatment was, also, sufficient to improve APP/PS1 mouse memory, suggesting an action against soluble AßOs. This was confirmed in an AßO-induced mouse model, where the AßO-mediated memory impairment was abolished by a DOXY pretreatment. Although AßOs induce memory impairment through glial activation, assessing the anti-inflammatory action of DOXY, we found that in both the AßO-treated and APP/PS1 mice, the memory recovery was associated with a lower neuroinflammation. Our data promote DOXY as a hopeful repositioned drug counteracting crucial neuropathological AD targets.