Everolimus improves memory and learning while worsening depressive- and anxiety-like behavior in an animal model of depression

Everolimus (EVR) is an orally-administered rapamycin analog that selectively inhibits the mammalian target of rapamycin (mTOR) kinase (mainly mTORC1 and likely mTORC2) and the related signaling pathway. mTOR is a serine/threonine protein kinase regulating multiple important cellular functions; dysfunction of mTOR signaling has also been implicated in the pathophysiology of several neurological, neurodegenerative, developmental and cognitive disorders. EVR is widely used as an anti-neoplastic therapy and more recently in children with tuberous sclerosis complex (TSC). However, no clear correlation exists between EVR use and development of central side effects e.g. depression, anxiety or cognitive impairment. We studied the effects of a 3 weeks administration of EVR in mice chronically treated with betamethasone 21-phosphate disodium (BTM) as a model of depression and cognitive decline. EVR treatment had detrimental effects on depressive- and anxiety-like behavior while improving cognitive performance in both control (untreated) and BTM-treated mice. Such effects were accompanied by an increased hippocampal neurogenesis and synaptogenesis. Our results therefore might support the proposed pathological role of mTOR dysregulation in depressive disorders and confirm some previous data on the positive effects of mTOR inhibition in cognitive decline. We also show that EVR, possibly through mTOR inhibition, may be linked to the development of anxiety. The increased hippocampal neurogenesis by EVR might explain its ability to improve cognitive function or protect from cognitive decline. Our findings suggest some caution in the use of EVR, particularly in the developing brain; patients should be carefully monitored for their psychiatric/neurological profiles in any clinical situation where an mTOR inhibitor and in particular EVR is used e.g. cancer treatment, TSC or immunosuppression.     

Hypothalamic hormones and metabolism

The ketogenic diet is an effective treatment for medically intractable epilepsy and may have antiepileptogenic, neuroprotective, and antitumor properties. While on a ketogenic diet, the body obtains most of its calories from fat rather than carbohydrates. This dramatic change in caloric composition results in a unique metabolic state. In turn, these changes in caloric composition and metabolism alter some of the neurohormones that participate in the complex neuronal network regulating energy homeostasis. Two observed changes are an increase in serum leptin and a decrease in serum insulin. These opposing changes in leptin and insulin are unique compared to other metabolic stimuli and may modify the activity of several cell signaling cascades including phosphoinositidyl-3 kinase (PI3K), adenosine monophosphate activated protein kinase (AMPK), and mammalian target of rapamycin (mTOR). These cell signaling pathways may mediate the anticonvulsant and other beneficial effects of the diet, though the neurohormonal changes induced by the ketogenic diet and the physiological consequences of these changes remain poorly characterized.     

Erythropoietin and Wnt1 govern pathways of mTOR, Apaf-1, and XIAP in inflammatory microglia

Inflammatory microglia modulate a host of cellular processes in the central nervous system that include neuronal survival, metabolic fluxes, foreign body exclusion, and cellular regeneration. Elucidation of the pathways that oversee microglial survival and integrity may offer new avenues for the treatment of neurodegenerative disorders. Here we demonstrate that erythropoietin (EPO), an emerging strategy for immune system modulation, prevents microglial early and late apoptotic injury during oxidant stress through Wnt1, a cysteine-rich glycosylated protein that modulates cellular development and survival. Loss of Wnt1 through blockade of Wnt1 signaling or through the gene silencing of Wnt1 eliminates the protective capacity of EPO. Furthermore, endogenous Wnt1 in microglia is vital to preserve microglial survival since loss of Wnt1 alone increases microglial injury during oxidative stress. Cellular protection by EPO and Wnt1 intersects at the level of protein kinase B (Akt1), the mammalian target of rapamycin (mTOR), and p70S6K, which are necessary to foster cytoprotection for microglia. Downstream from these pathways, EPO and Wnt1 control "anti-apoptotic" pathways of microglia through the modulation of mitochondrial membrane permeability, the release of cytochrome c, and the expression of apoptotic protease activating factor-1 (Apaf-1) and X-linked inhibitor of apoptosis protein (XIAP). These studies offer new insights for the development of innovative therapeutic strategies for neurodegenerative disorders that focus upon inflammatory microglia and novel signal transduction pathways.     

Acetaldehyde promotes rapamycin-dependent activation of p70(S6K) and glucose uptake despite inhibition of Akt and mTOR in dopaminergic SH-SY5Y human neuroblastoma cells

Alcohol intake is one of the important lifestyle factors for the risk of insulin resistance and type 2 diabetes. Acetaldehyde, the major ethanol metabolite which is far more reactive than ethanol, has been postulated to participate in alcohol-induced tissue injury although its direct impact on insulin signaling is unclear. This study was designed to examine the effect of acetaldehyde on glucose uptake and insulin signaling in human dopaminergic SH-SY5Y cells. Akt, mammalian target of rapamycin (mTOR), ribosomal-S6 kinase (p70(S6K)), the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and insulin receptor substrate (IRS)-2 were evaluated by Western blot analysis. Glucose uptake and apoptosis were measured using [(3)H]-2-deoxyglucose uptake and caspase-3 assay, respectively. Short-term exposure (12 h) of acetaldehyde (150 muM) facilitated glucose uptake in a rapamycin-dependent manner without affecting apoptosis, IRS-2 expression and insulin-stimulated glucose uptake in SH-SY5Y cells. Acetaldehyde suppressed basal and insulin-stimulated Akt phosphorylation without affecting total Akt expression. Acetaldehyde inhibited mTOR phosphorylation without affecting total mTOR and insulin-elicited response on mTOR phosphorylation. Rapamycin, which inhibits mTOR leading to inactivation of p70(S6K), did not affect acetaldehyde-induced inhibition on phosphorylation of Akt and mTOR. Interestingly, acetaldehyde enhanced p70(S6K) activation and depressed 4E-BP1 phosphorylation, the effect of which was blunted and exaggerated, respectively, by rapamycin. Collectively, these data suggested that acetaldehyde did not adversely affect glucose uptake despite inhibition of insulin signaling cascade at the levels of Akt and mTOR, possibly due to presence of certain mechanism(s) responsible for enhanced p70(S6K) phosphorylation.     

PINK1 enhances insulin-like growth factor-1-dependent Akt signaling and protection against apoptosis

Mutations in the PARK6 gene coding for PTEN-induced kinase 1 (PINK1) cause recessive early-onset Parkinsonism. Although PINK1 and Parkin promote the degradation of depolarized mitochondria in cultured cells, little is known about changes in signaling pathways that may additionally contribute to dopamine neuron loss in recessive Parkinsonism. Accumulating evidence implicates impaired Akt cell survival signaling in sporadic and familial PD (PD). IGF-1/Akt signaling inhibits dopamine neuron loss in several animal models of PD and both IGF-1 and insulin are neuroprotective in various settings. Here, we tested whether PINK1 is required for insulin-like growth factor 1 (IGF-1) and insulin dependent phosphorylation of Akt and the regulation of downstream Akt target proteins. Our results show that embryonic fibroblasts from PINK1-deficient mice display significantly reduced Akt phosphorylation in response to both IGF-1 and insulin. Moreover, phosphorylation of glycogen synthase kinase-3β (GSK-3β) and nuclear exclusion of FoxO1 are decreased in IGF-1 treated PINK1-deficient cells. In addition, phosphorylation of ribosomal protein S6 is reduced indicating decreased activity of mitochondrial target of rapamycin (mTOR) in IGF-1 treated PINK1^−/− cells. Importantly, the protection afforded by IGF-1 against staurosporine-induced metabolic dysfunction and apoptosis is abrogated in PINK1-deficient cells. Moreover, IGF-1-induced Akt phosphorylation is impaired in primary cortical neurons from PINK1-deficient mice. Inhibition of cellular Ser/Thr phosphatases did not increase the amount of phosphorylated Akt in PINK1^−/− cells, suggesting that components upstream of Akt phosphorylation are compromised in PINK1-deficient cells. Our studies show that PINK1 is required for optimal IGF-1 and insulin dependent Akt signal transduction, and raise the possibility that impaired IGF-1/Akt signaling is involved in PINK1-related Parkinsonism by increasing the vulnerability of dopaminergic neurons to stress-induced cell death.     

Pentoxifylline and propentofylline prevent proliferation and activation of the mammalian target of rapamycin and mitogen activated protein kinase in cultured spinal astrocytes

Astrocyte activation is an important feature in many disorders of the central nervous system, including chronic pain conditions. Activation of astrocytes is characterized by a change in morphology, including hypertrophy and increased size of processes, proliferation, and an increased production of proinflammatory mediators. The xanthine derivatives pentoxifylline and propentofylline are commonly used experimentally as glial inhibitors. These compounds are generally believed to attenuate glial activity by raising cyclic AMP (cAMP) levels and inhibiting glial tumor necrosis factor (TNF) production. In the present study, we show that these substances inhibit TNF and serum‐induced astrocyte proliferation and signaling through the mammalian target of rapamycin (mTOR) pathway, demonstrated by decreased levels of phosphorylated S6 kinase (S6K), commonly used as a marker of mTOR complex (mTORC) activation. Furthermore, we show that pentoxifylline and propentofylline also inhibit JNK and p38, but not ERK, activation induced by TNF. In addition, the JNK antagonist SP600125, but not the p38 inhibitor SB203580, prevents TNF‐induced activation of S6 kinase, suggesting that pentoxifylline and propentofylline may regulate mTORC activity in spinal astrocytes partially through inhibition of the JNK pathway. Our results suggest that pentoxifylline and propentofylline inhibit astrocyte activity in a broad fashion by attenuating flux through specific pathways. © 2012 Wiley Periodicals, Inc.     

Mechanistic target of rapamycin is necessary for changes in dendritic spine morphology associated with long-term potentiation

Alterations in the strength of excitatory synapses in the hippocampus is believed to serve a vital function in the storage and recall of new information in the mammalian brain. These alterations involve the regulation of both functional and morphological features of dendritic spines, the principal sites of excitatory synaptic contact. New protein synthesis has been implicated extensively in the functional changes observed following long-term potentiation (LTP), and changes to spine morphology have similarly been documented extensively following synaptic potentiation. However, mechanistic links between de novo translation and the structural changes of potentiated spines are less clear. Here, we assess explicitly the potential contribution of new protein translation under control of the mechanistic target of rapamycin (mTOR) to LTP-associated changes in spine morphology. Utilizing genetic and pharmacological manipulations of mTORC1 function in combination with confocal microscopy in live dissociated hippocampal cultures, we demonstrate that chemically-induced LTP (cLTP) requires do novo protein synthesis and intact mTORC1 signaling. We observed a striking diversity in response properties across morphological classes, with mushroom spines displaying a particular sensitivity to altered mTORC1 signaling across varied levels of synaptic activity. Notably, while pharmacological inhibition of mTORC1 signaling significantly diminished glycine-induced changes in spine morphology, transient genetic upregulation of mTORC1 signaling was insufficient to produce spine enlargements on its own. In contrast, genetic upregulation of mTORC1 signaling promoted rapid expansion in spine head diameter when combined with otherwise sub-threshold synaptic stimulation. These results suggest that synaptic activity-derived signaling pathways act in combination with mTORC1-dependent translational control mechanisms to ultimately regulate changes in spine morphology. As several monogenic neurodevelopmental disorders with links to Autism and Intellectual Disability share a common feature of dysregulated mTORC1 signaling, further understanding of the role of this signaling pathway in regulating synapse function and morphology will be essential in the development of novel therapeutic interventions.     

mTOR and neuronal cell cycle reentry: How impaired brain insulin signaling promotes Alzheimer's disease

A major obstacle to presymptomatic diagnosis and disease-modifying therapy for Alzheimer's disease (AD) is inadequate understanding of molecular mechanisms of AD pathogenesis. For example, impaired brain insulin signaling is an AD hallmark, but whether and how it might contribute to the synaptic dysfunction and neuron death that underlie memory and cognitive impairment has been mysterious. Neuron death in AD is often caused by cell cycle reentry (CCR) mediated by amyloid-β oligomers (AβOs) and tau, the precursors of plaques and tangles. We now report that CCR results from AβO-induced activation of the protein kinase complex, mTORC1, at the plasma membrane and mTORC1-dependent tau phosphorylation, and that CCR can be prevented by insulin-stimulated activation of lysosomal mTORC1. AβOs were also shown previously to reduce neuronal insulin signaling. Our data therefore indicate that the decreased insulin signaling provoked by AβOs unleashes their toxic potential to cause neuronal CCR, and by extension, neuron death.     

mTOR inhibitor rapamycin suppresses striatal post-ischemic LTP

The two complexes of the mammalian target of rapamycin (mTOR), mTORC1 and mTORC2, have central functions in the integration of both extracellular and intracellular signals that are also critical players in the induction of post-ischemic long-term potentiation (i-LTP), a pathological form of plasticity inducible in striatal medium spiny neurons (MSNs) after a brief episode of in vitro ischemia. To evaluate the involvement of mTOR complexes during ischemia we analyzed the time course of i-LTP by intracellular recordings of MSNs from corticostriatal slices incubated with 1 μM mTOR inhibitor rapamycin. Although rapamycin did not affect the amplitude and duration of ischemia-induced membrane depolarization it fully prevented i-LTP, leaving unaffected the capability to undergo activity-dependent LTP following high-frequency stimulation of corticostriatal fibers. The present results argue for a role of mTOR complex in i-LTP and suggest that rapamycin, by selectively blocking i-LTP, represents a promising therapeutic tool to limit cellular damage after ischemic brain insult.     

Chronic alcohol consumption alters mammalian target of rapamycin (mTOR), reduces ribosomal p70s6 kinase and p4E-BP1 levels in mouse cerebral cortex

Reduced insulin sensitivity following chronic alcohol consumption may contribute to alcohol-induced brain damage although the underlying mechanism(s) has not been elucidated. This study was designed to examine the effect of chronic alcohol intake on insulin signaling in mouse cerebral cortex. FVB mice were fed with a 4% alcohol diet for 16 weeks. Insulin receptor substrates (IRS-1, IRS-2) and post-receptor signaling molecules Akt, mammalian target of rapamycin (mTOR), ribosomal p70s6 kinase (p70s6k) and the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) as well as the apoptotic marker caspase-3 were evaluated using Western blot analysis. Chronic alcohol intake significantly dampened whole body glucose tolerance, enhanced expression of caspase-3 and mTOR, reduced p70s6k and 4E-BP1 with little effect on Akt signaling in alcohol-consuming mice. These data suggest that chronic alcohol intake may contribute to cerebral cortex dysfunction through mechanisms related, at least in part, to dampened post insulin receptor signaling at the levels of mTOR, p70s6k and 4E-BP1.     

Transient activation of mTOR following forced treadmill exercise in rats

The beneficial effect of exercise on hippocampal plasticity is possibly mediated by increased angiogenesis and neurogenesis. In angiogenesis, insulin‐like growth factor‐1 (IGF‐1), vascular endothelial growth factor (VEGF), and hypoxia‐inducible factor 1, alpha subunit (HIF1α) are important factors, while the induction of neurogenesis requires signaling through the VEGF receptor, Flk‐1 (VEGFR‐2). VEGF expression is believed to be regulated by two distinct mTOR (mammalian target of Rapamycin)‐containing multiprotein complexes mTORC1 and mTORC2, respectively. This study was initiated to investigate the effect of exercise on the expression of VEGF, cognate receptors, HIF1α, mTORC1, and mTORC2 in hippocampus and frontal cortex. To this end, we measured messenger RNA (mRNA) levels in rat brain using quantitative real‐time polymerase chain reaction (real‐time qPCR) after forced treadmill exercise for 1 day, 2 weeks, and 8 weeks. Rats were euthanized either immediately (0 h) or 24 h after last exercise session. Here, we show that exercise affected mRNA levels of VEGF, VEGFR2, and the coreceptor neuropilin 2 (NRP2) when the rats were euthanized immediately, whereas at 24 h only the expression of mTOR was regulated after a single bout of exercise. In conclusion, the effect of treadmill exercise on the VEGF system is acute rather than chronic and there is a transient activation of mTOR. More studies are needed to understand whether this could be beneficial in the treatment of neuropsychiatric disorders. Synapse 67:620–625, 2013. © 2013 Wiley Periodicals, Inc.     

Novel applications of trophic factors,Wnt and WISP for neuronal repair and regeneration in metabolic disease

Diabetes mellitus affects almost 350 million individuals throughout the globe resulting in significant morbidity and mortality. Of further concern is the growing population of individuals that remain undiagnosed but are susceptible to the detrimental outcomes of this disorder. Diabetes mellitus leads to multiple complications in the central and peripheral nervous systems that include cognitive impairment, retinal disease, neuropsychiatric disease, cerebral ischemia, and peripheral nerve degeneration. Although multiple strategies are being considered, novel targeting of trophic factors, Wnt signaling, Wnt1 inducible signaling pathway protein 1, and stem cell tissue regeneration are considered to be exciting prospects to overcome the cellular mechanisms that lead to neuronal injury in diabetes mellitus involving oxidative stress, apoptosis, and autophagy. Pathways that involve insulin-like growth factor-1, fibroblast growth factor, epidermal growth factor, and erythropoietin can govern glucose homeostasis and are intimately tied to Wnt signaling that involves Wnt1 and Wnt1 inducible signaling pathway protein 1(CCN4) to foster control over stem cell proliferation, wound repair, cognitive decline, β-cell proliferation, vascular regeneration, and programmed cell death. Ultimately, cellular metabolism through Wnt signaling is driven by primary metabolic pathways of the mechanistic target of rapamycin and AMP activated protein kinase. These pathways offer precise biological control of cellular metabolism, but are exquisitely sensitive to the different components of Wnt signaling. As a result, unexpected clinical outcomes can ensue and therefore demand careful translation of the mechanisms that govern neural repair and regeneration in diabetes mellitus.     

NRF2-dependent glutamate-L-cysteine ligase catalytic subunit expression mediates insulin protection against hyperglycemia- induced brain endothelial cell apoptosis

Increased oxidative stress and susceptibility of brain endothelium are contributing factors in the development of central nervous system complications in neuro-degenerative disorders in diabetes, Alzheimer's and Parkinson's disease. The molecular mechanisms underpinning the vulnerability of brain endothelial cells to chronic oxidative challenge have not been elucidated. Here, we investigated the oxidative susceptibility of human brain endothelial cells (IHEC) to chronic hyperglycemic stress and insulin signaling and cytoprotection. Chronic hyperglycemia exacerbated IHEC apoptosis in accordance with exaggerated cytosolic and mitochondrial glutathione and protein-thiol redox imbalance, and actin/Keap-1 S-glutathionylation. Insulin attenuated hyperglycemia-induced apoptosis via restored cytosolic and mitochondrial redox. Insulin stimulated glutamate-L-cysteine ligase (GCL) activity by activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling, increased serine phosphorylation and nuclear translocation of nuclear NF-E2-related factor 2 (Nrf2), and upregulation of Nrf2-dependent GCL-catalytic (GCLc) subunit expression. Expression of the GCL-modulatory subunit (GCLm) was unchanged. Inhibitors of insulin receptor tyrosine kinase, PI3K, Akt and mTOR abrogated insulin-induced Nrf2-mediated GCLc expression, redox balance, and IHEC survival. Collectively, these results demonstrate that human brain endothelial cells exhibit vulnerability to hyperglycemic stress which is associated with marked cytosolic and mitochondrial redox shifts. Activation of insulin signaling through PI3K/Akt/mTOR/Nrf2/ GCLc pathway affords significant cell protection by maintaining cellular redox balance.     

Sirt1 overexpression in neurons promotes neurite outgrowth and cell survival through inhibition of the mTOR signaling

The mammalian nicotinamide-adenine dinucleotide (NAD)-dependent deacetylase Sirt1 impacts different processes involved in the maintenance of brain integrity and in the pathogenic pathways associated with several neurodegenerative disorders, including Alzheimer's disease. Here we used human Sirt1 transgenic mice to demonstrate that neuron-specific Sirt1 overexpression promoted neurite outgrowth and improved cell viability under normal and nutrient-limiting conditions in primary culture systems and that Sirt1-overexpressing neurons exhibited higher tolerance to cell death or degeneration induced by amyloid-β1-42 oligomers. Coincidentally, we found that enhanced Sirt1 expression in neurons downregulated the mammalian target of rapamycin (mTOR) protein levels and its phosphorylation without changes in its mRNA levels, which was accompanied by concomitant inhibition of the mTOR downstream signaling activity as revealed by decreased p70S6 kinase (p70S6K) phosphorylation at Thr389. Consistently with this, using a Sirt1 siRNA transfection approach, we observed that reduction of endogenous mouse Sirt1 led to increased levels of mTOR and phosphorylation of itself and p70S6K as well as impaired cell survival and neurite outgrowth in wild-type mouse primary neurons, corroborating a suppressing effect of mTOR by Sirt1. Correspondingly, the mTOR inhibitor rapamycin markedly improved neuronal cell survival in response to nutrient deprivation and significantly enhanced neurite outgrowth in wild-type mouse neurons. The protective effect of rapamycin was extended to neurons even with Sirt1 siRNA knockdown that displayed developmental abnormalities compared with siRNA control-treated cells. Collectively, our findings suggest that Sirt1 may act to promote growth and survival of neurons in the central nervous system via its negative modulation of mTOR signaling.     

The emerging role of autophagic-lysosomal dysfunction in Gaucher disease and Parkinson's disease

Gaucher disease(GD),the commonest lysosomal storage disorder,results from the lack or functional deficiency of glucocerebrosidase(GCase) secondary to mutations in the GBA1 gene.There is an established association between GBA1 mutations and Parkinson's disease(PD),and indeed GBA1 mutations are now considered to be the greatest genetic risk factor for PD.Impaired lysosomal-autophagic degradation of cellular proteins,including α-synuclein(α-syn),is implicated in the pathogenesis of PD,and there is increasing evidence for this also in GD and GBA1-PD.Indeed we have recently shown in a Drosophila model lacking neuronal GCase,that there are clear lysosomal-autophagic defects in association with synaptic loss and neurodegeneration.In addition,we demonstrated alterations in mechanistic target of rapamycin complex 1(mTORC1) signaling and functional rescue of the lifespan,locomotor defects and hypersensitivity to oxidative stress on treatment of GCase-deficient flies with the mT OR inhibitor rapamycin.Moreover,a number of other recent studies have shown autophagy-lysosomal system(ALS) dysfunction,with specific defects in both chaperone-mediated autophagy(CMA),as well as macroautophagy,in GD and GBA1-PD model systems.Lastly we discuss the possible therapeutic benefits of inhibiting mT OR using drugs such as rapamycin to reverse the autophagy defects in GD and PD.     

Urolithin A alleviates blood-brain barrier disruption and attenuates neuronal apoptosis following traumatic brain injury in mice

Urolithin A(UA)is a natural metabolite produced from polyphenolics in foods such as pomegranates,berries,and nuts.UA is neuroprotective against Parkinson’s disease,Alzheimer’s disease,and cerebral hemorrhage.However,its effect against traumatic brain injury remains unknown.In this study,we established adult C57BL/6J mouse models of traumatic brain injury by controlled cortical impact and then intraperitoneally administered UA.We found that UA greatly reduced brain edema;increased the expression of tight junction proteins in injured cortex;increased the immunopositivity of two neuronal autophagy markers,microtubule-associated protein 1A/B light chain 3A/B(LC3)and p62;downregulated protein kinase B(Akt)and mammalian target of rapamycin(mTOR),two regulators of the phosphatidylinositol 3-kinase(PI3K)/Akt/mTOR signaling pathway;decreased the phosphorylation levels of inhibitor of NFκB(IκB)kinase alpha(IKKα)and nuclear factor kappa B(NFκB),two regulators of the neuroinflammation-related Akt/IKK/NFκB signaling pathway;reduced blood-brain barrier permeability and neuronal apoptosis in injured cortex;and improved mouse neurological function.These findings suggest that UA may be a candidate drug for the treatment of traumatic brain injury,and its neuroprotective effects may be mediated by inhibition of the PI3K/Akt/mTOR and Akt/IKK/NFκB signaling pathways,thus reducing neuroinflammation and enhancing autophagy.     

mTORC1 and mTORC2 have largely distinct functions in Purkinje cells

The mammalian target of rapamycin (mTOR) is a key regulator of cellular growth which associates with other proteins to form two multi‐protein complexes called mTORC1 and mTORC2. Dysregulation of mTORC1 signalling in brain is implicated in neuropathological conditions such as autism spectrum or neurodegenerative disorders. Accordingly, allosteric mTOR inhibitors are currently in clinical trials for the treatment of such disorders. Here, we ablated either mTORC1 or mTORC2 conditionally in Purkinje cells of the mouse cerebellum to dissect their role in the development, function and survival of these neurons. We find that the two mouse models largely differ from each other by phenotype and cellular responses. Inactivation of mTORC2, but not of mTORC1, led to motor coordination deficits at an early age. This phenotype correlated with developmental deficits in climbing fibre elimination and impaired dendritic self‐avoidance in mTORC2‐deficient Purkinje cells. In contrast, inactivation of mTORC1, but not of mTORC2, affected social interest of the mice and caused a progressive loss of Purkinje cells due to apoptosis. This cell loss was paralleled by age‐dependent motor deficits. Comparison of mTORC1‐deficient Purkinje cells with those deficient for the mTORC1 inhibitor TSC1 revealed a striking overlap in Purkinje cell degeneration and death, which included neurofilamentopathy and reactive gliosis. Altogether, our study reveals distinct roles of mTORC1 and mTORC2 in Purkinje cells for mouse behaviour and the survival of neurons. Our study also highlights a convergence between the phenotypes of Purkinje cells lacking mTORC1 activity and those expressing constitutively active mTORC1 due to TSC1 deficiency.     

Arrest of myelination and reduced axon growth when Schwann cells lack mTOR

In developing peripheral nerves, differentiating Schwann cells sort individual axons from bundles and ensheath them to generate multiple layers of myelin. In recent years, there has been an increased understanding of the extracellular and intracellular factors that initiate and stimulate Schwann cell myelination, together with a growing appreciation of some of the signaling pathways involved. However, our knowledge of how Schwann cell growth is regulated during myelination is still incomplete. The mammalian target of rapamycin (mTOR) is a core kinase in two major complexes, mTORC1 and mTORC2, that regulate cell growth and differentiation in a variety of mammalian cells. Here we show that elimination of mTOR from murine Schwann cells prevented neither radial sorting nor the initiation of myelination. However, normal postnatal growth of myelinating Schwann cells, both radially and longitudinally, was highly retarded. The myelin sheath in the mutant was much thinner than normal; nevertheless, sheath thickness relative to axon diameter (g-ratio) remained constant in both wild-type and mutant nerves from P14 to P90. Although axon diameters were normal in the mutant at the initiation of myelination, further growth as myelination proceeded was retarded, and this was associated with reduced phosphorylation of neurofilaments. Consistent with thinner axonal diameters and internodal lengths, conduction velocities in mutant quadriceps nerves were also reduced. These data establish a critical role for mTOR signaling in both the longitudinal and radial growth of the myelinating Schwann cell.