Alkaline intracellular pH (pHi) activates AMPK-mTORC2 signaling to promote cell survival during growth factor limitation.

The mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) signaling controls cell metabolism, promotes cell survival, and contributes to tumorigenesis, yet its upstream regulation remains poorly defined. Although considerable evidence supports the prevailing view that amino acids activate mTOR complex 1 but not mTORC2, several studies reported paradoxical activation of mTORC2 signaling by amino acids. We noted that after amino acid starvation of cells in culture, addition of an amino acid solution increased mTORC2 signaling. Interestingly, we found the pH of the amino acid solution to be alkaline, ∼pH 10. These observations led us to discover and demonstrate here that alkaline intracellular pH (pHi) represents a previously unknown activator of mTORC2. Using a fluorescent pH-sensitive dye (cSNARF1-AM) coupled with live-cell imaging, we demonstrate that culturing cells in media at an alkaline pH induces a rapid rise in the pHi, which increases mTORC2 catalytic activity and downstream signaling to the pro-growth and pro-survival kinase Akt. Alkaline pHi also activates AMPK, a canonical sensor of energetic stress. Functionally, alkaline pHi activates AMPK-mTOR signaling, which attenuates apoptosis caused by growth factor withdrawal. Collectively, these findings reveal that alkaline pHi increases mTORC2- and AMPK-mediated signaling to promote cell survival during conditions of growth factor limitation, analogous to the demonstrated ability of energetic stress to activate AMPK-mTORC2 and promote cell survival. As an elevated pHi represents an underappreciated hallmark of cancer cells, we propose that the alkaline pHi stress sensing by AMPK-mTORC2 may contribute to tumorigenesis by enabling cancer cells at the core of a growing tumor to evade apoptosis and survive.

Magnetization transfer ratio: a quantitative imaging biomarker for 5q spinal muscular atrophy.

BACKGROUND AND PURPOSE: We quantified peripheral nerve lesions in adults with 5q-linked spinal muscular atrophy (SMA) type 3 by analysing the magnetization transfer ratio (MTR) of the sciatic nerve, and tested its potential as a novel biomarker for macromolecular changes. METHODS: Eighteen adults with SMA 3 (50% SMA 3a, 50% SMA 3b) and 18 age-/sex-matched healthy controls prospectively underwent magnetization transfer contrast imaging in a 3-Tesla magnetic resonance scanner. Two axial three-dimensional gradient echo sequences, with and without an off-resonance saturation rapid frequency pulse, were performed at the right distal thigh. Sciatic nerve regions of interest were manually traced on 10 consecutive axial slices in the images generated without off-resonance saturation, and then transferred to corresponding slices generated by the sequence with the off-resonance saturation pulse. Subsequently, MTR and cross-sectional areas (CSAs) of the sciatic nerve were analysed. In addition, detailed neurologic, physiotherapeutic and electrophysiologic examinations were conducted in all patients. RESULTS: Sciatic nerve MTR and CSA reliably differentiated between healthy controls and SMA 3, 3a or 3b. MTR was lower in the SMA 3 (P < 0.0001), SMA 3a (P < 0.0001) and SMA 3b groups (P = 0.0020) than in respective controls. In patients with SMA 3, MTR correlated with all clinical scores, and arm nerve compound motor action potentials (CMAPs). CSA was lower in the SMA 3 (P < 0.0001), SMA 3a (P < 0.0001) and SMA 3b groups (P = 0.0006) than in controls, but did not correlate with clinical scores or electrophysiologic results. CONCLUSIONS: Magnetization transfer ratio is a novel imaging marker that quantifies macromolecular nerve changes in SMA 3, and positively correlates with clinical scores and CMAPs.

Diabetes regulates mitochondrial biogenesis and fission in mouse neurons.

AIMS/HYPOTHESIS: Normal mitochondrial activity is a critical component of neuronal metabolism and function. Disruption of mitochondrial activity by altered mitochondrial fission and fusion is the root cause of both neurodegenerative disorders and Charcot-Marie-Tooth type 2A inherited neuropathy. This study addressed the role of mitochondrial fission in the pathogenesis of diabetic neuropathy. METHODS: Mitochondrial biogenesis and fission were assayed in both in vivo and in vitro models of diabetic neuropathy. Gene, protein, mitochondrial DNA and ultrastructural analyses were used to assess mitochondrial biogenesis and fission. RESULTS: There was greater mitochondrial biogenesis in dorsal root ganglion neurons from diabetic compared with non-diabetic mice. An essential step in mitochondrial biogenesis is mitochondrial fission, regulated by the mitochondrial fission protein dynamin-related protein 1 (DRP1). Evaluation of diabetic neurons in vivo indicated small, fragmented mitochondria, suggesting increased fission. In vitro studies revealed that short-term hyperglycaemic exposure increased levels of DRP1 protein. The influence of hyperglycaemia-mediated mitochondrial fission on cell viability was evaluated by knockdown of Drp1 (also known as Dnm1l). Knockdown of Drp1 resulted in decreased susceptibility to hyperglycaemic damage. CONCLUSIONS/INTERPRETATION: We propose that: (1) mitochondria undergo biogenesis in response to hyperglycaemia, but the increased biogenesis is insufficient to accommodate the metabolic load; (2) hyperglycaemia causes an excess of mitochondrial fission, creating small, damaged mitochondria; and (3) reduction of aberrant mitochondrial fission increases neuronal survival and indicates an important role for the fission-fusion equilibrium in the pathogenesis of diabetic neuropathy.

Notch-Delta signaling is required for spatial patterning and Müller glia differentiation in the zebrafish retina.

Notch-Delta signaling has been implicated in several alternative modes of function in the vertebrate retina. To further investigate these functions, we examined retinas from zebrafish embryos in which bidirectional Notch-Delta signaling was inactivated either by the mind bomb (mib) mutation, which disrupts E3 ubiquitin ligase activity, or by treatment with gamma-secretase inhibitors, which prevent intramembrane proteolysis of Notch and Delta. We found that inactivating Notch-Delta signaling did not prevent differentiation of retinal neurons, but it did disrupt spatial patterning in both the apical-basal and planar dimensions of the retinal epithelium. Retinal neurons differentiated, but their laminar arrangement was disrupted. Photoreceptor differentiation was initiated normally, but its progression was slowed. Although confined to the apical retinal surface as in normal retinas, the planar organization of cone photoreceptors was disrupted: cones of the same spectral subtype were clumped rather than regularly spaced. In contrast to neurons, Müller glia failed to differentiate suggesting an instructive role for Notch-Delta signaling in gliogenesis.

Neurotrophins support the development of diverse sensory axon morphologies.

The initial outgrowth of peripheral axons in developing embryos is thought to occur independently of neurotrophins. However, the degree to which peripheral neurons can extend axons and elaborate axonal arborizations in the absence of these molecules has not been studied directly because of exquisite survival requirements for neurotrophins at early developmental stages. We show here that embryonic sensory neurons from BAX-deficient mice survived indefinitely in the absence of neurotrophins, even in highly dissociated cultures, allowing assessment of cell autonomous axon outgrowth. At embryonic day 11 (E11)-E13, stages of rapid axon growth toward targets in vivo, Bax-/- sensory neurons cultured without neurotrophins were almost invariably unipolar and extended only a rudimentary axon. Addition of neurotrophins caused outgrowth of a second axon and a marked, dose-dependent elongation of both processes. Surprisingly, morphological responses to individual neurotrophins differed substantially. Neurotrophin-3 (NT-3) supported striking terminal arborization of subsets of Bax-/- neurons, whereas NGF produced predominantly axon elongation in a different subset. We conclude that axon growth in vitro is neurotrophin dependent from the earliest stages of sensory neuron development. Furthermore, neurotrophins support the appearance of distinct axonal morphologies that characterize different sensory neuron subpopulations.

The laminin alpha chains: expression, developmental transitions, and chromosomal locations of alpha1-5, identification of heterotrimeric laminins 8-11, and cloning of a novel alpha3 isoform.

Laminin trimers composed of alpha, beta, and gamma chains are major components of basal laminae (BLs) throughout the body. To date, three alpha chains (alpha1-3) have been shown to assemble into at least seven heterotrimers (called laminins 1-7). Genes encoding two additional alpha chains (alpha4 and alpha5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant alpha4 and alpha5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that alpha4 and alpha5 assemble into four novel laminin heterotrimers (laminins 8-11: alpha4beta1gamma1, alpha4beta2gamma1, alpha5beta1gamma1, and alpha5beta2gamma1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of alpha1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, alpha4 and alpha5 exhibited the broadest patterns of expression, while expression of alpha1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the alpha chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one alpha chain, all alpha chains were present in multiple BLs, and some BLs contained two or three alpha chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different alpha chains and two different beta chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

Distribution of the ten known laminin chains in the pathways and targets of developing sensory axons.

Laminins are heterotrimers of alpha, beta, and gamma chains. At present, five alpha, three beta, and two gamma chains have been described. The best characterized laminin (laminin 1 = alpha 1, beta 1, gamma 1) promotes neurite outgrowth from virtually all classes of developing neurons, implying that laminins may serve as axon guidance molecules in vivo. Moreover, different laminin trimers exert distinct effects on subsets of laminin-1-responsive cells, suggesting that isoform diversity may underlie some axonal choices in vivo. As a first step toward evaluating these hypotheses, we have documented the expression patterns of all 10-known laminin chains in the peripheral nervous system and spinal cord of the murine embryo. The alpha 2, alpha 4, beta 1, and gamma 1 chains are expressed in peripheral axonal pathways by embryonic day (E) 11.5, when sensory and motor axonal outgrowth is underway. Thus, laminins (but not laminin 1) may promote peripheral axonal outgrowth. By E 13.5, laminin chains are differentially expressed in the limb-bud, with prominent expression of alpha 2 and alpha 4 in muscle and of alpha 3 and alpha 5 in skin. This pattern raises the possibility that laminin isoform diversity contributes to the ability of cutaneous and muscle sensory axons to distinguish their targets. Later in development, some chains (e.g., alpha 2, alpha 4, and beta 1) are downregulated in peripheral nerve while others (e.g., gamma 1), continue to be expressed by Schwann cells into adulthood. In contrast to peripheral nerves and ganglia, laminin chains are expressed at low levels, if at all, in the developing spinal cord gray matter.

Cloning and expression of a novel murine semaphorin with structural similarity to insect semaphorin I.

We describe a novel semaphorin family member, Sema VIa, with 25-36% sequence identity at the amino acid level in the semaphorin domain to previously published mouse homologues. This novel family member shares considerable homology with the best characterized murine semaphorin, Sema III (also known as SemD), at the 5' end but is divergent from Sema III near the 3' end because it contains a putative transmembrane domain. Remarkably, of the known semaphorins, Sema VIa bears the greatest structural similarity to insect Sema I, although it contains a much larger intracellular domain. We propose, therefore, that Sema VIa is the prototype of a new class (class VI) of semaphorins. In order to gain insights into potential functions of Sema VIa, we have compared mRNA expression of Sema VIa to that of Sema III during development. In the nervous system, Sema VIa is expressed in strikingly localized and transient patterns that are markedly different from those of Sema III. Interestingly, Sema VIa and Sema III frequently exhibit complementary or adjacent loci of expression. We suggest that Sema VIa may be important to nervous system development via a mechanism that involves cell-cell communication.

Tetrahydrobiopterin biosynthesis in the rat brain: heterogeneity of GTP cyclohydrolase I mRNA expression in monoamine-containing neurons.

GTP cyclohydrolase I is the first and rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. A quantitative in situ hybridization technique was used to study the expression of GTP cyclohydrolase I mRNA in the rat brain at the cellular level. Coronal sections between the diencephalon and myelencephalon were exposed to a 35S-labelled antisense GTP cyclohydrolase I cRNA probe. Sections serial to these were hybridized with a 35S-labelled antisense cRNA probe complementary to tyrosine hydroxylase mRNA. Tyrosine hydroxylase and GTP cyclohydrolase I mRNAs were found to colocalize within catecholamine neurons located throughout the brain. The overall distribution of neurons expressing GTP cyclohydrolase I mRNA was observed to correspond exactly to the known distribution of the dopamine, norepinephrine/epinephrine and serotonin-containing cell groups. Overall, a 30-fold range of GTP cyclohydrolase I mRNA expression was observed, with the transcript being significantly more abundant in serotonin than in dopamine or norepinephrine/epinephrine neurons. Comparisons across serotonin cell groups indicated that neurons of the median raphe nucleus, caudal linear nucleus raphe (B8) and the dorsal raphe (B6/B7) expressed the highest levels of GTP cyclohydrolase I mRNA. Comparisons across dopamine cell groups indicated that the transcript was more abundant in neurons of the ventral tegmental area (A10) than in neurons of the substantia nigra pars compacta (A9) and that both A9 and A10 dopamine neurons exhibited higher levels of expression than the DA neurons of the hypothalamus (A11-A14). Norepinephrine neurons of the locus coeruleus (A6) and subcoeruleus (A6v) exhibited significantly higher levels of GTP cyclohydrolase I mRNA than did neurons in other norepinephrine (A1 and A2) or epinephrine (C1 and C2) cell groups. GTP cyclohydrolase I mRNA could not be detected unequivocally in neurons known to contain nitric oxide synthase. Heterogeneity in the level of expression of GTP cyclohydrolase I mRNA by monoamine-containing neurons may play an important role in determining steady state levels of tetrahydrobiopterin and, ultimately, the regulation of monoamine biosynthesis.

Tetrahydrobiopterin cofactor biosynthesis: GTP cyclohydrolase I mRNA expression in rat brain and superior cervical ganglia.

GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin, the reduced pteridine cofactor required for catecholamine (CA), indoleamine, and nitric oxide biosynthesis. We have used the reverse transcription-polymerase chain reaction technique, based on the published cDNA sequence for rat liver GTPCH, to clone a portion of the GTPCH transcript from rat adrenal gland mRNA and have used this clone for the analysis of GTPCH mRNA in brain and other tissues of the rat by northern blot, nuclease protection assay, and in situ hybridization. Two GTPCH mRNA transcripts of 1.2 and 3.8 kb in length were detected by northern blot, with the 1.2-kb form predominating in the liver and the 3.8-kb form in the pineal gland, adrenal gland, brainstem, and hypothalamic neurons maintained in culture. In situ hybridization studies localized GTPCH mRNA to CA-containing perikarya in the locus ceruleus, ventral tegmental area, and substantia nigra, pars compacta. Levels of GTPCH mRNA in central and peripheral catecholamine neurons determined by nuclease protection assay were increased twofold 24 h after a single injection of the CA-depleting drug reserpine; both the 1.2- and 3.8-kb transcripts were increased in the adrenal gland. Low levels of GTPCH mRNA were also detected by nuclease protection assay in the striatum, hippocampus, and cerebellum, brain regions that do not contain monoaminergic perikarya.

Substance P gene expression in sympathetic neurons is regulated by neuron/support cell interaction.

In agreement with previous findings, the presence of support cells was found to increase the level of preprotachykinin (i.e. substance P-encoding) mRNA in cultures of sympathetic neurons. Treatment of neuron-only cultures, which did not express detectable levels of preprotachykinin mRNA, with conditioned medium from support cell-only cultures, also increased the level of preprotachykinin mRNA. This elevation in substance P gene expression was reflected in a 2-fold increase in the number of substance P-like immunoreactive neurons. In contrast, treatment of neuron-only cultures with conditioned medium from co-cultures of sympathetic neurons and support cells did not increase the level of preprotachykinin mRNA or the number of neurons containing substance P-like immunoreactivity. These observations suggest that while support cells release a soluble factor(s) capable of inducing substance P expression in sympathetic neurons, the production or action of this factor(s) is inhibited by the interaction between support cells and sympathetic neurons. Thus, by interacting with non-neuronal cells in their environment, sympathetic neurons appear to play an active role in determining which neurotransmitter phenotype they express.

Differential metabolism of tetrahydrobiopterin in monoamine neurons: a hypothesis based upon clinical and basic research.

This chapter has attempted to describe and integrate some of the clinical and basic research that support our hypothesis that the metabolism of BH4 is normally heterogeneous across different populations of monoamine-containing neurons. Based upon this hypothesis, there may now be reason to support the idea that certain neuropsychiatric illnesses, which are though to be the result (at least in part) of altered monoamine metabolism, might find their roots in an abnormal metabolism of BH4 within specific monoaminergic cell groups. Such a specific dysfunction might not be apparent in the rest of the brain or peripheral nervous system, thereby being difficult to detect. Perhaps the application of molecular biological techniques to studies of BH4 metabolism in man will shed new light on these problems.

Release of cholecystokinin from rat midbrain slices and modulatory effect of D2DA receptor stimulation.

Cholecystokinin octapeptide (CCK-8) is colocalized within a majority of dopamine (DA)-containing neurons of the rat midbrain. Exogenous CCK-8 can modulate the electrophysiological activity of DA neurons, at least in part, by direct actions on the somatodendritic region of these cells. If CCK-8 is somatodendritically released from DA neurons, it may influence DA cell function as has been shown for DA itself. In the present study, radioimmunoassay was used to determine if CCK-8 is released in vitro from slices of rat midbrain under basal and depolarizing (30 mM potassium) conditions. Low levels of CCK-8 were detected in the basal incubation medium. Thirty mM potassium caused about a 3-fold increase in the release of CCK-8. This stimulated release was abolished in calcium-free medium. The D2 receptor agonist quinpirole, but not the D1 agonist SKF 38393, attenuated the potassium-stimulated release of CCK-8 but did not affect basal release. These results show that CCK-8, like DA, can be released from midbrain slices, presumably from DA/CCK-8-containing neurons. This finding is in accordance with the possibility that CCK-8 plays a role in the regulation of DA neuronal function at the level of the cell body, where it might influence the excitability of the DA cell membrane.

Motor effects of globus pallidus stimulation in the rat: lesions to corticofugal fibers block the motor effects.

Unilateral electrical stimulation of the globus pallidus (GP) in anesthetized male rats was used to determine the nature of the activity driven in muscles of the neck and shoulder by GP output. In 6 groups of animals stimulation was coupled with lesions to sites that interrupted corticofugal fibers or GP output. Interruption of corticofugal fibers blocked the driven activity while lesions that compromised GP output left the activity unaffected.