Effects of rapamycin on growth hormone receptor knockout mice.

It is well documented that inhibition of mTORC1 (defined by Raptor), a complex of mechanistic target of rapamycin (mTOR), extends life span, but less is known about the mechanisms by which mTORC2 (defined by Rictor) impacts longevity. Here, rapamycin (an inhibitor of mTOR) was used in GHR-KO (growth hormone receptor knockout) mice, which have suppressed mTORC1 and up-regulated mTORC2 signaling, to determine the effect of concurrently decreased mTORC1 and mTORC2 signaling on life span. We found that rapamycin extended life span in control normal (N) mice, whereas it had the opposite effect in GHR-KO mice.

Leucyl-tRNA Synthetase Activates Vps34 in Amino Acid-Sensing mTORC1 Signaling.

Amino acid availability activates signaling by the mammalian target of rapamycin (mTOR) complex 1, mTORC1, a master regulator of cell growth. The class III PI-3-kinase Vps34 mediates amino acid signaling to mTORC1 by regulating lysosomal translocation and activation of the phospholipase PLD1. Here, we identify leucyl-tRNA synthetase (LRS) as a leucine sensor for the activation of Vps34-PLD1 upstream of mTORC1.

Single-Molecule Analysis of Lipid-Protein Interactions in Crude Cell Lysates.

Recognition of signaling phospholipids by proteins is a critical requirement for the targeting and initiation of many signaling cascades. Most biophysical methods for measuring protein interactions with signaling phospholipids use purified proteins, which do not take into account the effect of post-translational modifications and other cellular components on these interactions. To potentially circumvent these problems, we have developed a single-molecule fluorescence approach to analyzing lipid-protein interactions in crude cell extracts.

Stoichiometry and assembly of mTOR complexes revealed by single-molecule pulldown.

The mammalian target of rapamycin (mTOR) kinase is a master regulator of cellular, developmental, and metabolic processes. Deregulation of mTOR signaling is implicated in numerous human diseases including cancer and diabetes. mTOR functions as part of either of the two multisubunit complexes, mTORC1 and mTORC2, but molecular details about the assembly and oligomerization of mTORCs are currently lacking.

Transplanted glial restricted precursor cells improve neurobehavioral and neuropathological outcomes in a mouse model of neonatal white matter injury despite limited cell survival.

Objective: Neonatal white matter injury (NWMI) is the leading cause of cerebral palsy and other neurocognitive deficits in prematurely-born children, and no restorative therapies exist. Our objective was to determine the fate and effect of glial restricted precursor cell (GRP) transplantation in an ischemic mouse model of NWMI.

Methods: Neonatal CD-1 mice underwent unilateral carotid artery ligation on postnatal-Day 5 (P5).

Ischemia-induced neuroinflammation is associated with disrupted development of oligodendrocyte progenitors in a model of periventricular leukomalacia.

Microglial activation in crossing white matter tracts is a hallmark of noncystic periventricular leukomalacia (PVL), the leading pathology underlying cerebral palsy in prematurely born infants. Recent studies indicate that neuroinflammation within an early time window can produce long-lasting defects in oligodendroglial maturation, myelination deficit, as well as disruption of transcription factors important in oligodendroglial maturation. We recently reported an ischemic mouse model of PVL, induced by unilateral neonatal carotid artery ligation, leading to selective long-lasting bilateral myelination deficits, ipsilateral thinning of the corpus callosum, ventriculomegaly, as well as evidence of axonopathy.

Derivation of glial restricted precursors from E13 mice.

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases.

Phosphatidic acid activates mammalian target of rapamycin complex 1 (mTORC1) kinase by displacing FK506 binding protein 38 (FKBP38) and exerting an allosteric effect.

Phosphatidic acid (PA) is a critical mediator of mitogenic activation of mammalian target of rapamycin complex 1 (mTORC1) signaling, a master regulator of mammalian cell growth and proliferation. The mechanism by which PA activates mTORC1 signaling has remained unknown. Here, we report that PA selectively stimulates mTORC1 but not mTORC2 kinase activity in cells and in vitro.

Probing cellular protein complexes using single-molecule pull-down.

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions.

In vivo magnetization transfer MRI shows dysmyelination in an ischemic mouse model of periventricular leukomalacia.

Periventricular leukomalacia, PVL, is the leading cause of cerebral palsy in prematurely born infants, and therefore more effective interventions are required. The objective of this study was to develop an ischemic injury model of PVL in mice and to determine the feasibility of in vivo magnetization transfer (MT) magnetic resonance imaging (MRI) as a potential monitoring tool for the evaluation of disease severity and experimental therapeutics. Neonatal CD-1 mice underwent unilateral carotid artery ligation on postnatal day 5 (P5); at P60, in vivo T2-weighted (T2w) and MT-MRI were performed and correlated with postmortem histopathology.