PMID- 32781001
OWN - NLM
STAT- MEDLINE
DCOM- 20210310
LR  - 20211203
IS  - 1090-2430 (Electronic)
IS  - 0014-4886 (Print)
IS  - 0014-4886 (Linking)
VI  - 334
DP  - 2020 Dec
TI  - Dynamic analysis of 4E-BP1 phosphorylation in neurons with Tsc2 or Depdc5 
      knockout.
PG  - 113432
LID - S0014-4886(20)30263-6 [pii]
LID - 10.1016/j.expneurol.2020.113432 [doi]
AB  - TSC1 or TSC2 mutations cause Tuberous Sclerosis Complex (TSC), and lead to 
      mechanistic target of rapamycin (mTOR) hyperactivation evidenced by 
      hyperphosphorylation of ribosomal S6 protein and 4-elongation factor binding 
      protein 1 (4E-BP1). Amino acid (AA) levels modulate mTOR-dependent S6 and 4E-BP1 
      phosphorylation in non-neural cells, but this has not been comprehensively 
      investigated in neurons. The effects of AA levels on mTOR signaling and S6 and 
      4E-BP1 phosphorylation were analyzed in Tsc2 and Depdc5 (a distinct mTOR 
      regulatory gene associated with epilepsy) CRISPR-edited Neuro2a (N2a) cells and 
      differentiated neurons. Tsc2 or Depdc5 knockout (KO) led to S6 and 4E-BP1 
      hyperphosphorylation and cell soma enlargement, but while Tsc2 KO N2a cells 
      exhibited reduced S6 phosphorylation (Ser240/244) and cell soma size after 
      incubation in AA free (AAF) media, Depdc5 KO cells did not. Using a CFP/YFP 
      FRET-biosensor coupled to 4E-BP1, we assayed 4E-BP1 phosphorylation in living N2a 
      cells and differentiated neurons following Tsc2 or Depdc5 KO. AAF conditions 
      reduced 4E-BP1 phosphorylation in Tsc2 KO N2a cells but had no effect in Depdc5 
      KO cells. Rapamycin blocked S6 protein phosphorylation but had no effect on 
      4E-BP1 phosphorylation, following either Tsc2 or Depdc5 KO. Confocal imaging 
      demonstrated that AAF media promoted movement of mTOR off the lysosome, 
      functionally inactivating mTOR, in Tsc2 KO but not Depdc5 KO cells, demonstrating 
      that AA levels modulate lysosomal mTOR localization and account, in part, for 
      differential effects of AAF conditions following Tsc2 versus Depdc5 KO. AA levels 
      and rapamycin differentially modulate S6 and 4E-BP1 phosphorylation and mTOR 
      lysosomal localization in neurons following Tsc2 KO versus Depdc5 KO. Neuronal 
      mTOR signaling in mTOR-associated epilepsies may have distinct responses to mTOR 
      inhibitors and to levels of cellular amino acids.
CI  - Copyright (c) 2020 Elsevier Inc. All rights reserved.
FAU - Iffland, Philip H 2nd
AU  - Iffland PH 2nd
AD  - Department of Neurology, University of Maryland School of Medicine, Baltimore, 
      MD, United States of America.
FAU - Barnes, Allan E
AU  - Barnes AE
AD  - Department of Neurology, University of Maryland School of Medicine, Baltimore, 
      MD, United States of America.
FAU - Baybis, Marianna
AU  - Baybis M
AD  - Department of Neurology, University of Maryland School of Medicine, Baltimore, 
      MD, United States of America.
FAU - Crino, Peter B
AU  - Crino PB
AD  - Department of Neurology, University of Maryland School of Medicine, Baltimore, 
      MD, United States of America. Electronic address: pcrino@som.umaryland.edu.
LA  - eng
GR  - R01 NS094596/NS/NINDS NIH HHS/United States
GR  - R01 NS099452/NS/NINDS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20200808
PL  - United States
TA  - Exp Neurol
JT  - Experimental neurology
JID - 0370712
RN  - 0 (Adaptor Proteins, Signal Transducing)
RN  - 0 (Cell Cycle Proteins)
RN  - 0 (Depdc5 protein, mouse)
RN  - 0 (EIF4EBP1 protein, human)
RN  - 0 (GTPase-Activating Proteins)
RN  - 0 (Immunosuppressive Agents)
RN  - 0 (Tsc2 protein, mouse)
RN  - 0 (Tuberous Sclerosis Complex 2 Protein)
RN  - W36ZG6FT64 (Sirolimus)
SB  - IM
MH  - Adaptor Proteins, Signal Transducing/*metabolism
MH  - Animals
MH  - Cell Cycle Proteins/*metabolism
MH  - Cell Line, Tumor
MH  - GTPase-Activating Proteins/*deficiency/genetics
MH  - Gene Knockout Techniques/methods
MH  - Immunosuppressive Agents/pharmacology
MH  - Mice
MH  - Neurons/drug effects/*metabolism
MH  - Phosphorylation/drug effects/physiology
MH  - Sirolimus/pharmacology
MH  - Tuberous Sclerosis Complex 2 Protein/deficiency/genetics
PMC - PMC8170708
MID - NIHMS1633698
OTO - NOTNLM
OT  - Cortical malformations
OT  - Epilepsy
OT  - GATOR1
OT  - Tuberous sclerosis complex
OT  - mTORopathies
EDAT- 2020/08/12 06:00
MHDA- 2021/03/11 06:00
PMCR- 2021/12/01
CRDT- 2020/08/12 06:00
PHST- 2020/03/06 00:00 [received]
PHST- 2020/07/13 00:00 [revised]
PHST- 2020/08/04 00:00 [accepted]
PHST- 2020/08/12 06:00 [pubmed]
PHST- 2021/03/11 06:00 [medline]
PHST- 2020/08/12 06:00 [entrez]
PHST- 2021/12/01 00:00 [pmc-release]
AID - S0014-4886(20)30263-6 [pii]
AID - 10.1016/j.expneurol.2020.113432 [doi]
PST - ppublish
SO  - Exp Neurol. 2020 Dec;334:113432. doi: 10.1016/j.expneurol.2020.113432. Epub 2020 
      Aug 8.