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Cited by 82Neuroscience and Neuropharmacology ResearchGenetics and Neurodevelopmental DisordersProtein Kinase Regulation and GTPase Signaling

AMPA receptor and GEF-H1/Lfc complex regulates dendritic spine development through RhoA signaling cascade

Open access
Kang, Myoung-Goo, Guo, Yurong, Huganir, Richard L.·2009·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.0812861106PMC PMC2638734
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Abstract

AMPA receptors (AMPA-R) are major mediators of synaptic transmission and plasticity in the developing and adult central nervous system. Activity-dependent structural plasticity mediated by dynamic changes in the morphology of spines and dendrites is also essential for the formation and tuning of neuronal circuits. RhoA and Rac1 are known to play important roles in the regulation of spine and dendrite development in response to neuronal activity. These Rho GTPases are activated by guanine nucleotide exchange factors (GEFs). In this study, we identified GEF-H1/Lfc as a component of the AMPA-R complex in the brain. GEF-H1 is enriched in the postsynaptic density and is colocalized with GluR1 at spines. GEF-H1 activity negatively regulates spine density and length through a RhoA signaling cascade. In addition, AMPA-R-dependent changes in spine development are eliminated by down-regulation of GEF-H1. Altogether, these results strongly suggest that GEF-H1 is an important mediator of AMPA-R activity-dependent structural plasticity in neurons.

AI snapshot

Written from the paper’s full text.

TL;DR

GEF-H1/Lfc was identified as a component of the AMPA receptor complex in rat brain and shown to negatively regulate dendritic spine density and length through RhoA-ROCK signaling. AMPA-R blockade with NBQX decreases spine density via GEF-H1-dependent activation of RhoA and inhibition of Rac1.

Key findings

  • GEF-H1 co-purified with AMPA-R complexes from rat brain by WGA chromatography and large-scale immunoprecipitation, was identified by LC-MS/MS, and was confirmed by Western blot and co-IP in cortical neurons and heterologous cells; it is enriched in the postsynaptic density and colocalizes with GluR1 at spines.
  • Dominant-negative GEF-H1 and lentiviral shRNA knockdown of GEF-H1 both significantly increased spine density and length in cultured hippocampal neurons; GEF-H1 overexpression significantly decreased spine length without changing spine density.
  • GEF-H1 shRNA knockdown reduced endogenous GEF-H1 by 86 ± 1.0% (n = 4) and also down-regulated GluR1 by 21 ± 3.0% (n = 4); knockdown decreased active RhoA and increased active Rac1.
  • The RhoA inhibitor C3T and the ROCK inhibitor Y27632 increased spine density and length and eliminated the spine-length decrease caused by GEF-H1 overexpression; NBQX (20 µM, 7 days) decreased spine density, increased RhoA activity, and decreased Rac1 activity, and these NBQX effects were eliminated by GEF-H1 shRNA.

Methods

  • AMPA-R complexes were purified from rat brains using wheat germ agglutinin chromatography and large-scale immunoprecipitation, then analyzed by LC-MS/MS and Western blot; co-IP was also performed in cortical neuronal cultures and HEK293T cells expressing GFP-GEF-H1 with myc-tagged GluR subunits.
  • Cultured cortical and hippocampal neurons were transfected at 13 DIV or infected with lentivirus at 9 DIV, fixed at 16 DIV, and stained for GFP/mCherry, GluR1, and MAP2; spine density was counted per 10 µm of dendrite and spine length distributions were measured.
  • Manipulations included dominant-negative GEF-H1 (T247A), lentiviral GEF-H1 shRNA, C3T (1.0 µg/ml overnight for 14–16 h at 15 DIV), Y27632 (100 µM for 3 days), and NBQX (20 µM for 7 days); RhoA and Rac1 activities were measured by pull-down assays.

Limitations

  • Stated: heterologous-cell co-IP efficiency for GEF-H1 and AMPA-R subunits was much lower than with brain or neuronal lysates, suggesting the interaction may require other proteins or posttranslational modifications.
  • Stated: overexpressed GFP-GEF-H1 was not observed in spines but in dendritic shafts, unlike endogenous GEF-H1; the authors note this may reflect differences in expression timing and cDNA length compared with a previous study, and they selected neurons with microtubule-like GEF-H1 expression for quantification.
  • Evident: the functional conclusions rely heavily on cultured neurons with overexpression, dominant-negative, shRNA knockdown, and pharmacological inhibitors; the text does not report in vivo spine or behavioral rescue experiments.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1073/pnas.0812861106

Cites

37 of 37 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

4 of 38 cited sources checked · 2 supported · 0 flagged · accuracy not yet measured

  • Premisecitation
    § Discussion · p. 5

    The previous study used rat GEF-H1 cDNA (encode 958 aa) that is shorter than our rat GEF-H1 cDNA (encode 985 aa).

    “In addition, the previous study (26) used rat GEF-H1 cDNA (encode 958 aa) that is shorter than our rat GEF-H1cDNA(encode985aa).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [26] Ryan XP, et al. (2005) The Rho-specific GEF Lfc interacts with neurabin and spinophilin to regulate dendritic spine morphology. Neuron 47(1):85-100. source

      The Results section (page unavailable) says the study used rat Lfc and describes probes and antibodies targeting amino acids 701–958 of the full-length protein. This supports the attribution of a rat Lfc/GEF-H1 construct extending to residue 958, but the source does not state the length of the citing authors’ 985-amino-acid cDNA or make that comparison. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
  • Findingown data
    § Discussion · p. 5

    Endogenous GEF-H1 is highly enriched in PSD III fraction from the brains.

    “Our Western blot analysis showed that endogenous GEF-H1 is highly enriched in PSD III fraction from the brains”
    Quote in the paperSubstantial claim
  • Findingown data
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 4

    The negative effect of GEF-H1 over-expression on spine length is eliminated by an inhibitor of RhoA or ROCK.

    “The negative effect of GEF-H1 over-expression on spine length is eliminated by an inhibitor of RhoA or ROCK.”
    Quote in the paperSubstantial claim
  • Findingown data
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 5

    The activity change of Rho GTPases by NBQX was completely eliminated by down-regulation of GEF-H1 expression.

    “This activity change of Rho GTPases by NBQX was completely eliminated by the down-regulation of GEF-H1 expression (see Fig. 5 F and Table S2).”
    Quote in the paperSubstantial claim
  • Findingown data
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 4

    The effect of Y27632 on spine length was significantly stronger than that of C3T.

    “In our experiments, the effect of Y27632 on spine length was significantly stronger than that of C3T (see Fig. 4 E and F , and Table S1)”
    Quote in the paperNot a substantial claim
  • Classified as probably not a substantial claim. 24
  • May combine two claims. 21
  • The quote only partly states this claim. 1

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion · p. 5

    A previous study showed that GFP-GEF-H1 was localized in the dendritic shaft and translocated to spines only after KCl-dependent depolarization or electrical stimulation.

    “A previous study of GEF-H1 in neurons showed that GFPGEF-H1 was localized in the dendritic shaft and translocated to spines only after KCl-dependent depolarization or electrical stimulation (26).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [26] Ryan XP, et al. (2005) The Rho-specific GEF Lfc interacts with neurabin and spinophilin to regulate dendritic spine morphology. Neuron 47(1):85-100. source

      The Results (page unavailable) report that GFP-Lfc was mainly in dendritic shafts and moved into spines after KCl depolarization or electrical stimulation; the Discussion identifies human Lfc as GEF-H1. But “only after” is too restrictive: the Results also report translocation after glutamate or NMDA stimulation, and some unstimulated neurons already had Lfc staining in spines. (LLM, conf=0.91)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 4

    Rac1 can positively regulate spine density.

    “and that Rac1 could positively regulate spine density (11, 19, 20).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [11]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [19] Nakayama AY, Harms MB, Luo L (2000) Small GTPases Rac and Rho in the maintenance of dendritic spines and branches in hippocampal pyramidal neurons. J Neurosci 20:5329-5338. source
    • cites [20] Penzes P, et al. (2003) Rapid induction of dendritic spine morphogenesis by transsynaptic ephrinB-EphB receptor activation of the Rho-GEF kalirin. Neuron 37:263-274. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion · p. 6

    AMPA-Rs regulate experience-dependent dendritic arbor growth in vivo.

    “Furthermore, it has been reported that AMPA-Rs regulate experience-dependent dendritic arbor growth in vivo (27).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [27] Haas K, Li J, Cline HT (2006) AMPA receptors regulate experience-dependent dendritic arbor growth in vivo. Proc Natl Acad Sci USA 103:12127-12131. source

  • May combine two claims.

    Findingown data
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 4

    Rac1 activity was significantly increased by the RhoA inhibitor but not by the ROCK inhibitor.

    “Furthermore, Rac1 activity was significantly increased by the RhoA inhibitor but not by the ROCK inhibitor.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 4

    Treatment of neurons with C3T significantly increased spine density and length of cultured neurons.

    “Treatment of neurons with a RhoA inhibitor, C3T, significantly increased the spine density and length of cultured neurons (see Fig. 4 B , D , and E , and Table S1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 3

    Knock-down of GEF-H1 significantly increases active RhoA and decreases active Rac1.

    “Compared with the infection of lentivirus carrying GFP only (Vec), the infection of lentivirus carrying GFP and GEF-shRNA (Ri) significantly increased and decreased active RhoA and Rac1, respectively. See Table S2 for values and statistical analysis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 4

    Treatment with C3T or Y27632 significantly increases spine density.

    “Compared with untreated neurons (Cont), neurons treated with C3T (C3T) or Y27632 (Y27) showed significant increases in spine density.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § Discussion · p. 5

    Our full-length cDNA encodes 27 aa more in the N-terminal region.

    “ComparedtotheirshortercDNA, our full-length cDNA encode 27 aa more in the N-terminal region.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § AMPA receptor and GEF-H1/Lfc complex regulates dendritic spine development through RhoA signaling cascade · p. 1

    GEF-H1 is enriched in the postsynaptic density and colocalized with GluR1 at spines.

    “GEF-H1 is enriched in the postsynaptic density and is colocalized with GluR1 at spines.”
    • Also stated in: § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 2 · plain
      “Our biochemical and immunocytochemical analyses of GEF-H1 showed localization of GEF-H1 in postsynaptic density (PSD) and spines (Figs. S3 A and S4 C ).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § Identification of GEF-H1 as a Component of the AMPA-R Complex. · p. 2

    GEF-H1 was detected in solubilized brain lysates at around 120 kDa, similar in size to its human and mouse homologs.

    “GEF-H1 was detected in solubilized brain lysates at around 120 kDa, similar in size to its human and mouse homologous.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 4

    Over-expression of GEF-H1 showed a dosage effect on spine development as well as on dendritic arbor development.

    “Over-expression of GEF-H1 showed a dosage effect on spine development as well as on dendritic arbor development.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 2

    Over-expression of GEF-H1 does not increase spine number or density.

    “without increase in spine number (see Fig. 4 D and Table S1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 4

    C3T treatment significantly decreased RhoA activity and increased Rac1 activity in cortical neurons.

    “C3T treatment significantly decreased and increased the activity of RhoA and Rac1, respectively.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 3

    Knock-down of endogenous GEF-H1 by shRNA significantly increases spine length in cultured hippocampal neurons.

    “and spine length (see Fig. 3 D and Table S1).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Findingown data
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 3

    Four days after viral infection, GFP signals begin to appear and reached a plateau 7 days after infection.

    “Four days after viral infection, GFP signals begin to appear and reached a plateau 7 days after the infection.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 5

    NBQX treatment significantly increased RhoA activity and decreased Rac1 activity.

    “NBQX treatment significantly increased RhoA activity and decreased Rac1 activity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Identification of GEF-H1 as a Component of the AMPA-R Complex. · p. 2

    The procedure specifically isolated a major protein band with a molecular weight of 105 kDa, which contains the AMPA-R subunits GluR1-4, and several other protein bands.

    “As shown in Fig. 1 A , this procedure specifically isolated a major protein band with a molecular weight of 105 kDa, which contains the AMPA-R subunits GluR1-4, and several other protein bands detected by silver-staining.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 4

    The stronger effect of Y27632 on spine length may be because of the relatively longer treatment of neurons with Y27632 (3 days) than with C3T (14-16 h).

    “which may be because of the relatively longer treatment of neurons with Y27632 (3 days) than that of C3T (14-16 h).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing). May combine two claims.

    Premisecitation
    § Discussion · p. 6

    Synaptic Ca2+ influx through NMDA receptors during long-term potentiation induction triggers Rho GTPase-mediated actin polymerization, resulting in AMPA-R trafficking to synapses.

    “It has been suggested that synaptic Ca 2 + influx through NMDA receptors during long-term potentiation induction triggers Rho GTPase-meditated actin polymerization, resulting in AMPA-R trafficking to synapses (28).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [28] Matus A (2005) Growth of dendritic spines: a continuing story. Curr Opin Neurobiol 15(1):67-72. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 3

    Early studies suggested GEF-H1 could activate both RhoA and Rac1.

    “Early studies of GEF-H1 suggested that GEF-H1 could activate both RhoA and Rac1 (7, 12, 13).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [7]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [12]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [13]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 4

    The slight increase in RhoA activity by Y27632 may be because of negative feedback.

    “possibly because of negative feedback.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § Discussion · p. 5

    Full-length human GEF-H1 and mouse Lfc have the same size as our GEF-H1 cDNA.

    “Previous studies showed that full-length human GEF-H1 and mouse Lfc have the same size as our GEF-H1 cDNA (9, 15).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [9]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [15]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § AMPA receptor and GEF-H1/Lfc complex regulates dendritic spine development through RhoA signaling cascade · p. 1

    GEF-H1 binding to microtubules regulates its enzymatic activity.

    “GEF-H1 binding to microtubule regulates its enzymatic activity, suggesting GEF-H1 could mediate cross-talk between microtubules and actin (9, 10).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [9]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [10]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Interpretationown data
    § Identification of GEF-H1 as a Component of the AMPA-R Complex. · p. 2

    The efficiency of co-IP between GEF-H1 and AMPA-R subunits in heterologous cell lysate was much lower than that with brain or neuronal lysates, suggesting the interaction may be stabilized by other proteins or posttranslational modifications in neurons.

    “However, the efficiency of this co-IP with heterologous cell lysate was much lower than that with brain or neuronal lysates, suggesting that the interaction may be stabilized by other proteins or by posttranslational modifications in neurons.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 5

    AMPA-R activity is involved in the stabilization of spines.

    “Previous studies also demonstrated that AMPA-R activity is involved in the stabilization of spines (2, 22).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [2]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [22] Nimchinsky EA, Sabatini BL, Svoboda K (2002) Structure and function of dendritic spines. Annu Rev Physiol Annu Rev Physiol 64:313-353. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning
    § Discussion · p. 5

    The difference in expression time of exogenous GFP-GEF-H1 might be responsible for the different subcellular location of GEF-H1.

    “This difference in expression time of exogenous GFP-GEF-H1 might be responsible for the different subcellular location of GEF-H1.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 5

    Inhibition of AMPA-R activity results in the activation of GEF-H1 and the modulation of spine development.

    “All together, these results strongly suggest that inhibition of AMPA-R activity results in the activation of GEF-H1 and the modulation of spine development.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 2

    Dendritic spine development starts after 14 days in vitro in cultured dissociated neurons.

    “Because previous studies showed that dendritic spine development starts after 14 days in vitro (DIV) in cultured dissociated neurons (11)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [11]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § AMPA receptor and GEF-H1/Lfc complex regulates dendritic spine development through RhoA signaling cascade · p. 1

    Actin dynamics is regulated by Rho GTPases in response to external cues in various cell types including neurons.

    “In various cell types including neurons, actin dynamics is regulated by Rho GTPases in response to external cues (1).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Interpretationreasoning
    § Discussion · p. 6

    GEF-H1 may be one of the common regulators of functional and structural plasticity because AMPA-R trafficking is regulated by actin dynamics that is regulated by GEF-H1 activity.

    “GEF-H1 may be one of the common regulators because AMPA-R trafficking is regulated by actin dynamics that is regulated by GEF-H1 activity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data
    § Identification of GEF-H1 as a Component of the AMPA-R Complex. · p. 2

    A GluR1 mutant lacking the cytoplasmic C-terminal (R1CtΔ) was coimmunoprecipitated with GFP-GEF-H1, indicating that the C-terminal of AMPA-R is not required for GEF-H1 binding.

    “Surprisingly, a GluR1 mutant lacking the cytoplasmic C-terminal (R1Ct 𝚫 ) was also coimmunoprecipitated with GFP-GEF-H1, indicating that the C-terminal of AMPA-R is not required for GEF-H1 binding (see Fig. 1 D ).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data
    § Identification of GEF-H1 as a Component of the AMPA-R Complex. · p. 2

    GluR6 was not coimmunoprecipitated with GFP-GEF-H1, indicating that the interaction between AMPA-Rs and GEF-H1 is specific.

    “On the other hand, GluR6 (a subunit of kainate receptors) was not coimmunoprecipitated with GFP-GEF-H1, indicating that the interaction between AMPA-Rs and GEF-H1 is specific (Fig. S2).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § AMPA receptor and GEF-H1/Lfc complex regulates dendritic spine development through RhoA signaling cascade · p. 1

    Expression of GEF proteins is tissue-specific, providing a molecular mechanism for tissue-specific modulation of Rho GTPases.

    “The expression of GEF proteins is tissue-specific, providing a molecular mechanism for tissue-specific modulation of Rho GTPases (3).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [3]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion · p. 5

    Neurological disorders such as mental retardation are associated with defects in dendritic spine morphology and number of dendritic spines.

    “Many numbers of neurological disorders, such as mental retardation, are associated with defects in dendritic spine morphology and number of dendritic spines (25).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [25] Halpain S, Spencer K, Graber S (2005) Dynamics and pathology of dendritic spines. Prog Brain Res 147:29-37. source

  • Classified as probably not a substantial claim.

    Study design and conductreasoning
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 4

    For quantification of spine development, only neurons showing microtubule-like structure of GEF-H1 were selected as neurons over-expressing GEF-H1.

    “Therefore, for our quantification of spine development shown in Fig. 4, only the neurons showing microtubule-like structure of GEF-H1 were selected as neurons over-expressing GEF-H1.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation
    § GEF-H1 Negatively Regulates the Development of Dendritic Spines. · p. 5

    AMPA-R activity regulates the development of dendrites through Rho GTPases.

    “It has been previously reported that AMPA-R activity regulates the development of dendrites through Rho GTPases (2, 22).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [2]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [22] Nimchinsky EA, Sabatini BL, Svoboda K (2002) Structure and function of dendritic spines. Annu Rev Physiol Annu Rev Physiol 64:313-353. source

  • May combine two claims.

    Interpretationreasoning
    § Discussion · p. 6

    AMPA-R activation by local presynaptic inputs could negatively regulate the RhoA signaling pathway, resulting in an increase in spine density and strengthening local connections with the presynaptic inputs.

    “Similarly, for spine development, AMPA-R activation by local presynaptic inputs could negatively regulate the RhoAsignaling pathway, resulting in an increase in spine density and strengthening local connections with the presynaptic inputs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data
    § Discussion · p. 5

    AMPA-R activity negatively regulates GEF-H1 activity, resulting in inhibition of the synaptic RhoA signaling cascades.

    “Our data also strongly indicate that AMPA-R activity negatively regulates GEF-H1 activity, resulting in inhibition of the synaptic RhoA signaling cascades.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation
    § Discussion · p. 6

    Glutamate receptor activation by visual stimulation negatively regulated the RhoA signaling pathway, resulting in dendritic arbor growth.

    “Glutamate receptor activation by visual stimulation negatively regulated the RhoA signaling pathway, resulting in dendritic arbor growth (2).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [2]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Premisecitation
    § AMPA receptor and GEF-H1/Lfc complex regulates dendritic spine development through RhoA signaling cascade · p. 1

    RhoA and Rac1 are key players for regulation of spine and dendrite development and dynamics.

    “Particularly, RhoA and Rac1 have been known as key players for the regulation of spine and dendrite development and dynamics (2).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [2]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § AMPA receptor and GEF-H1/Lfc complex regulates dendritic spine development through RhoA signaling cascade · p. 1

    Tiam1 is a neuronal GEF involved in NMDA receptor activity-dependent structural plasticity.

    “Tiam1 is known as a neuronal GEF involved in NMDA receptor activity-dependent structural plasticity (4).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [4]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning
    § Discussion · p. 6

    Regulation of the RhoA signaling pathway by GEF-H1 could also be involved in NMDA receptor-mediated AMPA-R trafficking in and out of synapses.

    “Regulation of the RhoA signaling pathway by GEF-H1 could also be involved in this NMDA receptor-mediated AMPA-R trafficking in and out of synapses.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation
    § AMPA receptor and GEF-H1/Lfc complex regulates dendritic spine development through RhoA signaling cascade · p. 1

    Modulation of trafficking and activity of AMPA-R by NMDA receptor activity is a major mechanism for synaptic plasticity.

    “Modulation of the trafficking and activity of AMPA-R by NMDA receptor activity is a major mechanism for synaptic plasticity (5).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [5]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

43 of 43

  • The paper relies on this without support.

    Neuronal GEFs involved in AMPA receptor activity-dependent structural plasticity have not been reported.

    “However, neuronal GEFs involved in AMPA receptor (AMPA-R) activity-dependent structural plasticity have not been reported.”

  • The authors state this as an assumption.

    The T247A mutant of GEF-H1 acts as a dominant-negative inhibitor of GEF-H1.

    “A dominant-negative form of GEF-H1 was generated by introducing a point mutation (T247A) on the catalytic domain (Rho GEF domain) of GEF-H1 (see SI Methods for detail).”
2 of 2

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure 3 caption reverses the reported RhoA response to GEF-H1 knock-down

    Major

    The Figure 3 caption says GEF-H1 shRNA increased active RhoA, whereas the Results text says the same knock-down significantly decreased RhoA activity. This reverses a result central to the proposed signaling pathway.

1 of 1

Cites

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Related

Authors
Kang, M.-G., Guo, Y., & Huganir, R. L.
Affiliations
  • Howard Hughes Medical Institute (US)
  • Johns Hopkins University (US)
Corresponding author
Huganir, Richard L.
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2009-03-03
Volume
106
Issue
9
Pages
3549-3554
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.0812861106
PMC
PMC2638734
OpenAlex
W2017885748
Cited by
82
References
36
Citations by year
2026: 4 · 2025: 2 · 2024: 2 · 2023: 2 · 2022: 2 · 2021: 3 · 2020: 5 · 2019: 2 · 2018: 10 · 2017: 5 · 2016: 7 · 2015: 4 · 2014: 5 · 2013: 8 · 2012: 7
FWCI
2.90
Type
journal article
Language
en
Open access
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • Howard Hughes Medical Institute
  • National Institutes of Health (n01hv28180-13-0-0, N01-HV-28180)
  • National Heart, Lung, and Blood Institute (N01 HV-28180, N01 HV028180)
  • National Institute of Neurological Disorders and Stroke (R01NS036715)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetics and Neurodevelopmental Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
Keywords
AMPA receptors, GEF-H1, dendritic spine development, RhoA signaling, Rho GTPases, Rac1, guanine nucleotide exchange factor, postsynaptic density, GluR1, synaptic plasticity, dendritic spine morphology
MeSH terms
Animals, Protein Binding, Signal Transduction, Down-Regulation, Receptors, AMPA, rhoA GTP-Binding Protein, RNA Interference, Dendritic Spines, Rats, Rho Guanine Nucleotide Exchange Factors

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-02.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-02.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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