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Cited by 43Neuroscience and Neuropharmacology ResearchMemory and Neural MechanismsGenetics and Neurodevelopmental Disorders

Arf4 Determines Dentate Gyrus-Mediated Pattern Separation by Regulating Dendritic Spine Development

Open access
Jain, Sachi, Yoon, Seo Yeon, Zhu, Lei, Brodbeck, Jens, Dai, Jessica, Walker, David, Huang, Yadong·2012·PLoS ONE
DOI 10.1371/journal.pone.0046340PMC PMC3457985
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Abstract

The ability to distinguish between similar experiences is a critical feature of episodic memory and is primarily regulated by the dentate gyrus (DG) region of the hippocampus. However, the molecular mechanisms underlying such pattern separation tasks are poorly understood. We report a novel role for the small GTPase ADP ribosylation factor 4 (Arf4) in controlling pattern separation by regulating dendritic spine development. Arf4(+/-) mice at 4-5 months of age display severe impairments in a pattern separation task, as well as significant dendritic spine loss and smaller miniature excitatory post-synaptic currents (mEPSCs) in granule cells of the DG. Arf4 knockdown also decreases spine density in primary neurons, whereas Arf4 overexpression promotes spine development. A constitutively active form of Arf4, Arf4-Q71L, promotes spine density to an even greater extent than wildtype Arf4, whereas the inactive Arf4-T31N mutant does not increase spine density relative to controls. Arf4's effects on spine development are regulated by ASAP1, a GTPase-activating protein that modulates Arf4 GTPase activity. ASAP1 overexpression decreases spine density, and this effect is partially rescued by concomitant overexpression of wildtype Arf4 or Arf4-Q71L. In addition, Arf4 overexpression rescues spine loss in primary neurons from an Alzheimer's disease-related apolipoprotein (apo) E4 mouse model. Our findings suggest that Arf4 is a critical modulator of DG-mediated pattern separation by regulating dendritic spine development.

AI snapshot

Written from the paper’s full text.

TL;DR

Arf4 haploinsufficiency in mice impairs dentate gyrus–dependent pattern separation and reduces DG granule-cell dendritic spines and mEPSC amplitude. In primary neurons, Arf4 promotes spine development through its activity state and is negatively regulated by ASAP1, and Arf4 overexpression rescues spine loss in an apoE4 model.

Key findings

  • Arf4+/− mice at 4–5 months showed no preference for an object in a novel context during pattern separation testing, unlike WT mice, but were not impaired in Morris water maze spatial learning/memory, open field activity, rotarod motor coordination, or elevated plus maze anxiety (12–13 mice per genotype per sex).
  • Arf4 protein was reduced by 49% in Arf4+/− hippocampus; DG granule cells showed significant decreases in total and mushroom spine density, while CA1 spine density/morphology was unchanged; mEPSC amplitude was 37% lower in Arf4+/− DG granule cells, with no change in mEPSC frequency (n = 9 Arf4+/− vs. 6 WT cells at 2 months).
  • In primary neurons, Arf4 overexpression increased spine density at DIV12, DIV14, and DIV19; constitutively active Arf4-Q71L promoted spine density more than WT Arf4, whereas inactive Arf4-T31N did not increase total spine density and reduced mushroom spine density. Arf4 shRNA knockdown decreased total spine density and all subtypes except filopodia, and human Arf4-HA rescued this effect.
  • ASAP1 overexpression decreased spine density and stubby/mushroom spines; co-expression with Arf4-WT or Arf4-Q71L partially rescued this. Arf4 overexpression fully restored spine loss in NSE-apoE4 neurons; Arf4 mRNA was 36% lower in hippocampi from 10-month-old female NSE-apoE4 vs. NSE-apoE3 mice (p = 0.015). BDNF levels were unchanged in Arf4+/− mice or Arf4-overexpressing neurons.

Methods

  • Arf4+/− mice were generated from a BayGenomics CSH658 gene-trapped ES cell line; Arf4−/− mice were embryonically lethal, so in vivo studies used Arf4+/− and WT littermates backcrossed at least 5 generations to C57Bl6. Behavioral testing at 4–5 months included pattern separation, Morris water maze, open field, rotarod, and elevated plus maze, with blinded analysis.
  • Spine morphology was assessed by Golgi-Cox staining in CA1 and DG (n = 4 mice/genotype; 28–30 neurons/genotype), and mEPSCs were recorded from DG granule cells at 2 months (n = 9 Arf4+/− vs. 6 WT cells). Primary mouse hippocampal/cortical neurons were transfected at DIV5 and imaged at DIV12, DIV14, or DIV19 for spine density and subtype analysis.
  • Statistical comparisons used mean ± SEM, t-test or one-way ANOVA with Bonferroni post-hoc test, with p < 0.05 considered significant.

Limitations

  • The paper does not state limitations explicitly. Evident from the text: Arf4−/− mice are embryonically lethal, so all in vivo work uses Arf4+/− mice; no conditional or inducible knockout is used, leaving developmental versus acute Arf4 functions unresolved.
  • Evident from the text: in vivo behavioral/spine data are from 4–5-month-old mice, while electrophysiology is from 2-month-old mice, and no in vivo rescue of the pattern separation deficit by Arf4 or ASAP1 manipulation is shown.
  • Evident from the text: the mechanistic spine findings rely largely on overexpression and shRNA knockdown in primary neurons, and the link between DG spine loss and pattern separation impairment remains correlative rather than causally established in vivo.

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

Integrity signals

Registry

Crossref · 10.1371/journal.pone.0046340

Cites

69 of 69 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

30 of 69 cited sources checked · 15 supported · 3 flagged · accuracy not yet measured

  • Premisecitation

    Some aged rodents that do not develop AD pathology nevertheless have memory deficits, such as a failure to encode novel information while navigating similar situations.

    “Some aged rodents that do not develop AD pathology nevertheless have memory deficits, such as a failure to encode novel information while navigating similar situations [69].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [69] Wilson IA, Ikonen S, Gureviciene I, McMahan RW, Gallagher M, et al (2004) Cognitive aging and the hippocampus: how old rats represent new environments. J. Neurosci 24: 3870– 3878. 15084668 PMC6729357 source

      The Experiment 1 Spatial learning section reports impaired spatial memory in some aged rats, and Representation of a novel environment reports that some initially retained a familiar spatial representation in a novel arena. But the arenas differed in shape and salient cues rather than being similar, the deficit was initially delayed encoding rather than a lasting failure, and the paper does not establish that the rats lacked AD pathology. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premisecitation

    Patients with amnestic mild cognitive impairment (MCI) showed an impaired ability to distinguish between a previously seen object and a very similar but unobserved object.

    “Patients with amnestic mild cognitive impairment (MCI), for instance, showed an impaired ability to distinguish between a previously seen object and a very similar but unobserved object [68].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [68] Yassa MA, Stark SM, Bakker A, Albert MS, Gallagher M, et al (2010) High-resolution structural and functional MRI of hippocampal CA3 and dentate gyrus in patients with amnestic mild cognitive impairment. NeuroImage 51: 1242– 1252. 20338246 PMC2909476 source
  • Findingcitation

    The dendritic spine density of NSE-apoE4 neurons was significantly less than that of either NSE-apoE3 or WT neurons.

    “Consistent with previous findings [21], the dendritic spine density of NSE-apoE4 neurons (Figs. 12E and E′) was significantly less than that of either NSE-apoE3 (Figs. 12D and D′) or WT neurons (Figs. 12C and C′).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [21] Brodbeck J, McGuire J, Liu Z, Meyer-Franke A, Balestra ME, et al (2011) Structure-dependent impairments of intracellular apolipoprotein E4 trafficking and its detrimental effects are rescued by small-molecule structure correctors. J Biol Chem 286: 17217– 17226. 21454574 PMC3089564 source

      The dendritic-spine development Results section reports significantly lower spine density in hippocampal neurons from NSE-apoE4 mice than in those from NSE-apoE3 mice (Fig. 11). The study does not report a dendritic-spine comparison with WT neurons, so that part of the attributed claim is unsupported by this source. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
  • Premisecitation

    Spine loss is strongly correlated with cognitive impairments in AD.

    “As spine loss is strongly correlated with cognitive impairments in AD [15], [66]”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [15] Penzes P, Cahil ME, Jones KA, VanLeeuwen JE, Wolfrey KM (2011) Dendritic spine pathology in neuropsychiatric disorders. Nat Neurosci 14: 285– 292. 21346746 PMC3530413 source
    • cites [66] Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, et al (1991) Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment. Ann Neurol 30: 572– 580. 1789684 source
  • Findingown data

    Arf4 knockdown decreases spine density in primary neurons.

    “Arf4 knockdown also decreases spine density in primary neurons”
    • Also stated in: § Introduction · plain
      “knocking down Arf4 decreases spine density in primary neurons”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 28
  • May combine two claims. 26
  • The quote only partly states this claim. 2

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

    Premisecitation

    Previous studies have reported seemingly contradictory roles for GDP-bound Arf proteins (Arf-T31N) as either inactive mutants whose physiological effects do not differ from wildtype Arfs, or as dominant-negative mutants with inhibitory effects on vesicular transport and other cellular functions.

    “Previous studies have reported seemingly contradictory roles for GDP-bound Arf proteins (Arf-T31N) as either inactive mutants whose physiological effects do not differ from wildtype Arfs [57], [58], or as dominant-negative mutants with inhibitory effects on vesicular transport and other cellular functions [59].”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [57] Chen YG, Shields D (1996) ADP-ribosylation factor-1 stimulates formation of nascent secretory vesicles from the trans-Golgi network of endocrine cells. J Biol Chem 271: 5297– 5300. 8621377 source

      Split into 2 atoms — 0 confirmed, 1 partially supported, 1 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [58] Islam A, Shen X, Hiroi T, Moss J, Vaughan M, et al (2007) The brefeldin A-inhibited guanine nucleotide-exchange protein, BIG2, regulates the constitutive release of TNFR1 exosome-like vesicles. J Biol Chem 282: 9591– 9599. 17276987 source

      Split into 2 atoms — 0 confirmed, 1 partially supported, 1 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [59] Dascher C, Balch WE (1994) Dominant inhibitory mutants of ARF1 block endoplasmic reticulum to Golgi transport and trigger disassembly of the Golgi apparatus. J Biol Chem 269: 1437– 1448. 8288610 source

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

    Premisecitation

    Decreases in both thin and mushroom spine types have been reported in animal models of cognitive decline.

    “Decreases in both spine types have been reported in animal models of cognitive decline [54], [55].”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [54] Dumitriu D, Hao J, Hara Y, Kaufmann J, Janssen WG, et al (2010) Selective changes in thin spine density and morphology in monkey prefrontal cortex correlate with aging-related cognitive impairment. J Neurosci 30: 7507– 7515. 20519525 PMC2892969 source

      The Results (Effects of aging on dendritic spine morphology) report a 45.6% decrease in thin spine density but no statistically significant difference in mushroom spine density. The Discussion (Synaptic basis for cognitive aging) explicitly says mushroom spine density was unchanged in aged versus young monkeys, contrary to the claim that both types decreased. (LLM, conf=0.90)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [55] Perez-Cruz C, Nolte MW, van Gaalen MM, Rustay NR, Termont A, et al (2011) Reduced spine density in specific regions of CA1 pyramidal neurons in two transgenic mouse models of Alzheimer's disease. J Neurosci 31: 3926– 3934. 21389247 PMC6622797 source

      The ‘Density of synaptic spines in CA1 of the hippocampus’ section reports reduced mushroom-like spine density in two transgenic mouse lines with cognitive deficits. The paper counted only mushroom-like spines, so it does not support a decrease in thin spines. (LLM, conf=0.90)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Members of the ARF family of small GTPases and their regulators are important regulators of spine density and morphology.

    “Members of the ADP-ribosylation factor (ARF) family of small GTPases and their regulators have gained increasing attention as important regulators of spine density and morphology [27], [28], [29].”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [27] Choi S, Ko J, Lee JR, Lee HW, Kim K, et al (2006) ARF6 and EFA6A regulate the development and maintenance of dendritic spines. J Neurosci 26: 4811– 4819. 16672654 PMC6674147 source
    • cites [28] Sakagami H, Sanda M, Fukaya M, Miyazaki T, Sukegawa J, et al (2008) IQ-Arf4GEF/BRAG1 is a guanine nucleotide exchange factor for Arf6 that interacts with PSD-95 at postsynaptic density of excitatory synapses. Neurosci Res 60: 199– 212. 18164504 source
    • cites [29] Moore CD, Thacker EE, Larimore J, Gaston D, Underwood A, et al (2007) The neuronal Arf GAP centaurin alpha1 modulates dendritic differentiation. J Cell Sci 120: 2683– 2693. 17635995 PMC2810648 source

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

    Premisecitation

    Aberrations in spine density and morphology are associated with a number of neurological disorders, including Alzheimer's disease.

    “Aberrations in spine density and morphology are associated with a number of neurological disorders, including Alzheimer's disease (AD) [15], [16], [17].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [15] Penzes P, Cahil ME, Jones KA, VanLeeuwen JE, Wolfrey KM (2011) Dendritic spine pathology in neuropsychiatric disorders. Nat Neurosci 14: 285– 292. 21346746 PMC3530413 source
    • cites [16] Fiala JC, Spacek J, Harris KM (2002) Dendritic spine pathology: cause or consequence of neurological disorders? Brain Res Rev 29: 29– 54. 12086707 source
    • cites [17] Tackenberg C, Ghori A, Brandt R (2009) Thin, stubby or mushroom: spine pathology in Alzheimer's disease. Curr Alzheimer Res 6: 261– 268. 19519307 source

  • May combine two claims.

    Premisecitation

    The DG subregion of the hippocampus mediates episodic memory formation and disambiguation of similar but discrete events (pattern separation).

    “The dentate gyrus (DG) subregion of the hippocampus mediates episodic memory formation and the disambiguation of similar but discrete events, a phenomenon known as pattern separation [3], [4], [5], [6].”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [3] Schmidt B, Marrone DF, Markus EJ (2011) Disambiguating the similar: the dentate gyrus and patern separation. Behav Brain Res 226: 56– 65. 21907247 source
    • cites [4] Aimone JB, Deng W, Gage FH (2011) Resolving new memories: a critical look at the dentate gyrus, adult neurogenesis, and pattern separation. Neuron 70: 589– 596. 21609818 PMC3240575 source
    • cites [5] Treves A, Tashiro A, Witter ME, Moser EI (2008) What is the mammalian dentate gyrus good for? Neuroscience 154: 1155– 1172. 18554812 source

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

    Premisecitation

    The hippocampus plays a central role in encoding and storage of spatial and episodic memory.

    “the hippocampus plays a central role in the encoding and storage of certain types of memory, including spatial and episodic memory [1], [2].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [1] Deng W, Aimone JB, Gage FH (2010) New neurons and new memories: how does adult hippocampal neurogenesis affect learning and memory? Nat Rev Neurosci 11: 339– 350. 20354534 PMC2886712 source
    • cites [2] Squire LR, Stark CE, Clark RE (2004) The medial temporal lobe. Annu Rev Neurosci 27: 279– 306. 15217334 source

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

    Premisecitation

    Apolipoprotein E4 is associated with spatial learning and memory impairments.

    “and is associated with spatial learning and memory impairments [22].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [22] Andrews-Zwilling Y, Bien-Ly N, Xu Q, Li G, Bernardo A, et al (2010) Apolipoprotein E4 causes age-and tau-dependent impairment of GABAergic interneurons, leading to learning and memory deficits in mice. J Neurosci 30: 13707– 13717. 20943911 PMC2988475 source

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

    Premisecitation

    Apolipoprotein E4 decreases spine density both in vivo and in vitro.

    “apolipoprotein (apo) E4–a major genetic risk factor for Alzheimer's disease–decreases spine density both in vivo [18], [19] and in vitro [20], [21]”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [18] Dumanis SB, Tesoriero JA, Babus LW, Nguyen MT, Trotter JH, et al (2009) ApoE4 decreases spine density and dendritic complexity in cortical neurons in vivo. J Neurosci 29: 15317– 15322. 19955384 PMC2846754 source
    • cites [19] Ji Y, Gong Y, Gan W, Beach T, Holtzman DM, et al (2003) Apolipoprotein E isoform-specific regulation of dendritic spine morphology in apolipoprotein E transgenic mice and Alzheimer's disease patients. Neuroscience 122: 305– 315. 14614898 source
    • cites [20] Brodbeck J, Balestra ME, Saunders AM, Roses AD, Mahley RW, et al (2008) Rosiglitazone increases dendritic spine density and rescues spine loss caused by apolipoprotein E4 in primary cortical neurons. Proc Natl Acad Sci USA 105: 1343– 1346. 18212130 PMC2234140 source

  • Classified as probably not a substantial claim.

    Premisecitation

    Small GTPases play a critical role in neuronal trafficking and spine morphogenesis.

    “small GTPases play a critical role in neuronal trafficking and spine morphogenesis [23], [24], [25], [26].”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [23] Tada T, Sheng M (2006) Molecular mechanisms of dendritic spine morphogenesis. Curr Opin Neurobiol 16: 95– 101. 16361095 source
    • cites [24] Lippman J, Dunaevsky A (2005) Dendritic spine morphogenesis and plasticity. J Neurobiol 64: 47– 57. 15884005 source
    • cites [25] Tolias KF, Duman JG, Um K (2011) Control of synapse development and plasticity by Rho GTPase regulatory proteins. Prog Neurobiol 94: 133– 148. 21530608 PMC3129138 source

  • May combine two claims.

    Premisecitation

    Overexpression of GFP-β-actin does not impair neuronal function or dendritic spine morphology and density.

    “Overexpression of GFP-β-actin does not impair neuronal function or dendritic spine morphology and density and can, therefore, be used to highlight dendritic spines for imaging and quantification [29], [45].”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [29] Moore CD, Thacker EE, Larimore J, Gaston D, Underwood A, et al (2007) The neuronal Arf GAP centaurin alpha1 modulates dendritic differentiation. J Cell Sci 120: 2683– 2693. 17635995 PMC2810648 source

      The Materials and Methods describe using eYFP to visualize dendrites and spines, not GFP-β-actin. The Results compare spine density after centaurin α1 expression, but do not assess whether GFP-β-actin overexpression affects neuronal function or spine morphology and density. Searches for GFP-β-actin and GFP-actin found no passage addressing that claim; page numbers were unavailable in our copy. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [45] Morales M, Colicos MA, Goda Y (2000) Actin-dependent regulation of neurotransmitter release at central synapses. Neuron 27: 539– 550. 11055436 source

      The Results (page unavailable) show that GFP–β-actin was expressed in hippocampal neurons and visualized puncta on their dendrites, supporting its use as an imaging marker. They do not establish that overexpression leaves neuronal function or dendritic spine morphology and density unimpaired; the reported electrophysiology and morphology comparisons concern latrunculin A treatment, not GFP–β-actin overexpression. (LLM, conf=0.79)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Granule cells of the DG are covered by dendritic spines that are important loci of synaptic plasticity.

    “granule cells of the DG are covered by dendritic spines that are important loci of synaptic plasticity [9], [10], [11], [12].”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [9] Yuste R (2011) Dendritic spines and distributed circuits. Neuron 71: 772– 780. 21903072 PMC4071954 source

      The section “Spines enable and regulate input-specific plasticity” describes spines as sites where changes in synaptic strength can occur. The Introduction describes dendritic trees covered with spines, but the paper does not establish that dentate gyrus granule cells specifically are covered with them. Page numbers were not provided in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [10] Hotulainen P, Hoogenraad CC (2010) Actin in dendritc spines: connecting dynamics to function. J Cell Biol 189: 619– 627. 20457765 PMC2872912 source

      The Introduction, under “Dendritic spine structure and function” (page unavailable), says hippocampal neurons can have thousands of dendritic spines and describes their role in regulating synaptic strength. It does not specifically identify dentate gyrus granule cells as covered by spines, so the cell-specific part of the claim is unsupported by this source. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [11] Hering H, Sheng M (2001) Dendritic spines: structure, dynamics, and regulation. Nat Rev Neurosci 2: 880– 888. 11733795 source

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

    Premisecitation

    Small GTPases modulate actin cytoskeletal rearrangement and dynamics in both neuronal and non-neuronal cells.

    “Small GTPases modulate actin cytoskeletal rearrangement and dynamics in both neuronal and non-neuronal cells [10], [60]”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [10] Hotulainen P, Hoogenraad CC (2010) Actin in dendritc spines: connecting dynamics to function. J Cell Biol 189: 619– 627. 20457765 PMC2872912 source
    • cites [60] Sekino Y, Kojima N, Shirao T (2007) Role of actin cytoskeleton in dendritic spine morphogenesis. Neurochem Int 51: 92– 104. 17590478 source

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

    Premisecitation

    BDNF plays critical roles in both pattern separation and dendritic spine development.

    “Since brain-derived neurotrophic factor (BDNF) plays critical roles in both pattern separation [47] and dendritic spine development [48], [49]”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [47] Beckinschtein P, Oomen CA, Saksida LM, Bussey TJ (2011) Effects of environmental enrichment and voluntary exercise on neurogenesis, learning and memory, and pattern separation: BDNF as a critical variable? Semin Cell Dev Biol 22: 536– 542. 21767656

      Not checked in the free run.

    • cites [48] Vigers AJ, Amin DS, Talley-Farnham T, Gorski JA, Xu B, et al (2012) Sustained expression of brain-derived neurotrophic factor is required for maintenance of dendritic spines and normal behavior. Neuroscience 212: 1– 18. 22542678 PMC4880414 source
    • cites [49] Kaneko M, Xie Y, An JJ, Stryker MP, Xu B (2012) Dendritic BDNF synthesis is required for late-phase spine maturation and recovery of cortical responses following sensory deprivation. J Neurosci 32: 4790– 4802. 22492034 PMC3356781 source

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

    Premisecitation

    Mossy fiber "detonator" synapses arising from DG granule cells strongly activate the CA3 region and are involved in a variety of neurological functions, including memory and spatial representations.

    “Mossy fiber “detonator” synapses arising from DG granule cells strongly activate the CA3 region and are involved in a variety of neurological functions, including memory and spatial representations [6], [64].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [6] Yassa MA (2011) Pattern separation in the hippocampus. Trends Neurosci 34: 515– 525. 21788086 PMC3183227 source
    • cites [64] Palmer A, Good M (2011) Hippocampal synaptic activity, patterns separation and episodic like memory: implications for mouse models of Alzheimer's disease pathology. Biochem Soc Trans 39: 902– 909. 21787321 source

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

    Premisecitation

    Spine head size is positively correlated with mEPSC amplitude, and the functional loss of NMDA receptors in DG granule cells has been associated with impairments in pattern separation.

    “Spine head size is positively correlated with mEPSC amplitude [65], and the functional loss of NMDA receptors in DG granule cells has been associated with impairments in pattern separation [8].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [65] Matsuzaki M, Ellis-Davies GC, Nemoto T, Miyashita Y, Lino M, et al (2001) Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. Nat Neurosci 4: 1086– 1092. 11687814 PMC4229049 source

      The Results report a positive correlation between spine-head volume and the amplitude of responses evoked by uncaged glutamate, not a measured correlation with mEPSC amplitude. The paper discusses mEPSCs separately but does not establish the specific correlation attributed to citation [65]. The DG granule-cell claim is attached to citation [8] and is not judged here. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [8] McHugh TJ, Jones MW, Quinn JJ, Balthasar N, Coppari R, et al (2007) Dentate gyrus NMDA receptors mediate rapid pattern separation in the hippocampal network. Science 317: 94– 99. 17556551 source

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

    Premisecitation

    Arf activation promotes recruitment of several actin-regulatory proteins, including cortactin and dynamin, to the vesicle-budding sites of the trans-Golgi network (TGN).

    “Recent studies have shown that Arf activation promotes recruitment of several actin-regulatory proteins, including cortactin and dynamin, to the vesicle-budding sites of the trans-Golgi network (TGN) [62], [63].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [62] Cao H, Weller S, Orth JD, Chen J, Huang B, et al (2005) Actin and Arf1-dependent recruitment of a cortactin-dynamin complex to the Golgi regulates post-Golgi transport. Nat Cell Biol 7: 483– 492. 15821732 source
    • cites [63] Carreno S, Enggvist-Goldstein AE, Zhang CX, McDonald KL, Drubin DG (2004) Actin dynamics coupled to clathrin-coated vesicle formation at the trans-Golgi network. J Cell Biol 165: 781– 788. 15210728 PMC2172402 source

      The Introduction (page not available in our copy) says that clathrin-coated vesicle formation at the TGN involves recruitment of dynamin. The Results and discussion place the actin-regulatory Arp2/3 complex on TGN-derived vesicles. Neither passage supports the more specific claim that Arf activation recruits cortactin and dynamin to those sites. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    NSE-apoE4 mice have impairments in learning and memory and a loss of dendritic spines in primary neurons and in the hippocampus and cortex.

    “These mice have impairments in learning and memory [50], as well as a loss of dendritic spines in primary neurons and in the hippocampus and cortex [21].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [50] Raber J, Wong D, Buttini M, Orth M, Bellosta S, et al (1998) Isoform-specific effects of human apolipoprotein E on brain function revealed in ApoE knockout mice: increased susceptibility of females. Proc Natl Acad Sci USA 95: 10914– 10919. 9724804 PMC27995 source
    • cites [21] Brodbeck J, McGuire J, Liu Z, Meyer-Franke A, Balestra ME, et al (2011) Structure-dependent impairments of intracellular apolipoprotein E4 trafficking and its detrimental effects are rescued by small-molecule structure correctors. J Biol Chem 286: 17217– 17226. 21454574 PMC3089564 source

      The dendritic-spine Results section (page not provided) reports significantly lower spine density in primary hippocampal neurons cultured from NSE-apoE4 mice than from NSE-apoE3 mice. It also refers to spine loss in the brains of transgenic mice, but does not specify loss in the hippocampus and cortex in vivo. The learning-and-memory clause is attached to a different citation and is not judged here. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Thin spines are more transient and motile than mushroom spines, and an increase in the proportion of thin spines represents a greater capacity to stabilize after LTP.

    “Previous studies have shown that thin spines are more transient and motile than mushroom spines, and an increase in the proportion of thin spines represents a greater capacity to stabilize after LTP [52], [53].”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [52] Holtmaat AJ, Trachtenberg JT, Wilbrecht L, Shepherd GM, Zhang X, et al (2005) Transient and persistent dendritic spines in the neocortex in vivo. Neuron 45: 279– 291. 15664179 source

      The spine-morphology passages (Figure 7B and 7D; page not supplied) associate thin spines with turnover and changing length, while persistent spines tend to be thicker, including mushroom-shaped spines. The paper only suggests that thin spines might stabilize and enlarge under some conditions; it does not show that a higher proportion of thin spines means greater capacity to stabilize after LTP. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [53] Zuo Y, Lin A, Chang P, Gan WB (2005) Development of long-term dendritic spine stability in diverse regions of cerebral cortex. Neuron 46: 181– 189. 15848798 source

      The Discussion (page unavailable) distinguishes rapidly turning-over, thin filopodia from spines; it does not compare thin spines with mushroom spines. A search for LTP and stabilization found no passage linking a higher proportion of thin spines to greater capacity to stabilize after LTP. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Arf4 is expressed in the hippocampi of 4.5-month-old mice, as well as in primary neurons and Neuro-2a neuroblastoma cells.

    “Arf4 is expressed in the hippocampi of 4.5-month-old mice, as well as in primary neurons and Neuro-2a (N2a) neuroblastoma cells (Figs. 6A–C), as determined by western blots.”
    Quote in the paperSubstantial claim

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

    Findingown data

    Similar increases in spine density were observed using Arf4-mCherry in place of Arf4-HA.

    “We observed similar increases in spine density in GFP-β-actin co-transfection experiments using Arf4-mCherry in the place of Arf4-HA.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Arf4+/− mice were not impaired in locomotor and exploratory activity, motor coordination, or anxiety-related behaviors.

    “Arf4+/− mice were also not impaired in locomotor and exploratory activity (open field test) (Figs. 2D–F), motor coordination (rotarod) (Fig. 2G), or anxiety-related behaviors (elevated plus maze) (Figs. 2H and I).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Arf4+/− mice at 4-5 months of age have significant dendritic spine loss in granule cells of the DG.

    “there was a significant decrease in total spine density in the granule cells of the DG (Figs. 5A and B)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Arf4+/− mice are fertile, viable and show no overt phenotype.

    “Arf4+/− mice were fertile, viable and showed no overt phenotype.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Overexpression of Arf4 did not significantly alter total dendrite number or length, nor the number or length of primary, secondary, or tertiary dendrites.

    “Overexpression of Arf4 did not significantly alter total dendrite number or length, nor did it change the number or length of primary, secondary, or tertiary dendrites (Figs. 6J–M).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Arf4+/− mice at 4-5 months of age have smaller mEPSCs in DG granule cells.

    “the amplitude of miniature excitatory postsynaptic currents (mEPSCs) was 37% lower in Arf4+/− DG granule cells than controls (Figs. 5D–F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Arf4 and its active mutant promoted stubby and thin spine development.

    “Arf4 and its active mutant also promoted stubby and thin spine development (Fig. 8C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The increase in spine density was significant across time points, whereas spine densities remained relatively constant throughout development for neurons transfected with GFP-β-actin alone.

    “Furthermore, this increase in spine density was significant across time points, whereas spine densities remained relatively constant throughout development for neurons transfected with GFP-β-actin alone (Fig. 7G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Arf4+/− mice did not show deficits in spatial learning or memory retention.

    “Arf4+/− mice did not show deficits in spatial learning (Fig. 2A) or memory retention (Figs. 2B and C)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mushroom spine numbers were significantly lower in the DG of Arf4+/− compared to WT mice, and this spine loss correlated with a reduction in mEPSC amplitude.

    “In our study, mushroom spine numbers were significantly lower in the DG of Arf4+/− compared to WT mice, and this spine loss correlated with a reduction in mEPSC amplitude.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Young (4-5 month old) Arf4+/− mice have pattern separation deficits that are similar to those found in pre-clinical MCI patients and aged rodents, but exhibit normal spatial learning and memory.

    “In our current study, it is intriguing that young (4–5 month old) Arf4+/− mice have pattern separation deficits that are similar to those found in pre-clinical MCI patients and aged rodents, but exhibit normal spatial learning and memory.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The loss of one copy of Arf4 in vivo leads to severe impairments in pattern separation, as well as a decrease in DG granule cell spine density and mEPSC amplitude.

    “The loss of one copy of Arf4 in vivo leads to severe impairments in pattern separation, as well as a decrease in DG granule cell spine density and mEPSC amplitude.”
    • Also stated in: § Role of Arf4 in dentate gyrus-dependent pattern separation tasks · plain
      “Our experiments showed that Arf4 heterozygosity in vivo leads to reductions in spine number and mEPSC amplitude in the DG, and these alterations accompany profound pattern separation impairments.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Neurons were selected randomly for spine analysis.

    “Neurons were selected randomly and 1 to 2 equivalent-length dendritic segments from each neuron were chosen for quantification of protrusions.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    All behavioral experiments and analyses were performed blind to genotype.

    “All experiments and analyses were performed blind to genotype.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Series resistance was constantly monitored and recordings discarded if changes >15%.

    “Series resistance (<30 MΩ) was constantly monitored and the recording was discarded if changes >15% occur.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Interpretationcitation

    Arf4 might play a role in DG-associated learning and memory tasks.

    “suggesting that Arf4 might play a role in DG-associated learning and memory tasks.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [31] Suzuki I, Owada Y, Suzuki R, Yoshimoto T, Kondo H (2001) Localization of mRNAs for six ARFs (ADP-ribosylation factors) in the brain of developing and adult rats and changes in the hypoglossal nucleus after its axotomy. Mol Brain Res 88: 124– 134. 11295238 source

  • May combine two claims.

    Interpretationown data

    Reducing Arf4 by 50% significantly impaired spine development and electrophysiological function of granule cells in the DG.

    “Thus, reducing Arf4 by 50% significantly impaired spine development and the electrophysiological function of granule cells in the DG.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Spine morphology correlates with synaptic strength and structural plasticity.

    “Spine morphology correlates with synaptic strength and structural plasticity; for instance, thin spines are highly motile and likely to respond to activity-induced changes, whereas mushroom spines have larger post-synaptic densities (PSDs) and are more stable [13], [14].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [13] Peebles CL, Yoo J, Thwin MT, Palop JJ, Noebels JL, et al (2010) Arc regulates spine morphology and maintains network stability in vivo. Proc Natl Acad Sci USA 107: 18173– 18178. 20921410 PMC2964216 source
    • cites [14] Bourne J, Harris KM (2007) Do thin spines learn to be mushroom spines that remember? Curr Opin Neurobiol 17: 381– 386. 17498943 source

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

    Study design and conductown data

    Golgi-stained brain sections were coded and analyzed in a blinded manner.

    “Images of the CA1 and DG were taken with a Leica CTR5000 brightfield 63X oil objective, coded, and analyzed in a blinded manner using ImageJ software.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    ASAP1 is a GAP that forms a complex with Arf4 and regulates GTP hydrolysis for Arf1, Arf4, and Arf5.

    “ASAP1, a GAP that was previously shown to form a complex with Arf4 and regulate GTP hydrolysis for Arf1, Arf4, and Arf5 [32], [33].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [32] Mazelova J, Astuto-Gribble L, Inoue H, Tam BM, Schonteich E, et al (2009) Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4. EMBO J 28: 183– 192. 19153612 PMC2637330 source
    • cites [33] Deretic D (2006) A role for rhodopsin in a signal transduction cascade that regulates membrane trafficking and photoreceptor polarity. Vision Res 46: 4427– 4433. 17010408 source

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

    Premisecitation

    Mushroom spines have larger postsynaptic densities (PSDs) and more glutamate receptors than thin spines, making the synapse functionally stronger and more stable.

    “Mushroom spines have larger postsynaptic densities (PSDs) and more glutamate receptors than thin spines, making the synapse functionally stronger and more stable [14], [23].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [14] Bourne J, Harris KM (2007) Do thin spines learn to be mushroom spines that remember? Curr Opin Neurobiol 17: 381– 386. 17498943 source
    • cites [23] Tada T, Sheng M (2006) Molecular mechanisms of dendritic spine morphogenesis. Curr Opin Neurobiol 16: 95– 101. 16361095 source

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

    Premisecitation

    ASAP1 functions as an Arf4 GAP and forms a complex with Arf4.

    “Previous studies have shown that ASAP1 functions as an Arf4 GAP and forms a complex with Arf4 [32].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [32] Mazelova J, Astuto-Gribble L, Inoue H, Tam BM, Schonteich E, et al (2009) Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4. EMBO J 28: 183– 192. 19153612 PMC2637330 source

  • May combine two claims.

    Premisecitation

    The GTP-bound form of Arf4 stably associates with intracellular organelle membranes and can activate downstream effectors.

    “The GTP-bound form of Arf4 stably associates with intracellular organelle membranes and can activate downstream effectors [56], [46].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [56] Duijsings D, Lanke KH, van Dooren SH, van Dommelen MM, Wetzels R, et al (2009) Differential membrane association properties and regulation of class I and class II Arfs. Traffic 10: 316– 323. 19170981 source
    • cites [46] Woo IM, Eun SY, Jang HS, Kang ES, Kim GH, et al (2009) Identification of ADP-ribosylation factor 4 as a suppressor of N-(4 hydroxyphenyl) retinamide-induced cell death. Cancer Letters 276: 53– 60. 19041174 source

  • Classified as probably not a substantial claim.

    Premisecitation

    Spine number and structural plasticity are tightly correlated with synaptic function in the mammalian brain.

    “Spine number and structural plasticity are tightly correlated with synaptic function in the mammalian brain [23], [51].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [23] Tada T, Sheng M (2006) Molecular mechanisms of dendritic spine morphogenesis. Curr Opin Neurobiol 16: 95– 101. 16361095 source
    • cites [51] Kasai H, Matsuzaki M, Noguchi J, Yasumatsu N, Nakahara H (2003) Structure-stability-function relationships of dendritic spines. Trends Neurosci 26: 360– 368. 12850432 source

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

    Premisecitation

    ASAP1 regulates the actin cytoskeleton.

    “ASAP1 is known to regulate the actin cytoskeleton [61]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [61] Randazzo PA, Andrade J, Miura K, Brown MT, Long YQ, et al (2000) The Arf GTPase-activating protein ASAP1 regulates the actin cytoskeleton. Proc Natl Acad Sci USA 97: 4011– 4016. 10725410 PMC18133 source

  • May combine two claims.

    Interpretationown data

    Arf4-T31N acts as an inactive mutant with respect to its effects on total spine density, but displays some dominant-negative characteristics by specifically reducing the density of mushroom spines.

    “Interestingly, our results suggest that Arf4-T31N acts as an inactive mutant with respect to its effects on total spine density, but displays some dominant-negative characteristics by specifically reducing the density of mushroom spines.”
    Quote in the paperSubstantial claim

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

    Interpretationcitation

    Actin-binding proteins or their regulators might serve as effectors of Arf4 in spine morphogenesis.

    “suggesting that actin-binding proteins or their regulators might serve as effectors of Arf4 in spine morphogenesis.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Mushroom spines contain more AMPA and NMDA receptors than other spine types, and these receptors are critical for strengthening synaptic connections.

    “Mushroom spines contain more AMPA and NMDA receptors than other spine types, and these receptors are critical for strengthening synaptic connections [14].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [14] Bourne J, Harris KM (2007) Do thin spines learn to be mushroom spines that remember? Curr Opin Neurobiol 17: 381– 386. 17498943 source
47 of 47

  • The paper relies on this without support.

    Dendritic spines are largely made up of actin.

    “Since dendritic spines are largely made up of actin, we considered the possibility that Arf4 might be involved in spine development.”

  • The paper relies on this without support.

    Arf4-Q71L is constitutively active and Arf4-T31N is inactive.

    “A constitutively active form of Arf4, Arf4-Q71L, promotes spine density to an even greater extent than wildtype Arf4, whereas the inactive Arf4-T31N mutant does not increase spine density relative to controls.”

  • The authors state this as an assumption.

    Arf4-T31N is functionally inactive in neurons.

    “In contrast, Arf4-T31N remains bound to GDP and is therefore theoretically inactive.”
3 of 3

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.

  • Abstract attributes two-month electrophysiology results to four- to five-month-old mice

    Minor

    The abstract describes the smaller mEPSCs as a finding in Arf4+/− mice aged 4-5 months, whereas Figure 5 specifies that the electrophysiological recordings were made at 2 months of age.

1 of 1

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Authors
Jain, S., Yoon, S. Y., Zhu, L., Brodbeck, J., Dai, J., Walker, D., & Huang, Y.
Affiliations
Gladstone Institutes (US)
Corresponding author
Huang, Yadong
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2012-09-25
Volume
7
Issue
9
Pages
e46340
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0046340
PMC
PMC3457985
OpenAlex
W2039531611
Semantic Scholar
47b6e554200f3026083fcc68da5bf7ca4fc03f36
Cited by
43
References
69
Citations by year
2026: 1 · 2025: 3 · 2024: 2 · 2023: 3 · 2022: 5 · 2021: 9 · 2019: 1 · 2018: 3 · 2017: 5 · 2016: 3 · 2015: 2 · 2014: 2 · 2013: 3
FWCI
0.92
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • National Science Foundation
  • S. D. Bechtel, Jr. Foundation and Stephen Bechtel Fund
  • Gladstone Institutes (AG022074)
  • Hellman Foundation
  • National Institutes of Health (1c06rr018928-01)
  • National Institute on Aging (P01AG022074)
  • National Institute of Neurological Disorders and Stroke (P30NS065780)
  • National Center for Research Resources (C06RR18928, C06-RR-018928)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Neuroscience › Cognitive Neuroscience › Memory and Neural Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetics and Neurodevelopmental Disorders
Keywords
ARF4, pattern separation, dentate gyrus, dendritic spine development, ASAP1, GTPase-activating protein, hippocampus, granule cells, miniature excitatory postsynaptic currents, episodic memory, Alzheimer's disease, APOE ε4, small GTPases
MeSH terms
Animals, Cells, Cultured, Electrophysiology, Immunohistochemistry, Mice, Mutant Strains, Dentate Gyrus, ADP-Ribosylation Factors, Adaptor Proteins, Signal Transducing, Dendritic Spines, Mice

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 2026-11-04.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-04.

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Statistics and arithmetic are recomputed, not judged.

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