Endosomal protein DENND10 promotes developmental competence of neurite extension

In the paper

“Previous studies reported that CCDC22 interacts with CAMSAP2, a microtubule-organizing protein involved in dendrite development,25”

What the sources say

• Supported

  The Results section (‘Commander assembles into a hetero-hexadecameric complex’) explicitly says that CCDC22 interacts with CAMSAP2 and describes CAMSAP2 as a microtubule-organizing protein that functions in dendrite development. (LLM, conf=0.95)

  cites [25] Laulumaa S. Kumpula E.P. Huiskonen J.T. Varjosalo M. Structure and interactions of the endogenous human Commander complex Nat. Struct. Mol. Biol. 31 2024 925 938 10.1038/s41594-024-01246-1 38459129 PMC11189303 source

In the paper

“This region is inherently flexible and exhibits high conformational heterogeneity, suggesting its potential role as a regulatory site.25”

What the sources say

• Supported

  The Results section (‘DENND10 and Retriever are scaffolded by CCDC22 and CCDC93’; page unavailable) calls the region around DENND10’s putative Rab-binding site inherently flexible, and another Results passage reports conformational heterogeneity in this region. The Discussion (page unavailable) speculates that COMMD1 may regulate the site’s availability through allostery, consistent with the claim’s tentative wording. (LLM, conf=0.88)

  cites [25] Laulumaa S. Kumpula E.P. Huiskonen J.T. Varjosalo M. Structure and interactions of the endogenous human Commander complex Nat. Struct. Mol. Biol. 31 2024 925 938 10.1038/s41594-024-01246-1 38459129 PMC11189303 source

In the paper

“its depletion has been shown to impair the sorting of Retromer cargoes.32”

What the sources say

• Partly supported

  The Results section (‘CCC loss leads to retriever-independent recycling defects’; page unavailable) reports that FAM45A deletion causes the retromer cargo GLUT1 to accumulate in early endosomes, supporting impaired sorting. The Discussion also describes an effect of FAM45A on retromer-mediated recycling. Our copy does not identify FAM45A as DENND10, so it does not establish that the reported depletion was of the protein named in the claim. (LLM, conf=0.78)

  cites [32] Singla A. Fedoseienko A. Giridharan S.S.P. Overlee B.L. Lopez A. Jia D. Song J. Huff-Hardy K. Weisman L. Burstein E. Billadeau D.D. Endosomal PI(3)P regulation by the COMMD/CCDC22/CCDC93 (CCC) complex controls membrane protein recycling Nat. Commun. 10 2019 4271 10.1038/s41467-019-12221-6 31537807 PMC6753146 sourceExperimental — accuracy not yet measured

In the paper

“Interestingly, it was reported that DENND10/FAM45A knockout in HeLa cells did not affect the trafficking of integrin β1, a typical Retriever cargo, but instead perturbed cargo recycling of the related Retromer complex.32”

What the sources say

• Supported

  The Results section, “CCC loss leads to retriever-independent recycling defects,” reports that FAM45A deletion did not affect integrin β1 trafficking in HeLa cells. The same section reports that FAM45A deletion caused accumulation of GLUT1, a retromer cargo, in early endosomes. Page numbers were not provided in our copy. (LLM, conf=0.94)

  cites [32] Singla A. Fedoseienko A. Giridharan S.S.P. Overlee B.L. Lopez A. Jia D. Song J. Huff-Hardy K. Weisman L. Burstein E. Billadeau D.D. Endosomal PI(3)P regulation by the COMMD/CCDC22/CCDC93 (CCC) complex controls membrane protein recycling Nat. Commun. 10 2019 4271 10.1038/s41467-019-12221-6 31537807 PMC6753146 source

In the paper

“Endosomal recruitment of the Retromer requires Rab5 and Rab7.73”

What the sources say

• Supported

  The Introduction states that Rab5 and Rab7 act sequentially to enable retromer recruitment to endosomes. The Abstract likewise says they act in concert to regulate that recruitment. (LLM, conf=0.94)

  cites [73] Rojas R. van Vlijmen T. Mardones G.A. Prabhu Y. Rojas A.L. Mohammed S. Heck A.J.R. Raposo G. van der Sluijs P. Bonifacino J.S. Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7 J. Cell Biol. 183 2008 513 526 10.1083/jcb.200804048 18981234 PMC2575791 source

In the paper

“For instance, a previous RNA-seq analysis on CAD differentiation identified an up-regulation of Acss2 mRNA, which produces a nuclear pool of acetyl-CoA and promotes histone acetylation, facilitating the expression of neuron-specific genes.45”

What the sources say

• Partly supported

  The Results (‘ACSS2 regulates neuronal gene expression’) report increased nuclear ACSS2 protein during CAD differentiation, while the Results (‘ACSS2 is recruited to transcriptionally active chromatin’) link ACSS2 to increased histone acetylation and neuronal-gene expression. The paper also describes ACSS2 as a source of nuclear acetyl-CoA. However, its RNA-seq account and Extended Data Figure 2 do not identify an up-regulation of Acss2 mRNA specifically; that part of the claim is unsupported by our copy. (LLM, conf=0.88)

  cites [45] Mews P. Donahue G. Drake A.M. Luczak V. Abel T. Berger S.L. Acetyl-CoA synthetase regulates histone acetylation and hippocampal memory Nature 546 2017 381 386 10.1038/nature22405 28562591 PMC5505514 sourceExperimental — accuracy not yet measured

In the paper

“It should be added that low levels of peptides from the C-terminal DENND10 sequence were detected in sgDennd10_No2 by mass spectrometry (see below, Table S1), likely due to truncated protein expression from cryptic alternative splicing events, a phenomenon observed in approximately 50% of CRISPR-edited cells.50”

What the sources say

• Partly supported

  The Results section (‘Unanticipated gene expression outcomes with CRISPR editing’; page unavailable) reports unexpected protein-expression or mRNA-splicing changes in approximately 50% of the CRISPR-edited cell lines studied. It does not say cryptic alternative splicing occurred in 50%: some novel proteins were attributed to alternative translation initiation. The citation supports the general phenomenon, not the DENND10-specific mass-spectrometry finding or the stated frequency for splicing. (LLM, conf=0.88)

  cites [50] Tuladhar R. Yeu Y. Tyler Piazza J. Tan Z. Rene Clemenceau J. Wu X. Barrett Q. Herbert J. Mathews D.H. Kim J. CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation Nat. Commun. 10 2019 4056 10.1038/s41467-019-12028-5 31492834 PMC6731291 sourceExperimental — accuracy not yet measured

In the paper

“The GDP-locked Rab27a-TN and Rab27b-TN mutants exhibited uniform diffusion throughout the cytoplasm, whereas the GTP-locked Rab27a-QL and Rab27b-QL mutants were enriched at the tip of neurites (Figure S5A)”

In the paper

“In total, 6997 proteins were identified, of which 6910 proteins were quantifiable (Table S1).”

In the paper

“We further focused on DEPs with FC greater than 1.3, which resulted in 363 DEPs for Genotype and 181 DEPs for Differentiation (Table S2).”

In the paper

“While the number of neurites tended to decrease in Ccdc22-deficient cells, the change did not reach statistical significance (Figure S7B)”

In the paper

“In sgDennd10_No1, a large deletion encompassing most of Exon_1 was observed, whereas sgDennd10_No2 showed disruption of the 5′ splicing donor site of the subsequent intron (Figure 1B).”

In the paper

“we observed a significant down-regulation of proteins involved in DNA biosynthesis and repair (POLD1, POLD3, POLE, and POLE2)”

• Also stated in: § Changes in proteomic landscape during neurite outgrowth · plain

  “Conversely, DEPs that were up-regulated after differentiation were primarily related to lipid metabolism, including enzymes for the biosynthesis of phospholipid (LPCAT3, LPIN1, and PNPLA8), sterol lipids (NSDHL, MSMO1, DHCR7, LSS, and TM7SF2) and long chain fatty acid (TECR, ACSL4, HSD17B8, and AACS)”

In the paper

“Additionally, in HeLa cells, mCherry-tagged DENND10 co-localized with Citrine-tagged CCDC22 on cytoplasmic vesicles, a subset of which were positive for endosomal marker CD63 (Figure S6C).”

In the paper

“While the number of neurites increased slightly after CCDC22 expression, this change did not reach statistical significance (Figure S7C).”

In the paper

“we identified 4046 proteins that were significantly up- or downregulated (FDR <0.05) for the Genotype factor, 3683 proteins significantly altered for the Differentiation factor, and 139 proteins that exhibited significant interaction effects between Genotype and Differentiation (Table S1).”

In the paper

“It is worth mentioning that the up-regulation of ACSS2 protein was also observed in our proteomic dataset (Table S3).”

In the paper

“Intriguingly, these clusters formed a rectangle-like shape on the PCA plot.”

In the paper

“Additionally, the number of neurites increased to varying degrees upon expression of Rab27-QL mutants (Figure S5B), with Rab27b-QL in sgDennd10 cells showing statistically significant increases”

In the paper

“The first principal component (PC1) displayed a strong correlation with the genotypes (NC vs. sgDennd10), while the second principal component (PC2) was associated with the differentiation condition (Non-diff vs. diff), separating the samples into four distinct clusters.”

In the paper

“In contrast, no significant enhancement was observed in NC cells, likely due to a ceiling effect for Rab27 activation under normal conditions.”

In the paper

“In primary cortical neurons, loss of DENND10 or CCDC22 led to shortened dendrites and impaired axon development.”

In the paper

“On average, neurite lengths in differentiated sgDennd10_No1 and No2 cells were approximately 34% and 24% of those in NC cells, respectively (Figures 1C and 1D).”

In the paper

“TUBB3, a neuron-specific cytoskeletal protein required for neurite extension,51 was predominantly localized to the neurites in NC cells but was mainly confined to the soma in sgDennd10 cells (Figure 1F).”

What the sources say

• Not checked yetcites [51] Latremoliere A. Cheng L. DeLisle M. Wu C. Chew S. Hutchinson E.B. Sheridan A. Alexandre C. Latremoliere F. Sheu S.H. Neuronal-Specific TUBB3 Is Not Required for Normal Neuronal Function but Is Essential for Timely Axon Regeneration Cell Rep. 24 2018 1865 1879 10.1016/j.celrep.2018.07.029 30110642 PMC6155462

In the paper

“From left to right: n = 29, 34, 46, 38, and 41 neurons, respectively, from three independent experiments.”

In the paper

“FDR of both protein identification and PSM identification; 1%.”

In the paper

“strongly suggesting that Dennd10 deficiency diminishes Rab27a and Rab27b activation and impairs their transport into neurites in CAD cells.”

In the paper

“Remarkably, a human-specific allele of the TKTL1 gene, which promotes the synthesis of specific membrane lipids, has been linked to increased neocortical neurogenesis in the human lineage compared to Neanderthals.71”

What the sources say

• Not checked yetcites [71] Pinson A. Xing L. Namba T. Kalebic N. Peters J. Oegema C.E. Traikov S. Reppe K. Riesenberg S. Maricic T. Human TKTL1 implies greater neurogenesis in frontal neocortex of modern humans than Neanderthals Science 377 2022 eabl6422 10.1126/science.abl6422 36074851

In the paper

“All animal protocols were approved by the Animal Care and Use Committee at Soochow University (202410A0190).”

In the paper

“While DENND10 interacts with Rab27 in a nucleotide-dependent manner in vitro,24”

What the sources say

• ?Not checked — no full text of the sourcecites [24] Zhang J. Zhang K. Qi L. Hu Q. Shen Z. Liu B. Deng J. Zhang C. Zhang Y. DENN domain-containing protein FAM45A regulates the homeostasis of late/multivesicular endosomes Biochim. Biophys. Acta Mol. Cell Res. 1866 2019 916 929 10.1016/j.bbamcr.2019.02.006 30771381 source

In the paper

“TBC1D5, a GTPase-activating protein (GAP) for Rab7, is a sub-stoichiometric component of the Retromer, linking endosomal recruitment of the Retromer with the maturation of the endosomes.29”

What the sources say

• ?Not checked — no full text of the sourcecites [29] Chen K.E. Healy M.D. Collins B.M. Towards a molecular understanding of endosomal trafficking by Retromer and Retriever Traffic 20 2019 465 478 10.1111/tra.12649 30993794 source

In the paper

“Rab27 is known to be critical for anterograde transport within neurites and synaptic vesicle exocytosis.65,66”

What the sources say

• Not checked yetcites [65] Arimura N. Kimura T. Nakamuta S. Taya S. Funahashi Y. Hattori A. Shimada A. Ménager C. Kawabata S. Fujii K. Anterograde transport of TrkB in axons is mediated by direct interaction with Slp1 and Rab27 Dev. Cell 16 2009 675 686 10.1016/j.devcel.2009.03.005 19460344
• Not checked yetcites [66] Yu E. Kanno E. Choi S. Sugimori M. Moreira J.E. Llinás R.R. Fukuda M. Role of Rab27 in synaptic transmission at the squid giant synapse Proc. Natl. Acad. Sci. USA 105 2008 16003 16008 10.1073/pnas.0804825105 18840683 PMC2562534

In the paper

“It has been shown that disruption in phospholipid synthesis leads to defective dendrite growth and maintenance in fruit flies.69”

What the sources say

• Not checked yetcites [69] Meltzer S. Bagley J.A. Perez G.L. O'Brien C.E. DeVault L. Guo Y. Jan L.Y. Jan Y.N. Phospholipid Homeostasis Regulates Dendrite Morphogenesis in Drosophila Sensory Neurons Cell Rep. 21 2017 859 866 10.1016/j.celrep.2017.09.089 29069593 PMC5687885

In the paper

“Equal masses of proteins from each sample were precipitated with cold acetone at −20°C.”

In the paper

“Membrane expansion during neurite outgrowth likely necessitates the de novo synthesis of membrane lipids.68”

What the sources say

• Not checked yetcites [68] Roy D. Tedeschi A. The Role of Lipids, Lipid Metabolism and Ectopic Lipid Accumulation in Axon Growth, Regeneration and Repair after CNS Injury and Disease Cells 10 2021 1078 10.3390/cells10051078 PMC8147289 34062747

In the paper

“These results suggest that both DENND10 and CCDC22 are critical for neurite growth and development in vivo.”

In the paper

“Six to eight random fields were imaged per coverslip with a 63× objective (N.A. = 1.4).”

In the paper

“Another study indeed identified DENND10/FAM45A following the IP of the Retromer subunit.36”

What the sources say

• Not checked yetcites [36] McMillan K.J. Gallon M. Jellett A.P. Clairfeuille T. Tilley F.C. McGough I. Danson C.M. Heesom K.J. Wilkinson K.A. Collins B.M. Cullen P.J. Atypical parkinsonism–associated retromer mutant alters endosomal sorting of specific cargo proteins J. Cell Biol. 214 2016 389 399 10.1083/jcb.201604057 27528657 PMC4987296

In the paper

“Protein concentrations of the supernatant were measured by BCA assay and adjusted to the same level.”

In the paper

“Paradoxically, a genome-wide screening identified Rab27 and Rab3 as negative regulators of neuronal regeneration.72”

What the sources say

• Not checked yetcites [72] Sekine Y. Lin-Moore A. Chenette D.M. Wang X. Jiang Z. Cafferty W.B. Hammarlund M. Strittmatter S.M. Functional Genome-wide Screen Identifies Pathways Restricting Central Nervous System Axonal Regeneration Cell Rep. 23 2018 415 428 10.1016/j.celrep.2018.03.058 29642001 PMC5937716

In the paper

“It is also important to recognize that the role of Rab27 in neurite growth could be context-dependent.”

In the paper

“A decrease in malonyl-CoA, the precursor of fatty acid biosynthesis, results in impaired axonal growth and reduced accumulation of late endolysosomes in axon terminals.70”

What the sources say

• Not checked yetcites [70] Palomo-Guerrero M. Fadó R. Casas M. Pérez-Montero M. Baena M. Helmer P.O. Domínguez J.L. Roig A. Serra D. Hayen H. Sensing of nutrients by CPT1C regulates late endosome/lysosome anterograde transport and axon growth Elife 8 2019 e51063 10.7554/eLife.51063 PMC6927751 31868590

In the paper

“In a previous study, we found that DENND10 is mainly localized in endosomes and lysosomes, where it regulates the positioning, maturation, and secretion of endosomes.24”

What the sources say

• ?Not checked — no full text of the sourcecites [24] Zhang J. Zhang K. Qi L. Hu Q. Shen Z. Liu B. Deng J. Zhang C. Zhang Y. DENN domain-containing protein FAM45A regulates the homeostasis of late/multivesicular endosomes Biochim. Biophys. Acta Mol. Cell Res. 1866 2019 916 929 10.1016/j.bbamcr.2019.02.006 30771381 source

In the paper

“From left to right: n = 81, 86, and 94 cells, respectively, from three independent experiments.”

In the paper

“indicating that they are essential for neuronal differentiation but not directly related to the neurite shortening phenotype observed in sgDennd10 cells.”

In the paper

“Rab11 and Rab35, two small GTPases found on recycling endosomes, promote neurite outgrowth and axonal elongation.7,13”

What the sources say

• Not checked yetcites [7] Quiroga S. Bisbal M. Cáceres A. Regulation of plasma membrane expansion during axon formation Dev. Neurobiol. 78 2018 170 180 10.1002/dneu.22553 29090510
• Not checked yetcites [13] Petrova V. Nieuwenhuis B. Fawcett J.W. Eva R. Axonal Organelles as Molecular Platforms for Axon Growth and Regeneration after Injury Int. J. Mol. Sci. 22 2021 1798 10.3390/ijms22041798 PMC7918155 33670312

In the paper

“Raw p values were adjusted for multi-comparison with the false discovery rate (FDR) method using the “p.adjust” function.”

In the paper

“Equal amounts of the supernatant were incubated with anti-FLAG M2 affinity gel (Sigma A2220) overnight at 4°C on a rotator.”

In the paper

“From left to right: n = 231, 203, 214, 226, 218, and 196 cells, respectively, from six independent experiments.”

In the paper

“Missense mutations in CCDC22 have been associated with intellectual disability.37,38”

What the sources say

• Not checked yetcites [37] Voineagu I. Huang L. Winden K. Lazaro M. Haan E. Nelson J. McGaughran J. Nguyen L.S. Friend K. Hackett A. CCDC22: a novel candidate gene for syndromic X-linked intellectual disability Mol. Psychiatr. 17 2012 4 7 10.1038/mp.2011.95 PMC3586744 21826058 source
• Not checked yetcites [38] Kolanczyk M. Krawitz P. Hecht J. Hupalowska A. Miaczynska M. Marschner K. Schlack C. Emmerich D. Kobus K. Kornak U. Missense variant in CCDC22 causes X-linked recessive intellectual disability with features of Ritscher-Schinzel/3C syndrome Eur. J. Hum. Genet. 23 2015 633 638 10.1038/ejhg.2014.109 24916641 PMC4402616

In the paper

“The Rpl32 gene was used as the internal control.”

In the paper

“Mechanistically, DENND10 exerts its influence by regulating both Rab GTPases and the CCC/Retriever complex.”

In the paper

“Conversely, loss-of-function mutations or down-regulation of Rab5, Rab4, Rab35, Rab33a or Rab10 result in impaired axonal growth.13”

What the sources say

• Not checked yetcites [13] Petrova V. Nieuwenhuis B. Fawcett J.W. Eva R. Axonal Organelles as Molecular Platforms for Axon Growth and Regeneration after Injury Int. J. Mol. Sci. 22 2021 1798 10.3390/ijms22041798 PMC7918155 33670312

In the paper

“On the one hand, differentiation treatment halts cell cycle and promotes lipid biosynthesis, producing surplus membrane material, as represented by Edge 3 and Edge 4 in the PCA plot (Figure 2A).”