The hereditary spastic paraplegia protein strumpellin: Characterisation in neurons and of the effect of disease mutations on WASH complex assembly and function

Mutations in the gene encoding strumpellin cause autosomal dominant hereditary spastic paraplegia (HSP).

“Mutations in the gene encoding strumpellin cause autosomal dominant hereditary spastic paraplegia (HSP), in which there is degeneration of corticospinal tract axons.”

• cites [8] Valdmanis P.N. Meijer I.A. Reynolds A. Lei A. MacLeod P. Schlesinger D. Zatz M. Reid E. Dion P.A. Drapeau P. Rouleau G.A. Mutations in the KIAA0196 gene at the SPG8 locus cause hereditary spastic paraplegia Am. J. Hum. Genet. 80 2007 152 161 17160902 PMC1785307 sourceSupported

Mammalian cells lacking strumpellin have an increased number of SNX1- and VPS26-positive endosomal tubules.

“Mammalian cells lacking strumpellin have an increased number of SNX1- and VPS26-positive endosomal tubules [9,10,13].”

• cites [9] Derivery E. Sousa C. Gautier J.J. Lombard B. Loew D. Gautreau A. The Arp2/3 activator WASH controls the fission of endosomes through a large multiprotein complex Dev. Cell 17 2009 712 723 19922875 sourcePartly supported

  The Results (page unavailable) report more transferrin-positive endosomal tubules after WASH depletion and tubules after depletion of another WASH-complex subunit, KIAA1033. They identify KIAA0196 as a complex member, but do not report tubulation after its depletion or establish that the tubules are SNX1- and VPS26-positive. The specific strumpellin-depletion and marker claims are therefore unsupported by this paper. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [10] Gomez T.S. Billadeau D.D. A FAM21-containing WASH complex regulates retromer-dependent sorting Dev. Cell 17 2009 699 711 19922874 PMC2803077 sourcePartly supported

  The Results section, “Retromer-mediated tubulation is exaggerated in the absence of WASH” (page unavailable), reports elongated VPS29-rich endosomal tubules after WASH depletion and describes SNX1 localization to endosomes. It does not establish the claimed increase in SNX1- and VPS26-positive tubules in cells lacking strumpellin; the tested depletion and VPS marker differ. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [13] Harbour M.E. Breusegem S.Y. Antrobus R. Freeman C. Reid E. Seaman M.N. The cargo-selective retromer complex is a recruiting hub for protein complexes that regulate endosomal tubule dynamics J. Cell Sci. 123 2010 3703 3717 20923837 PMC2964111 sourceNot checked — no full text of the source

Human wild-type, but not mutant, strumpellin could rescue the curly tail phenotype seen in zebrafish lacking strumpellin.

“Consistent with the idea that complexes containing mutant strumpellin are non-functional, human wild-type, but not mutant, strumpellin could rescue the curly tail phenotype seen in zebrafish lacking strumpellin [8].”

• cites [8] Valdmanis P.N. Meijer I.A. Reynolds A. Lei A. MacLeod P. Schlesinger D. Zatz M. Reid E. Dion P.A. Drapeau P. Rouleau G.A. Mutations in the KIAA0196 gene at the SPG8 locus cause hereditary spastic paraplegia Am. J. Hum. Genet. 80 2007 152 161 17160902 PMC1785307 sourceSupported

Models of the stoichiometry of the WASH complex suggest that there is 1 strumpellin molecule in the complex.

“Models of the stoichiometry of the WASH complex suggest that there is 1 strumpellin molecule in the complex [9,15].”

• cites [9] Derivery E. Sousa C. Gautier J.J. Lombard B. Loew D. Gautreau A. The Arp2/3 activator WASH controls the fission of endosomes through a large multiprotein complex Dev. Cell 17 2009 712 723 19922875 sourcePartly supported

  The Results (page unavailable) include KIAA0196 among the proposed WASH-complex subunits and say that a model with one of each subunit per complex already exceeds the measured mass. This supports the proposed one-per-complex stoichiometry, but our copy does not identify KIAA0196 as strumpellin, so the strumpellin-specific attribution cannot be verified from it. (LLM, conf=0.79)

  Experimental — accuracy not yet measured
• cites [15] Jia D. Gomez T.S. Metlagel Z. Umetani J. Otwinowski Z. Rosen M.K. Billadeau D.D. WASH and WAVE actin regulators of the Wiskott–Aldrich syndrome protein (WASP) family are controlled by analogous structurally related complexes Proc. Natl. Acad. Sci. U. S. A. 107 2010 10442 10447 20498093 PMC2890800 sourcePartly supported

  The Results and Discussion identify Strumpellin as part of a five-protein WASH core complex. They do not specify the stoichiometry or state that the complex contains exactly one Strumpellin molecule (no page number is available in our copy). (LLM, conf=0.83)

  Experimental — accuracy not yet measured

The WASH complex assembles in the brain.

“Here we demonstrate that the WASH complex assembles in the brain, where it interacts with retromer.”

• Also stated in: § The WASH complex assembles in the brain and interacts with retromer · plain

  “We concluded that brain tissue has a WASH complex that interacts with retromer.”

Strumpellin disease mutations do not have a dominant effect on transferrin receptor traffic.

“However, strumpellin disease mutations do not affect its incorporation into the WASH complex or its subcellular localisation, nor do they have a dominant effect on functions of the WASH complex, including regulation of endosomal tubulation, transferrin receptor traffic or β-2-adrenergic receptor localisation.”

Strumpellin disease mutations do not have a dominant effect on regulation of endosomal tubulation.

“However, strumpellin disease mutations do not affect its incorporation into the WASH complex or its subcellular localisation, nor do they have a dominant effect on functions of the WASH complex, including regulation of endosomal tubulation, transferrin receptor traffic or β-2-adrenergic receptor localisation.”

Members of the WASH and retromer complexes co-localise with each other in primary cortical neurons.

“Members of both complexes co-localise with each other and with endosomes in primary cortical neurons, and are present in somato-dendritic and axonal compartments.”

Strumpellin disease mutations do not have a dominant effect on β-2-adrenergic receptor localisation.

“However, strumpellin disease mutations do not affect its incorporation into the WASH complex or its subcellular localisation, nor do they have a dominant effect on functions of the WASH complex, including regulation of endosomal tubulation, transferrin receptor traffic or β-2-adrenergic receptor localisation.”

Strumpellin is required for the correct subcellular distribution of the β-2-adrenergic receptor.

“We show that strumpellin is not required for normal transferrin receptor traffic, but is required for the correct subcellular distribution of the β-2-adrenergic receptor.”

Strumpellin disease mutations do not affect its incorporation into the WASH complex.

“However, strumpellin disease mutations do not affect its incorporation into the WASH complex or its subcellular localisation, nor do they have a dominant effect on functions of the WASH complex, including regulation of endosomal tubulation, transferrin receptor traffic or β-2-adrenergic receptor localisation.”

• Also stated in: § Disease mutation of strumpellin does not prevent its participation in the WASH complex or interaction with retromer · plain

  “We concluded that mutation of strumpellin does not affect its incorporation into the WASH complex, the ability of the WASH complex to interact with retromer, or the subcellular distribution of the complex.”

The WASH complex interacts with retromer in the brain.

“Here we demonstrate that the WASH complex assembles in the brain, where it interacts with retromer.”

• Also stated in: § The WASH complex assembles in the brain and interacts with retromer · plain

  “We concluded that brain tissue has a WASH complex that interacts with retromer.”

WASH and retromer complex members are present in somato-dendritic compartments in primary cortical neurons.

“Members of both complexes co-localise with each other and with endosomes in primary cortical neurons, and are present in somato-dendritic and axonal compartments.”

• Also stated in: § WASH and retromer are present in somato-dendritic and axonal compartments in primary cortical neurons · plain

  “We found that both endogenous Wash1 (Fig. 3A) and VPS26 (Fig. 3B) labelling was widespread in the cell body and extended into the dendrites.”

Strumpellin is not required for normal transferrin receptor traffic.

“We show that strumpellin is not required for normal transferrin receptor traffic, but is required for the correct subcellular distribution of the β-2-adrenergic receptor.”

Strumpellin disease mutations do not affect its subcellular localisation.

“However, strumpellin disease mutations do not affect its incorporation into the WASH complex or its subcellular localisation, nor do they have a dominant effect on functions of the WASH complex, including regulation of endosomal tubulation, transferrin receptor traffic or β-2-adrenergic receptor localisation.”

• Also stated in: § Disease mutation of strumpellin does not prevent its participation in the WASH complex or interaction with retromer · plain

  “We concluded that mutation of strumpellin does not affect its incorporation into the WASH complex, the ability of the WASH complex to interact with retromer, or the subcellular distribution of the complex.”

Members of the WASH and retromer complexes co-localise with endosomes in primary cortical neurons.

“Members of both complexes co-localise with each other and with endosomes in primary cortical neurons, and are present in somato-dendritic and axonal compartments.”

WASH and retromer complex members are present in axonal compartments in primary cortical neurons.

“Members of both complexes co-localise with each other and with endosomes in primary cortical neurons, and are present in somato-dendritic and axonal compartments.”

• Also stated in: § WASH and retromer are present in somato-dendritic and axonal compartments in primary cortical neurons · hedged

  “Wash1 and VPS26 puncta were also visible in non-MAP2-labelled processes, indicating that these proteins are present in axons (magnified images in Fig. 3A and B).”

The steady state subcellular distribution of the TfnR was not altered in HeLa cells lacking strumpellin.

“In addition, the steady state subcellular distribution of the TfnR was not altered in HeLa cells lacking strumpellin (Fig. 9C).”

HA-tagged SNX27 co-localised in puncta with WASH and retromer complex markers.

“We began by confirming that in our hands HA-tagged SNX27 co-localised in puncta with WASH and retromer complex markers (Supplementary Fig. 4).”

The WASH and retromer complexes are present on punctate structures in both the somatodendritic and axonal compartments.

“we have extended these earlier studies with novel data to show that in the brain the WASH complex interacts with retromer, and that the WASH and retromer complexes are present on punctate structures in both the somatodendritic and axonal compartments [15].”

Wash1 co-localises extensively with EEA1 in primary cortical neurons.

“Wash1 co-localised extensively with EEA1.”

Wash1 and VPS26 puncta are visible in non-MAP2-labelled processes.

“Wash1 and VPS26 puncta were also visible in non-MAP2-labelled processes, indicating that these proteins are present in axons (magnified images in Fig. 3A and B).”

In cells lacking strumpellin, the distribution of β2AR was altered in a similar way to that seen with SNX27.

“Consistent with the known role of SNX27 in traffic of β2AR, we also found that in cells lacking strumpellin the distribution of this receptor was altered in a similar way to that seen with SNX27.”

FAM21 co-localises with Wash1.

“Thus, for example, strumpellin-myc strongly co-localised with endogenous Wash1 and VPS26 (Fig. 4D, E), and FAM21 colocalised with Wash1 (Supplementary Fig. 1).”

Both wild-type and mutant strumpellin show similar co-localisation with Wash1, VPS26, FAM21 and FKBP15 in HeLa cells.

“Consistent with these results, strumpellin mutation did not affect the co-localisation of the protein with other WASH or retromer complex components, as both wild-type and mutant strumpellin showed similar co-localisation with Wash1 (Fig. 6A, B), VPS26, FAM21 and FKBP15 (Supplementary Fig. 2) in HeLa cells and with Wash1 and VPS26 in primary cortical neurons (Fig. 6C, D).”

Wild-type and mutant strumpellin co-localised with both SNX27 and β2AR.

“Although wild-type and mutant strumpellin co-localised with both proteins, expression of disease mutant strumpellin did not cause the altered subcellular distribution that we observed after strumpellin depletion (Fig. 8E–H).”

Both wild-type and mutant strumpellin-myc show strong co-localisation with SNX1.

“Both wild-type and mutant strumpellin-myc showed strong co-localisation with SNX1 (Fig. 7A, B), and in some cases this co-localisation was on SNX1-positive tubules (arrowheads in Fig. 7B).”

Expression of disease mutant strumpellin did not cause the altered subcellular distribution of SNX27 and β2AR observed after strumpellin depletion.

“Although wild-type and mutant strumpellin co-localised with both proteins, expression of disease mutant strumpellin did not cause the altered subcellular distribution that we observed after strumpellin depletion (Fig. 8E–H).”

VPS26 displays a restricted location on domains of sorting endosomes in primary cortical neurons.

“VPS26 displayed a similar restricted location on domains of sorting endosomes (Fig. 4B).”

The clustered SNX27 puncta strongly co-localised with SNX1.

“The clustered SNX27 puncta strongly co-localised with SNX1 (Fig. 8B).”

Strumpellin-myc strongly co-localises with endogenous Wash1 and VPS26 in primary cortical neurons.

“Thus, for example, strumpellin-myc strongly co-localised with endogenous Wash1 and VPS26 (Fig. 4D, E), and FAM21 colocalised with Wash1 (Supplementary Fig. 1).”

Wash1 is often visible as peripheral spots on EEA1-positive puncta.

“It was often visible as peripheral spots on EEA1-positive puncta, consistent with reports in several non-neuronal cell types showing that Wash1 localises to restricted domains of the sorting endosomes (Fig. 4A) [9,10].”

Mutant strumpellin could be co-immunoprecipitated with a retromer component.

“However, our experiments demonstrated that mutant strumpellin could be co-immunoprecipitated with a retromer component.”

Disease mutant forms of strumpellin still allow the interaction between the WASH complex and the retromer complex.

“We next examined whether disease mutant forms of strumpellin still allowed the interaction between the WASH complex and the retromer complex.”

Wild-type and mutant strumpellin show similar patterns of co-localisation with EEA1 in primary cortical neurons and in HeLa cells.

“In addition, mutation of strumpellin did not affect its location on endosomes, as wild-type and mutant strumpellin showed similar patterns of co-localisation with EEA1, in primary cortical neurons (Figs. 4C and 6E) and in HeLa cells (Supplementary Fig. 3).”

The WASH complex and retromer are present on early endosomes in neurons.

“Furthermore, we show that the WASH complex and retromer are present on early endosomes in neurons.”

Endogenous Wash1 immunoprecipitated from rat brain lysate co-immunoprecipitates endogenous strumpellin.

“We immunoprecipitated endogenous Wash1 from rat brain lysate, and found that endogenous strumpellin was co-immunoprecipitated (Fig. 2A).”

Mutant strumpellin protein was localised appropriately on endosomes and endosomal tubules.

“However, we found no increase in endosomal tubulation in cells expressing mutant strumpellin, even though the mutant protein was localised appropriately on endosomes and endosomal tubules.”

Strumpellin mutation does not affect the co-localisation of the protein with other WASH or retromer complex components.

“Consistent with these results, strumpellin mutation did not affect the co-localisation of the protein with other WASH or retromer complex components, as both wild-type and mutant strumpellin showed similar co-localisation with Wash1 (Fig. 6A, B), VPS26, FAM21 and FKBP15 (Supplementary Fig. 2) in HeLa cells and with Wash1 and VPS26 in primary cortical neurons (Fig. 6C, D).”

When Wash1 is immunoprecipitated in HeLa cells transfected with myc-tagged wild-type or disease mutant strumpellin, all forms of strumpellin are co-immunoprecipitated in equal amounts.

“When Wash1 was immunoprecipitated in HeLa cells transfected with myc-tagged wild-type or disease mutant strumpellin, all forms of strumpellin were co-immunoprecipitated in equal amounts (Fig. 5a).”

Endogenous SNX1 immunoprecipitated in HeLa cells expressing wild-type or mutant strumpellin associates with all forms of strumpellin in apparently equal amounts.

“We immunoprecipitated endogenous SNX1 in HeLa cells expressing wild-type or mutant forms of strumpellin. All forms of strumpellin associated with SNX1 in apparently equal amounts (Fig. 5B).”

Mutation of strumpellin does not affect its location on endosomes.

“In addition, mutation of strumpellin did not affect its location on endosomes, as wild-type and mutant strumpellin showed similar patterns of co-localisation with EEA1, in primary cortical neurons (Figs. 4C and 6E) and in HeLa cells (Supplementary Fig. 3).”

In the brain, the WASH complex interacts with retromer.

“we have extended these earlier studies with novel data to show that in the brain the WASH complex interacts with retromer, and that the WASH and retromer complexes are present on punctate structures in both the somatodendritic and axonal compartments [15].”

In cells lacking strumpellin, SNX27-positive structures appeared larger and showed more peri-nuclear clustering than in control cells.

“We then examined the distribution of HA-SNX27 in cells lacking strumpellin and found that SNX27-positive structures appeared larger and showed more peri-nuclear clustering than in control cells (Fig. 8A, B, Supplementary Fig. 5).”

Strumpellin-myc co-localises with EEA1 in primary cortical neurons.

“Strumpellin-myc also showed co-localisation with EEA1 (Fig. 4C).”

VPS26 and Wash1 co-immunoprecipitate each other from rat brain lysate.

“VPS26 and Wash1 were immunoprecipitated in lysate prepared from rat brain tissue, using antibodies against the endogenous proteins (Fig. 2B). We found that each protein co-immunoprecipitated the other.”

The WASH complex assembles in brain tissue.

“We have shown biochemically that the WASH complex assembles in brain tissue.”

• Also stated in: § The pathological mechanism of strumpellin mutation · boosted

  “In summary, in this study we show that the WASH complex assembles in the brain, and that in primary neurons it is present in somatodendritic and axonal compartments and localises to endosomes.”

In some cases, co-localisation of wild-type and mutant strumpellin-myc with SNX1 is on SNX1-positive tubules.

“Both wild-type and mutant strumpellin-myc showed strong co-localisation with SNX1 (Fig. 7A, B), and in some cases this co-localisation was on SNX1-positive tubules (arrowheads in Fig. 7B).”

Mutant and wild-type forms of strumpellin co-immunoprecipitated with Wash1 with equal efficiency.

“since mutant and wild-type forms of strumpellin co-immunoprecipitated with Wash1 with equal efficiency, this is not likely.”

Both endogenous Wash1 and VPS26 labelling is widespread in the cell body and extends into dendrites of primary cortical neurons.

“We found that both endogenous Wash1 (Fig. 3A) and VPS26 (Fig. 3B) labelling was widespread in the cell body and extended into the dendrites.”

Quantitation of the number of SNX1 tubules revealed no significant difference in cells expressing wild-type strumpellin versus mutant forms of the protein.

“However, quantitation of the number of SNX1 tubules revealed no significant difference in cells expressing wild-type strumpellin versus mutant forms of the protein (Fig. 7C).”

Strumpellin depletion has no effect on transferrin uptake or recycling in HeLa cells.

“However, we found no effect of strumpellin depletion on transferrin uptake or recycling in HeLa cells (Fig. 9A, B).”

• Also stated in: § Strumpellin and the function of the WASH complex in the brain · plain

  “Wash1 depletion has a slight inhibitory effect upon recycling of TfnR [27,28], but we found no effect of strumpellin depletion on this pathway.”

Both wild-type and mutant strumpellin show similar co-localisation with Wash1 and VPS26 in primary cortical neurons.

“Consistent with these results, strumpellin mutation did not affect the co-localisation of the protein with other WASH or retromer complex components, as both wild-type and mutant strumpellin showed similar co-localisation with Wash1 (Fig. 6A, B), VPS26, FAM21 and FKBP15 (Supplementary Fig. 2) in HeLa cells and with Wash1 and VPS26 in primary cortical neurons (Fig. 6C, D).”

There is no increase in endosomal tubulation in cells expressing mutant strumpellin.

“However, we found no increase in endosomal tubulation in cells expressing mutant strumpellin, even though the mutant protein was localised appropriately on endosomes and endosomal tubules.”

In control cells, HA-tagged β2AR was visible as cytoplasmic puncta that showed minor colocalisation with SNX1.

“In control cells HA-tagged β2AR was visible as cytoplasmic puncta that showed minor colocalisation with SNX1 (Fig. 8C).”

Disease mutation of strumpellin has no obvious difference from wild-type strumpellin on transferrin uptake and recycling.

“We also examined the effect of disease mutation of strumpellin on transferrin uptake and recycling, and found no obvious difference between wild-type and mutant strumpellin (Fig. 9D, E).”

Strumpellin participates in the WASH complex via interactions with KIAA1033.

“Strumpellin participates in the complex via interactions with KIAA1033 (Fig. 1) [13].”

• cites [13] Harbour M.E. Breusegem S.Y. Antrobus R. Freeman C. Reid E. Seaman M.N. The cargo-selective retromer complex is a recruiting hub for protein complexes that regulate endosomal tubule dynamics J. Cell Sci. 123 2010 3703 3717 20923837 PMC2964111 sourceNot checked — no full text of the source

Strumpellin is a component of the WASH complex.

“Strumpellin is a component of the WASH complex, an actin-regulating complex that is recruited to endosomes by interactions with the retromer complex.”

• Also stated in: § Introduction · plain

  “However, the protein participates in the WASH complex, a recently described multimeric protein complex that acts at the interface between actin regulation and endosomal membrane dynamics [9,10].”

• cites [9] Derivery E. Sousa C. Gautier J.J. Lombard B. Loew D. Gautreau A. The Arp2/3 activator WASH controls the fission of endosomes through a large multiprotein complex Dev. Cell 17 2009 712 723 19922875 sourceNot checked yet
• cites [10] Gomez T.S. Billadeau D.D. A FAM21-containing WASH complex regulates retromer-dependent sorting Dev. Cell 17 2009 699 711 19922874 PMC2803077 sourceNot checked yet

In most cell types, having equal numbers of wild-type and mutant strumpellin complexes would provide sufficient functional WASH to allow normal cellular physiology.

“In most cell types this would provide sufficient functional WASH to allow normal cellular physiology.”

• AssumptionRests on an assumption: The WASH complex contains a single strumpellin molecule.

Corticospinal tract axonopathy is the main pathological feature of hereditary spastic paraplegias (HSPs).

“Corticospinal tract axonopathy is also the main pathological feature of a group of genetic disorders termed the hereditary spastic paraplegias (HSPs) [3,4].”

• cites [3] Reid E. Rugarli E. Valle D. Beaudet A. Vogelstein B. Kinzler K. Antonarakis S. Ballabio A. The Online Metabolic and Molecular Bases of Inherited Diseases 2010 McGraw Hill New YorkNot checked yet
• cites [4] Salinas S. Proukakis C. Crosby A. Warner T.T. Hereditary spastic paraplegia: clinical features and pathogenetic mechanisms Lancet Neurol. 7 2008 1127 1138 19007737 sourceNot checked — no full text of the source

Corticospinal neurons are especially vulnerable to reductions in functional WASH owing to the demands on membrane traffic imposed by their exceptionally long axons.

“However, owing to the demands on membrane traffic imposed by their exceptionally long axons, we suggest that corticospinal neurons are especially vulnerable to reductions in functional WASH.”

Three disease-causing mutations in strumpellin have been described: V626F, L619F and N471D.

“Three disease-causing mutations in strumpellin have been described, V626F, L619F and N471D, and all lie in highly conserved sequences [8].”

• cites [8] Valdmanis P.N. Meijer I.A. Reynolds A. Lei A. MacLeod P. Schlesinger D. Zatz M. Reid E. Dion P.A. Drapeau P. Rouleau G.A. Mutations in the KIAA0196 gene at the SPG8 locus cause hereditary spastic paraplegia Am. J. Hum. Genet. 80 2007 152 161 17160902 PMC1785307 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Cells lacking components of the WASH complex, including strumpellin, have increased endosomal tubulation.

“Thus cells lacking components of the WASH complex, including strumpellin, have increased endosomal tubulation, consistent with a defect in tubule fission from the endosome [9,10,13].”

• cites [9] Derivery E. Sousa C. Gautier J.J. Lombard B. Loew D. Gautreau A. The Arp2/3 activator WASH controls the fission of endosomes through a large multiprotein complex Dev. Cell 17 2009 712 723 19922875 sourceNot checked yet

  A check is scheduled.

• cites [10] Gomez T.S. Billadeau D.D. A FAM21-containing WASH complex regulates retromer-dependent sorting Dev. Cell 17 2009 699 711 19922874 PMC2803077 sourceNot checked yet

  A check is scheduled.

• cites [13] Harbour M.E. Breusegem S.Y. Antrobus R. Freeman C. Reid E. Seaman M.N. The cargo-selective retromer complex is a recruiting hub for protein complexes that regulate endosomal tubule dynamics J. Cell Sci. 123 2010 3703 3717 20923837 PMC2964111 sourceNot checked — no full text of the source

The three disease-causing mutations in strumpellin all lie in highly conserved sequences.

“Three disease-causing mutations in strumpellin have been described, V626F, L619F and N471D, and all lie in highly conserved sequences [8].”

• cites [8] Valdmanis P.N. Meijer I.A. Reynolds A. Lei A. MacLeod P. Schlesinger D. Zatz M. Reid E. Dion P.A. Drapeau P. Rouleau G.A. Mutations in the KIAA0196 gene at the SPG8 locus cause hereditary spastic paraplegia Am. J. Hum. Genet. 80 2007 152 161 17160902 PMC1785307 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

In patients with strumpellin mutations, complexes containing wild-type and mutant strumpellin should be present in equal numbers.

“Models of the WASH complex indicate that it contains a single strumpellin molecule, so in patients with strumpellin mutations, complexes containing wild-type and mutant strumpellin should be present in equal numbers.”

• AssumptionRests on an assumption: The WASH complex contains a single strumpellin molecule.

The WASH complex is recruited to endosomes by interactions with the retromer complex.

“Strumpellin is a component of the WASH complex, an actin-regulating complex that is recruited to endosomes by interactions with the retromer complex.”

• Also stated in: § Introduction · plain

  “The WASH complex is recruited to endosomes by the retromer complex [10,13].”

• cites [10] Gomez T.S. Billadeau D.D. A FAM21-containing WASH complex regulates retromer-dependent sorting Dev. Cell 17 2009 699 711 19922874 PMC2803077 sourceNot checked yet

  A check is scheduled.

• cites [13] Harbour M.E. Breusegem S.Y. Antrobus R. Freeman C. Reid E. Seaman M.N. The cargo-selective retromer complex is a recruiting hub for protein complexes that regulate endosomal tubule dynamics J. Cell Sci. 123 2010 3703 3717 20923837 PMC2964111 sourceNot checked — no full text of the source

The zebrafish curly tail phenotype caused by strumpellin depletion was rescued by expression of wild-type but not disease-mutant human strumpellin.

“In zebrafish, morpholino-mediated depletion of strumpellin caused a curly tail phenotype that was rescued by expression of wild-type but not disease-mutant human strumpellin, indicating that the mutants are non-functional.”

• cites [8] Valdmanis P.N. Meijer I.A. Reynolds A. Lei A. MacLeod P. Schlesinger D. Zatz M. Reid E. Dion P.A. Drapeau P. Rouleau G.A. Mutations in the KIAA0196 gene at the SPG8 locus cause hereditary spastic paraplegia Am. J. Hum. Genet. 80 2007 152 161 17160902 PMC1785307 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Corticospinal neurons are especially vulnerable to reductions in functional WASH.

“However, owing to the special demands on membrane traffic imposed by their exceptionally long axons, we suggest that corticospinal neurons are especially vulnerable to reductions in functional WASH.”

• AssumptionRests on an assumption: Membrane traffic in corticospinal neurons is subject to special demands because of their exceptionally long axons.

Cells depleted of other WASH complex components, such as Wash1, also have increased endosomal tubulation.

“Cells depleted of other WASH complex components, such as Wash1, also have increased endosomal tubulation, and this phenotype has been interpreted as a consequence of the loss of a WASH complex-generated pushing force involved in endosomal tubule fission [9,10].”

• cites [9] Derivery E. Sousa C. Gautier J.J. Lombard B. Loew D. Gautreau A. The Arp2/3 activator WASH controls the fission of endosomes through a large multiprotein complex Dev. Cell 17 2009 712 723 19922875 sourceNot checked yet
• cites [10] Gomez T.S. Billadeau D.D. A FAM21-containing WASH complex regulates retromer-dependent sorting Dev. Cell 17 2009 699 711 19922874 PMC2803077 sourceNot checked yet

The WASH complex is present on retromer-positive early endosomes in neurons.

“We concluded from these studies that the WASH complex is present on retromer-positive early endosomes in neurons.”

Expression of disease-mutant strumpellin on a wild-type background did not cause a phenotype in zebrafish.

“Expression of disease-mutant strumpellin on a wild-type background did not cause a phenotype, suggesting that the mutant proteins do not act by a dominant negative effect [8].”

• cites [8] Valdmanis P.N. Meijer I.A. Reynolds A. Lei A. MacLeod P. Schlesinger D. Zatz M. Reid E. Dion P.A. Drapeau P. Rouleau G.A. Mutations in the KIAA0196 gene at the SPG8 locus cause hereditary spastic paraplegia Am. J. Hum. Genet. 80 2007 152 161 17160902 PMC1785307 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Such processes are important throughout the neuron, including at the site of HSP pathology in the axon.

“As both the WASH complex and retromer were present in the somato-dendritic and axonal compartments, our work indicates that such processes are important throughout the neuron, including at the site of HSP pathology in the axon.”

Disease-causing strumpellin mutations do not have a dominant effect on the known functions of strumpellin in endosomal tubulation.

“Mutations do not have a dominant effect on the known functions of strumpellin in endosomal tubulation, although we speculate that strumpellin mutations have a deleterious effect on individual complexes.”

Knockdown of strumpellin causes mislocalisation and perinuclear clustering of other members of the WASH complex, notably WASH.

“Knockdown of strumpellin causes mislocalisation and perinuclear clustering of other members of the WASH complex, notably WASH [13].”

• cites [13] Harbour M.E. Breusegem S.Y. Antrobus R. Freeman C. Reid E. Seaman M.N. The cargo-selective retromer complex is a recruiting hub for protein complexes that regulate endosomal tubule dynamics J. Cell Sci. 123 2010 3703 3717 20923837 PMC2964111 sourceNot checked — no full text of the source

Jia et al. also found that mutant strumpellin could support WASH complex formation.

“These results support those of Jia et al., who also found that mutant strumpellin could support WASH complex formation [15].”

• cites [15] Jia D. Gomez T.S. Metlagel Z. Umetani J. Otwinowski Z. Rosen M.K. Billadeau D.D. WASH and WAVE actin regulators of the Wiskott–Aldrich syndrome protein (WASP) family are controlled by analogous structurally related complexes Proc. Natl. Acad. Sci. U. S. A. 107 2010 10442 10447 20498093 PMC2890800 sourceNot checked yet

The SPG8 mutational spectrum argues against a haplo-insufficiency pathological mechanism.

“This argues against a haplo-insufficiency pathological mechanism, with the caveat that additional mutational classes may be found as more HSP families are screened for strumpellin mutations.”

It is possible that sufficient normal WASH complexes remain to allow proper endosomal tubule fission.

“While incorporation of mutant strumpellin might have a deleterious effect on the individual complex involved, it is possible that sufficient “normal” WASH complexes remain to allow proper endosomal tubule fission.”

In most cell types, sufficient functional WASH complexes would be present to allow normal cellular physiology.

“We propose that in most cell types, sufficient functional WASH complexes would be present to allow normal cellular physiology.”

Expression of disease-mutant strumpellin in zebrafish in which endogenous strumpellin was present did not cause a phenotype.

“These results are consistent with findings in zebrafish, where expression of disease-mutant strumpellin in animals in which endogenous strumpellin was present did not cause a phenotype [8].”

• cites [8] Valdmanis P.N. Meijer I.A. Reynolds A. Lei A. MacLeod P. Schlesinger D. Zatz M. Reid E. Dion P.A. Drapeau P. Rouleau G.A. Mutations in the KIAA0196 gene at the SPG8 locus cause hereditary spastic paraplegia Am. J. Hum. Genet. 80 2007 152 161 17160902 PMC1785307 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

A functional consequence of defective endosomal tubule fission is altered traffic of receptors that normally transit endosomal tubular compartments.

“A functional consequence of defective endosomal tubule fission is altered traffic of receptors that normally transit endosomal tubular compartments, and this could cause HSP if receptors important for axonal survival are affected.”

The core WASH complex assembles in the brain.

“As the interaction between Wash1 and strumpellin occurs through at least one additional WASH complex component, KIAA1033, the demonstration of this interaction strongly suggests that the core WASH complex assembles in the brain.”

HeLa cells lacking strumpellin have increased tubulation of the SNX1 and retromer endosomal compartment.

“HeLa cells lacking strumpellin have increased tubulation of the SNX1 and retromer endosomal compartment [10,13].”

• cites [10] Gomez T.S. Billadeau D.D. A FAM21-containing WASH complex regulates retromer-dependent sorting Dev. Cell 17 2009 699 711 19922874 PMC2803077 sourceNot checked yet
• cites [13] Harbour M.E. Breusegem S.Y. Antrobus R. Freeman C. Reid E. Seaman M.N. The cargo-selective retromer complex is a recruiting hub for protein complexes that regulate endosomal tubule dynamics J. Cell Sci. 123 2010 3703 3717 20923837 PMC2964111 sourceNot checked — no full text of the source

The localisation and basic interactions of the WASH complex in neurons appear very similar to that described in other cell types.

“Thus, the localisation and basic interactions of the WASH complex in neurons appear very similar to that described in other cell types.”

Flies lacking Vps35 have NMJ synaptic overgrowth and satellite boutons.

“flies lacking Vps35, the retromer component that interacts with the WASH complex, have NMJ synaptic overgrowth and satellite boutons.”

• cites [33] Korolchuk V.I. Schutz M.M. Gomez-Llorente C. Rocha J. Lansu N.R. Collins S.M. Wairkar Y.P. Robinson I.M. O'Kane C.J. Drosophila Vps35 function is necessary for normal endocytic trafficking and actin cytoskeleton organisation J. Cell Sci. 120 2007 4367 4376 18057029 sourceNot checked — no full text of the source

In zebrafish, morpholino-mediated depletion of strumpellin caused a curly tail phenotype.

“In zebrafish, morpholino-mediated depletion of strumpellin caused a curly tail phenotype that was rescued by expression of wild-type but not disease-mutant human strumpellin, indicating that the mutants are non-functional.”

• cites [8] Valdmanis P.N. Meijer I.A. Reynolds A. Lei A. MacLeod P. Schlesinger D. Zatz M. Reid E. Dion P.A. Drapeau P. Rouleau G.A. Mutations in the KIAA0196 gene at the SPG8 locus cause hereditary spastic paraplegia Am. J. Hum. Genet. 80 2007 152 161 17160902 PMC1785307 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

The strumpellin mutant proteins do not act by a dominant negative effect.

“Expression of disease-mutant strumpellin on a wild-type background did not cause a phenotype, suggesting that the mutant proteins do not act by a dominant negative effect [8].”

• cites [8] Valdmanis P.N. Meijer I.A. Reynolds A. Lei A. MacLeod P. Schlesinger D. Zatz M. Reid E. Dion P.A. Drapeau P. Rouleau G.A. Mutations in the KIAA0196 gene at the SPG8 locus cause hereditary spastic paraplegia Am. J. Hum. Genet. 80 2007 152 161 17160902 PMC1785307 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

The interaction between Wash1 and strumpellin occurs through at least one additional WASH complex component, KIAA1033.

“As the interaction between Wash1 and strumpellin occurs through at least one additional WASH complex component, KIAA1033, the demonstration of this interaction strongly suggests that the core WASH complex assembles in the brain.”

• cites [13] Harbour M.E. Breusegem S.Y. Antrobus R. Freeman C. Reid E. Seaman M.N. The cargo-selective retromer complex is a recruiting hub for protein complexes that regulate endosomal tubule dynamics J. Cell Sci. 123 2010 3703 3717 20923837 PMC2964111 sourceNot checked — no full text of the source

The strumpellin disease mutants are non-functional.

“In zebrafish, morpholino-mediated depletion of strumpellin caused a curly tail phenotype that was rescued by expression of wild-type but not disease-mutant human strumpellin, indicating that the mutants are non-functional.”

• cites [8] Valdmanis P.N. Meijer I.A. Reynolds A. Lei A. MacLeod P. Schlesinger D. Zatz M. Reid E. Dion P.A. Drapeau P. Rouleau G.A. Mutations in the KIAA0196 gene at the SPG8 locus cause hereditary spastic paraplegia Am. J. Hum. Genet. 80 2007 152 161 17160902 PMC1785307 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

In the human disease, complexes containing wild-type and mutant strumpellin should be present in equal numbers.

“In the human disease, complexes containing wild-type and mutant strumpellin should be present in equal numbers.”

Cells transiently expressing mutant strumpellin will have a mixed population of WASH complexes, some containing endogenous wild-type strumpellin and others mutant strumpellin.

“Since mutant strumpellin is expressed and incorporated into the WASH complex, cells transiently expressing mutant strumpellin will have a mixed population of WASH complexes, some containing endogenous wild-type strumpellin and others mutant strumpellin.”

The WASH complex is implicated in β2AR recycling as it interacts with SNX27, an adapter that links β2AR to retromer-positive tubules.

“The WASH complex is also implicated in β2AR recycling as it interacts with SNX27, an adapter that links β2AR to retromer-positive tubules [29].”

• cites [29] Temkin P. Lauffer B. Jager S. Cimermancic P. Krogan N.J. von Zastrow M. SNX27 mediates retromer tubule entry and endosome-to-plasma membrane trafficking of signalling receptors Nat. Cell Biol. 13 2011 715 721 21602791 PMC3113693 sourceNot checked yet

The altered distribution of β2AR and SNX27 in cells lacking strumpellin is consistent with a failure to package β2AR into tubules for recycling to the plasma membrane.

“We found that β2AR and its cargo adaptor SNX27 had an altered subcellular distribution and increased localisation to retromer-positive compartments in cells lacking strumpellin, consistent with a failure to package β2AR into tubules for recycling to the plasma membrane.”