Regulation of Mitochondrial Transport and Inter-Microtubule Spacing by Tau Phosphorylation at the Sites Hyperphosphorylated in Alzheimer's Disease

May combine two claims.

Hyperphosphorylation of the tail domains would increase the inter-NF distances to expand the NF domain in axons, giving higher pressure to the MT domain.

“Hyperphosphorylation of the tail domains would increase the inter-NF distances to expand the NF domain in axons (Kumar et al., 2002;Kanungo et al., 2011), giving higher pressure to the MT domain.”

• cites Kumar et al., 2002 sourcePartly supported

  The Discussion reports that increased sidearm phosphorylation expands the sidearm brush and regulates spacing between neurofilaments. It does not establish that this expands a distinct NF domain in the axon. The subsequent suggestion of higher pressure on the MT domain is not addressed by the paper and appears to be the citing authors’ inference. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites Kanungo et al., 2011Not matched

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

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

Hyperphosphorylation of Tau is linked to neurodegeneration, with phosphorylation of more than 20 sites shown in degenerated brains of Alzheimer's patients.

“However, hyperphosphorylation is linked to neurodegeneration with phosphorylation of more than 20 sites shown in the degenerated brains of Alzheimer's patients (Watanabe et al., 1993;Morishima-Kawashima et al., 1995;Stoothoff and Johnson, 2005).”

• cites Watanabe et al., 1993 sourceNot checked — no full text of the source
• cites Morishima-Kawashima et al., 1995 sourcePartly supported

  The opening section reports abnormal phosphorylation of PHF-tau in Alzheimer’s disease and identifies 19 sites, not more than 20. It also describes neurofibrillary tangles as closely related to neuronal loss, while the Discussion presents a link between abnormal tau phosphorylation and neuronal degeneration as a possibility rather than an established finding. (LLM, conf=0.90)

  Experimental — accuracy not yet measured
• cites Stoothoff and Johnson, 2005Not matched

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

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

Detection by AT8 is commonly used as a marker of abnormal phosphorylation in brains of Alzheimer's patients.

“their detection by this antibody is commonly used as a marker of abnormal phosphorylation in brains of Alzheimer's patients.”

• cites Goedert et al., 1995 sourceNot checked — no full text of the source
• cites Wang et al., 2006 sourceCould not verify

  The Abstract links Alzheimer’s disease to tau hyperphosphorylation, but it does not identify AT8 or describe its use as a marker in patients’ brains. Searches for AT8 and for antibody-based detection found no passage addressing that point. (LLM, conf=0.74)

  Experimental — accuracy not yet measured
• cites Hanger et al., 2009 sourceNot checked — no full text of the source

May combine two claims.

Phosphorylation at the AT8 sites may extend the projection domain outwards from the surface of microtubules and affect mitochondrial transport.

“Phosphorylation at these sites may extend the projection domain outwards from the surface of MTs (Jeganathan et al., 2008) and affect mitochondrial transport (Mukhopadhyay and Hoh, 2001;Jeganathan et al., 2008;Shahpasand et al., 2008).”

• cites Jeganathan et al., 2008 sourcePartly supported

  The Results passage (Fig. 2C–D; page unavailable) reports that an AT8 phosphorylation mimic makes tau’s N-terminal domain swing away from its C-terminal domain. The introductory passage says the N-terminal domain projects away from the microtubule surface, but the study does not show that AT8 phosphorylation extends it farther from that surface. It discusses microtubule-based organelle transport generally, not an effect on mitochondrial transport. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites Mukhopadhyay and Hoh, 2001 sourcePartly supported

  The citation to Mukhopadhyay and Hoh is attached to the mitochondrial-transport clause. In Results and discussion (page unavailable), the paper says MAPs may regulate motor-protein interactions and reports that tau affects motor attachment and detachment, but not transport speed. It does not address mitochondrial transport or link phosphorylation at AT8 sites to a transport effect. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites Shahpasand et al., 2008 sourceNot checked — no full text of the source

May combine two claims.

The AT8 sites (Ser199/Ser202/Thr205) are physiological phosphorylation sites and markers of hyperphosphorylation in Alzheimer's disease.

“The AT8 sites are not only physiological phosphorylation sites (Kimura et al., 2007;Verwer et al., 2007) but also markers of hyperphosphorylation in Alzheimer's disease (Plattner et al., 2006).”

• cites Kimura et al., 2007 sourcePartly supported

  The Results section (page unavailable) reports AT8 phosphorylation in both adult and aged mice expressing wild-type human tau, without an age-related increase. This supports the narrower point that AT8 phosphorylation can occur without age-related hyperphosphorylation, but the study does not establish that the sites are physiological in normal animals; it also identifies the AT8 sites as Ser202 and Ser205, not Ser199/Ser202/Thr205. The Alzheimer's disease marker claim is attributed to Plattner et al., not Kimura et al., in the citing sentence. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites Verwer et al., 2007 sourceNot checked — no full text of the source
• cites Plattner et al., 2006 sourceSupported

May combine two claims.

The AT8 sites are physiological sites for phosphorylation, but they are not always phosphorylated.

“The AT8 sites are physiological sites for phosphorylation, but they are not always phosphorylated (Kimura et al., 2007;Verwer et al., 2007).”

• cites Kimura et al., 2007 sourcePartly supported

  The section on PHF1-site phosphorylation identifies AT8 as recognizing tau phosphorylated at Ser202 and Ser205, and reports AT8 immunoreactivity in the studied mice. The Discussion says AT8-site phosphorylation did not increase with aging in those mice, but it does not establish that phosphorylation at these sites is physiological or that the sites are sometimes unphosphorylated. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites Verwer et al., 2007 sourceNot checked — no full text of the source

May combine two claims.

The C-terminal tail domains of neurofilament M and H subunits extrude outward from core filaments, as does the projection domain of Tau, to make spaces between neurofilaments.

“The C-terminal tail domains of neurofilament M and H subunits extrude outward from core filaments, as does the projection domain of Tau, to make spaces between neurofilaments (NFs) (Hisanaga and Hirokawa, 1989).”

• cites Hisanaga and Hirokawa, 1989 sourcePartly supported

  The section “Structure of the projections of control and dephosphorylated NFs” (page unavailable) describes projections extending from NF core filaments, and the Discussion describes cross-bridges between NFs that seem to comprise NF-H and NF-M tail domains. The copy does not discuss Tau’s projection domain or establish that these projections create the spaces between neurofilaments. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

When Tau is overexpressed in cortical neurons, tubulin synthesis is upregulated and MTs become more numerous and densely packed, resulting in inhibition of mitochondrial movement.

“When Tau is overexpressed in cortical neurons, tubulin synthesis is upregulated and MTs become more numerous and densely packed, resulting in inhibition of mitochondrial movement.”

• cites Thies and Mandelkow, 2007 sourcePartly supported

  The Microtubule bundling section reports increased tubulin production and denser, more numerous microtubules after tau transfection; the mitochondrial-movement section reports strongly inhibited movement. However, Materials and Methods identifies the cells as hippocampal, not cortical, neurons, and the Discussion describes increased microtubule density and bundling as a possible contributor to the transport defect rather than its established sole cause. Page numbers were not available in our copy. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

May combine two claims.

Tau 3A and 3D constructs are not phosphorylated at the AT8 sites.

“Phosphorylated Tau WT was immunoreactive to AT8, whereas phosphorylated Tau 3A and 3D were not (Fig. 1 B)”

• Also stated in: § Phosphorylation of Tau mutants and their binding to MTs · boosted

  “Immunodetection of Tau WT but not 3A nor 3D clearly demonstrated that Tau WT was phosphorylated at the AT8 recognition sites in COS-7 cells (Fig. 1C, middle).”

May combine two claims.

In PC12 cells, the stationary phase of mitochondria was 23.0% in control, 47.6% in Tau WT, 44.1% in Tau 3A, and 61.3% in Tau 3D.

“The ratio of stationary phase was 23.0% in control PC12 cells but 47.6% in cells expressing Tau WT and 44.1% for 3A; for 3D, however, the percentage was higher still, 61.3% (Fig. 4B).”

The quote only partly states this claim.

Expression levels of Tau WT, 3A, and 3D were almost similar in PC12 cells and cultured neurons when 2.5 μg plasmids were used.

“Expression levels were almost similar among Tau WT, 3A, and 3D constructs (Fig. 3 B, C).”

• Also stated in: § Expression of Tau constructs in PC12 cells and cultured cortical neurons · plain

  “When 2.5 g of plasmids were used, the fluorescent intensities were ϳ1.7 and ϳ1.5 fold of untransfected PC12 cells (Fig. 3B) and cultured neurons (Fig. 3C), respectively.”

May combine two claims.

The inter-MT distance was the same (15-25 nm) in MT bundles formed by Tau WT, 3A, and 3D in Sf9 cell processes, but it was expanded to 35-45 nm in processes expressing Tau 3D when actin filaments were disrupted by latrunculin B.

“The inter-MT distance was the same (15-25 nm) in MT bundles formed by Tau WT, 3A, and 3D in Sf9 cell processes, but it was expanded to 35-45 nm in processes expressing Tau 3D when actin filaments were disrupted by latrunculin B.”

• Also stated in: § Discussion · plain

  “The mean distance was 15.7 Ϯ 2.2 nm for Tau WT (n ϭ 30), 17.5 Ϯ 2.7 nm for Tau 3A (n ϭ 27), and 16.3 Ϯ 2.2 nm for Tau 3D (n ϭ 28).”

• Also stated in: § Discussion · plain

  “The mean distance was 26.8 Ϯ 2.7 nm for Tau WT (n ϭ 60), 27.2 Ϯ 2.0 nm for Tau 3A (n ϭ 80), and 37.1 Ϯ 2.3 nm for Tau 3D (n ϭ 75).”

The quote only partly states this claim.

The ratio of mitochondria moving anterogradely and retrogradely was not different among Tau WT, Tau 3A, and Tau 3D in cortical neurons.

“However, the ratio of mitochondria moving anterogradely and retrogradely was not different among three Tau constructs, indicating that phosphorylation at the AT8 sites does not affect the direction of mitochondrial movements.”

May combine two claims.

In cortical neuron axons, the stationary phase of mitochondria was 31.6% in control, 48.5% in Tau WT, 46.6% in Tau 3A, and 64.4% in Tau 3D.

“The duration of stationary phase was 31.6% in control neurons, which was higher than that observed in PC12 cells, consistent with a previous report (Morel et al., 2010).A significant increase in stationary phase was observed after transfection with Tau WT (48.5%) or 3A (46.6%); however, a higher stationary state (64.4%) was caused by Tau 3D (Fig. 5B).”

• cites Morel et al., 2010 sourceNot checked — no full text of the source

May combine two claims.

In vitro MT pellets, the mean inter-MT distance was 11.7 ± 1.7 nm for Tau WT, 11.8 ± 2.1 nm for Tau 3A, and 11.9 ± 1.6 nm for Tau 3D.

“The mean distance was 11.7 Ϯ 1.7 nm for Tau WT (n ϭ 48), 11.8 Ϯ 2.1 nm for Tau 3A (n ϭ 51), and 11.9 Ϯ 1.6 nm for Tau 3D (n ϭ 69).”

May combine two claims.

Transfection with less than 2 μg Tau plasmid did not significantly affect mitochondrial movement, while more than 3 μg had a substantial and similar effect for all Tau constructs.

“While transfection with less than 2 g did not significantly affect mitochondrial movement, more than 3 g had a substantial (and similar) effect for all Tau constructs (data not shown).”

May combine two claims.

The ratio of anterogradely moving duration to total moving period was significantly different between control and Tau-overexpressing neurons (p < 0.01) but not different between the three Tau constructs (p = 0.55).

“The ratio was significantly different between control and Tau-overexpressing neurons (p Ͻ 0.01) but was not different between three Tau constructs(pϭ0.55).”

May combine two claims.

Tau overexpression increased the number of MTs and reduced the inter-MT spaces in neurites of PC12 cells.

“We observed here that Tau overexpression increased the number of MTs and reduced the inter-MT spaces in neurites of PC12 cells.”

• Also stated in: § Discussion · plain

  “The mean distance was 45.1 Ϯ 2.2 (n ϭ 54) for control PC12 cells and 33.1 Ϯ 2.4 nm (n ϭ 49), 34.1 Ϯ 2.6 nm (n ϭ 44), and 34.19 Ϯ 2.8 nm (n ϭ 62) for PC12 cells overexpressing Tau WT, 3A, and 3D, respectively.”

Probably not a substantial claim: may not pass S4 (based).

Tau 3A and 3D have similar phosphorylation profiles at other sites.

“Because Tau 3A and 3D have similar phosphorylation profiles at other sites”

May combine two claims.

In PC12 cell neurites, the mean inter-MT distance was 45.1 ± 2.2 nm for control and 33.1 ± 2.4 nm, 34.1 ± 2.6 nm, and 34.19 ± 2.8 nm for Tau WT, 3A, and 3D, respectively.

“The mean distance was 45.1 Ϯ 2.2 (n ϭ 54) for control PC12 cells and 33.1 Ϯ 2.4 nm (n ϭ 49), 34.1 Ϯ 2.6 nm (n ϭ 44), and 34.19 Ϯ 2.8 nm (n ϭ 62) for PC12 cells overexpressing Tau WT, 3A, and 3D, respectively.”

The quote only partly states this claim.

In latrunculin B-treated Sf9 cells, inter-microtubule distances were greater in Tau 3D-expressing cells than in Tau WT- or Tau 3A-expressing cells (mean 37.1 nm vs 26.8 and 27.2 nm).

“The inter-MT distances were greater in Tau 3Doverexpressing cells compared to those overexpressing WT or 3A (Fig. 7L).”

• Also stated in: § The phosphorylation mimic Tau 3D increases MT spacing in processes of Sf9 cells · plain

  “that range was 35-45 nm in Tau 3D-expressing cells (mean distance ϭ 37.1 Ϯ 2.3 nm)”

The quote only partly states this claim.

Tau overexpression reduced the velocity of mitochondria in axons of cortical neurons.

“Tau overexpression also reduced the velocity of mitochondria in axons.”

• Also stated in: § Tau phosphorylation reduces mitochondrial motility in neuritic processes of PC12 cells · plain

  “The mean anterograde and retrograde velocities were 0.55 Ϯ 0.08 and 0.45 Ϯ 0.03 m/s, respectively, in control cells, but they were reduced to 0.17 Ϯ 0.03 and 0.18 Ϯ 0.06 m/s in Tau WT-overexpressing neurons, 0.18 Ϯ 0.03 and 0.19 Ϯ 0.07 m/s in Tau 3A-overexpressing neurons, and 0.18 Ϯ 0.03 and 0.18 Ϯ 0.04 m/s in Tau 3Doverexpressing neurons.”

Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing). The quote only partly states this claim.

In cortical neurons, velocities faster than 1 μm/s were not observed in any Tau-overexpressing neurons.

“Velocities faster than 1 m/s, which accounted for 15 and 11% of anterograde and retrograde movements, respectively, in control neurons, were not observed in any Tau-overexpressing neurons.”

May combine two claims.

In vitro, inter-microtubule distances in microtubule pellets polymerized with Tau WT, 3A, and 3D were typically less than 10 nm, indicating that the phosphorylation-mimicking mutation at AT8 did not affect inter-microtubule spacing in pelleted microtubules.

“The distances were typically Ͻ10 nm in the MT pellets polymerized with Tau WT, 3A, and 3D (Fig. 7D), indicating that the phosphorylationmimicking mutation at Ser199/Ser202/Thr205 did not affect the inter-MT spacing in pelleted MTs.”

May combine two claims.

In untreated Sf9 cell processes, the mean inter-MT distance was 15.7 ± 2.2 nm for Tau WT, 17.5 ± 2.7 nm for Tau 3A, and 16.3 ± 2.2 nm for Tau 3D.

“The mean distance was 15.7 Ϯ 2.2 nm for Tau WT (n ϭ 30), 17.5 Ϯ 2.7 nm for Tau 3A (n ϭ 27), and 16.3 Ϯ 2.2 nm for Tau 3D (n ϭ 28).”

May combine two claims.

Phosphorylated Tau 3A and 3D had similar, but not identical, banding patterns, suggesting that these mutants underwent similar phosphorylation at other sites.

“Phosphorylated Tau 3A and 3D had similar, but not identical, banding patterns, suggesting that these mutants underwent similar phosphorylation at other sites (Fig. 1 D).”

May combine two claims.

Tau WT had a higher level of phosphorylation than Tau 3A, including at the AT8 site.

“As a whole, the decreased mobility of Tau WT compared to Tau 3A indicated the higher level of phosphorylation of Tau WT, including the AT8 site.”

May combine two claims.

The ratio of anterogradely moving time to total moving duration was significantly different between control and Tau-overexpressing PC12 cells (p < 0.01), but not significant between the three Tau constructs (p = 0.75).

“The ratio was significantly different between control and Tau-overexpressing PC12 cells (*p Ͻ 0.01), but was not significant between three Tau constructs ( p ϭ 0.75).”

May combine two claims.

In latrunculin B-treated Sf9 cell processes, the mean inter-MT distance was 26.8 ± 2.7 nm for Tau WT, 27.2 ± 2.0 nm for Tau 3A, and 37.1 ± 2.3 nm for Tau 3D.

“The mean distance was 26.8 Ϯ 2.7 nm for Tau WT (n ϭ 60), 27.2 Ϯ 2.0 nm for Tau 3A (n ϭ 80), and 37.1 Ϯ 2.3 nm for Tau 3D (n ϭ 75).”

May combine two claims.

Tau phosphorylation at the AT8 sites may have meaningful effects on mitochondrial movement, likely by controlling microtubule spacing.

“Thus, Tau phosphorylation at the AT8 sites may have meaningful effects on mitochondrial movement, likely by controlling microtubule spacing.”

• Also stated in: § Discussion · hedged

  “we would like to propose that phosphorylation of Tau at the AT8 sites affects the transport of mitochondria along MTs by changing the inter-MT spaces.”

• AssumptionRests on an assumption: Increased inter-microtubule spacing impairs mitochondrial transport.

May combine two claims.

AT8 recognizes Tau phosphorylated at Ser202 and Thr205 or at Ser199, Ser202, and Thr205.

“AT8, which recognizes Tau that is phosphorylated at Ser202 and Thr205 (Goedert et al., 1995) or Ser199, Ser202, and Thr205 (Hanger et al., 2009).”

• cites Goedert et al., 1995 sourceNot checked — no full text of the source
• cites Hanger et al., 2009 sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

Increased repulsive forces between MTs would generate a stronger reactive resistance against mitochondria moving inside MT bundles.

“Increased repulsive forces between MTs would generate a stronger reactive resistance against mitochondria moving inside MT bundles.”

May combine two claims.

Phosphorylation at major Ser-Pro/Thr-Pro sites reduces but does not abolish Tau binding to MTs, leading to more dynamic MTs.

“Phosphorylation at these sites reduces, but does not abolish, Tau binding to MTs, leading to more dynamic MTs (Wada et al., 1998;Liu et al., 2007).”

• cites Wada et al., 1998 sourceNot checked — no full text of the source
• cites Liu et al., 2007 sourceNot checked — no full text of the source

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

This would suppress mitochondrial transport along MTs by restricting radial displacement of MTs.

“This would suppress mitochondrial transport along MTs by restricting radial displacement of MTs.”

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

The force produced by outward extension of the projection domain may not be strong enough to push surrounding MTs against the membrane, which may explain why phosphorylation-induced expansion of the inter-MT distance has not been reported.

“The force produced by outward extension of the projection domain may not be strong enough to push surrounding MTs against the membrane, which may explain why phosphorylation-induced expansion of the inter-MT distance has not been reported.”

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

AT8 reactivity has been frequently used as an indicator of hyperphosphorylation of Tau in AD brains or other tauopathies.

“AT8 reactivity has been frequently used as an indicator of hyperphosphorylation of Tau in AD brains or other tauopathies (Stoothoff and Johnson, 2005;Hanger et al., 2009).”

• cites Stoothoff and Johnson, 2005Not matched

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

• cites Hanger et al., 2009 sourceNot checked — no full text of the source

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

All experiments were performed at least three times.

“All experiments were performed at least three times, and representative results are shown.”

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

According to the paperclip structural model, the site near Ser199/Ser202/Thr205 folds and the N-terminal region is situated close to the MT-binding repeats.

“According to the paperclip structural model of Tau (Jeganathan et al., 2006), the site near Ser199/Ser202/Thr205 folds and the N-terminal region is situated close to the MT-binding repeats.”

• cites Jeganathan et al., 2006 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing), S4 (based).

Tau impairs mitochondrial movement by inhibiting the interaction of kinesin with microtubules.

“Tau impairs mitochondrial movement by inhibiting the interaction of kinesin with MTs (Hagiwara et al., 1994).”

• cites Hagiwara et al., 1994 sourceNot checked — no full text of the source

May combine two claims.

Increased phosphorylation of the AT8 sites in brains of Alzheimer's patients decreases mitochondrial transport in axons, leading to axonal degeneration.

“Thus, our results suggest that the increased phosphorylation of the AT8 sites in brains of Alzheimer's patients decreases mitochondrial transport in axons, leading to axonal degeneration.”

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

Tau is a phosphoprotein whose functions can be regulated by phosphorylation.

“Tau is also a phosphoprotein, the functions of which can be regulated by phosphorylation (Stoothoff and Johnson, 2005;Hanger et al., 2009).”

• cites Stoothoff and Johnson, 2005Not matched

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

• cites Hanger et al., 2009 sourceNot checked — no full text of the source

May combine two claims.

Phosphorylation of Tau at the AT8 sites tends to increase the inter-MT distance, but under strong tension the inter-MT distance cannot expand, and instead the repulsive forces between MTs increase.

“Phosphorylation of Tau at the AT8 sites tends to increase the inter-MT distance, but under strong tension the inter-MT distance cannot expand (Fig. 8), and instead the repulsive forces between MTs increase (Fig. 9).”