Expression of Tau Produces Aberrant Plasma Membrane Blebbing in Glial Cells Through RhoA-ROCK-Dependent F-Actin Remodeling

Inclusion of NSC23766, a specific inhibitor of Rac1, did not affect the formation of PM blebs.

“However, we discarded this possibility because the inclusion of NSC23766, a specific inhibitor of Rac1 [65], did not affect the formation of PM blebs (data not shown).”

• cites [65] Gao, 2004, Rational design and characterization of a Rac GTPase-specific small molecule inhibitor, Proc Natl Acad Sci U S A sourceSupported

This result supports recent in vitro evidence that Tau can induce guided polymerization of Actin filaments along microtubule tracks.

“This result supports recent in vitro evidence that Tau can induce guided polymerization of Actin filaments along microtubule tracks [27, 68].”

• cites [27] Elie, 2015, Tau co-organizes dynamic microtubule and actin networks, Sci Rep sourceSupported
• cites [68] Mohan, 2015, Microtubule-associated proteins as direct crosslinkers of actin filaments and microtubules, IUBMB Life sourceNot checked — no full text of the source

Even after 72 h of transfection, no difference in cell viability was found between Tau-expressing and empty vector groups.

“Even after 72 h of transfection, no difference was found between these groups.”

Nocodazole but not Taxol treatments produced plasma membrane blebs in more than 80% of the cells analyzed.

“Nocodazole- but not Taxol-treatments produced PM blebs in more than 80% of the cells analyzed.”

After 30 min of Cytochalasin-D washing, approximately 85% of cells expressing Tau441 again developed plasma membrane blebs.

“After 30 min of Cytochalasin-D washing, approximately 85% of the cells expressing Tau441 again came to develop PM blebs.”

When C3-transferase was incubated while Tau expression began in a separate group of transfected cells, no evidence of PM blebbing was observed during 48 h of treatment.

“while Tau expression began in a separate group of transfected cells, no evidence of PM blebbing was observed during 48 h of treatment (green channel, Fig. 7C).”

Expression of Asp421-truncated and non-truncated Tau produced abnormal bundling of microtubules in C6-glial cells.

“in the present study we also corroborated that expression of Asp421-truncated and non-truncated Tau produced abnormal bundling of microtubules in C6-glial cells (Fig. 1A, B).”

After stimulation with high concentrations of FBS, a significant amount of empty-vector-transfected cells migrate to the wounded area to occupy the available space.

“As shown in Fig. 8A, after stimulation with high concentrations of FBS, significant amount of empty-vector-transfected cells migrate to the wounded area to occupy the available space (arrowheads).”

In the majority of Cytochalasin-D-treated Tau441-expressing cells, only a positive pool of Actin remains in polarized clusters.

“In the majority of cells, only a positive pool of Actin remains in polarized clusters (yellow arrow, Fig. 3D).”

When C6 cells expressing Tau441 were incubated with C3-transferase, Y-27632, or Blebbistatin, the number of cells showing plasma membrane blebs was significantly reduced compared with non-treated cells.

“When C6 cells expressing Tau441 were incubated with either C3-transferase (0.5 �g/ mL) (to inhibit RhoA-GTPase activity), Y-27632 (10 �M) (to inhibit ROCK), or Blebbistatin (10 �M) (to inhibit Myosin-II activity), the number of cells showing PM blebs was significantly reduced in comparison to non-treated cells. ∗p<0.0001, by one-way Analysis of variance (ANOVA) and consecutive Tukey's multiple”

The number of Tau-expressing C6-glial cells that developed plasma membrane blebs is reduced to 5% when Cytochalasin-D is incubated in the growth media for 30 min.

“The number of Tau-expressing C6-glial cells that developed PM blebs is reduced to 5% when Cytochalasin-D (20 �M) is incubated in the growth media for 30 min.”

Similar results were observed when Tau421-expressing cells were treated and washed with Cytochalasin-D.

“Similar results were observed when Tau421-expressing cells were treated and washed with this drug (data not shown).”

No evidence of free cytoplasmic GEF-H1 is found when the microtubule bundling effect was induced with Taxol in cells with no expression of Tau.

“No evidence of free cytoplasmic GEF-H1 is found when the microtubule bundling effect was induced with Taxol in cells with no expression of Tau (C: empty vector transfected cells; D: non-transfected cells [NTc]).”

When Tau441-expressing cells were stimulated, only few amounts of these cells migrate to the wounded area.

“By contrast, when Tau441-expressing cells were also stimulated, only few amounts of these cells migrate to the wounded area (arrows).”

Confirmation of F-Actin-myosin interaction during Tau-induced PM blebbing was obtained when cells were incubated with Blebbistatin, a specific inhibitor of Myosin-II activity, and neither F-Actin remodeling nor PM blebs formation were observed.

“Confirmation of F-Actin-myosin interaction during Tau-induced PM blebbing was obtained when cells were incubated with Blebbistatin, a specific inhibitor of Myosin-II activity [49], and neither F-Actin remodeling nor PM blebs formation were observed (Fig. 4C).”

• cites [49] Straight, 2003, Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor, Science sourceNot checked — no full text of the source

Immunoblot analysis with Tau-5 confirmed the lack of endogenous Tau expression in both normal cells and cells transfected only with the empty vector.

“Immunoblot analysis with the Tau-5 antibody, which recognizes residues 210–230 in the molecule of Tau (see Table 1) also confirmed the lack of endogenous expression of Tau in both normal cells and in cells only transfected with the empty vector (pcDNA3.1) (Fig. 1D).”

In C6-glial cells, expression of Tau produces some release of GEF-H1 from microtubules into the cytoplasmic space.

“In C6-glial cells, expression of Tau produces some release of GEF-H1 from microtubules into the cytoplasmic space.”

• Also stated in: § DISCUSSION · plain

  “when we analyzed GEF-H1 localization in Tau-expressing C6-glial cells, we found that some of this factor remains bound to the microtubule bundles; however, a free pool of this factor was also observed in the cytoplasm (Fig. 6B).”

By DIC microscopy, Tau-expressing cells acquired a deformed multilobular morphology compared with the flat shape of non-transfected cells.

“When Tau-expressing cells were viewed through differential interference contrast (DIC) microscopy, the manner in which these cells acquired deformed multilobular morphology was noticeable (arrows, Fig. 2A), in comparison with the flat shape displayed by non-transfected cells.”

The Asp214-cleaved PARP product is less detected in non-transfected cells than in Tau-expressing cells.

“This product is less detected in non-transfected cells.”

Incubation with C3-transferase (0.5 μg/mL) for 48 h did not show any alteration in the normal organization of the cytoskeleton components and cell morphology in non-Tau-expressing C6 cells.

“of Tau, incubation with C3-transferase (0.5 �g/mL) for 48 h did not show any alteration in the normal organization of the cytoskeleton components and cell morphology (Fig. 7A).”

Less than 20% of the cells that express Tau migrated to the wounded area.

“Quantitatively, less than 20% of the cells that express Tau migrated to this area.”

Tau-expressing cells showed negative TUNEL staining, indicating lack of induced apoptosis.

“The lack of induced apoptosis in Tau-expressing cells was evidenced by negative staining with the TUNEL assay (Fig. 2B, C).”

When C6 cells expressed either Tau441 or Tau421, severe morphological changes were observed, including formation of plasma membrane blebs along the entire cell surface.

“In contrast, when C6 cells expressed either Tau441 or Tau421, severe morphological changes were observed, such as the formation of PM blebs along the entire cell surface (yellow arrows, Fig. 1A, B).”

In the majority of Tau-expressing cells, morphological change was accompanied by abnormal bundling of the Tubulin cytoskeleton.

“In the majority of Tau-expressing cells, morphological change was also accompanied by abnormal bundling of the Tubulin cytoskeleton (arrowheads, Fig. 1A, B).”

In C6 cells with no Tau expression, the majority of GEF-H1 is observed in close association with the microtubule lattice.

“As depicted in Fig. 6A, in C6 cells with no Tau expression, the majority of the GEF-H1 is observed in close association with the microtubule lattice (arrows).”

Both the PM blebbing and F-Actin remodeling observed in Tau-expressing cells were reverted when Y-27632 was included in the incubation media.

“In contrast, both the PM blebbing and F-Actin remodeling observed in Tau-expressing cells (Fig. 7B) were reverted when Y-27632 was included in the incubation media (arrows, Fig. 7D).”

Cell migration in a scratch-wound assay was altered in Tau-expressing C6-glial cells.

“Cell migration in a scratch-wound assay was altered in Tau-expressing C6-glial cells.”

• AssumptionRests on an assumption: C6-glial cells are a valid model for glial cells in Alzheimer's disease and other tauopathies.
• AssumptionRests on an assumption: Transient expression of Tau441 or Tau421 in C6-glial cells reproduces the pathological accumulation of Tau in glial cells.

Abnormal bundling of microtubules specifically induced by Tau promotes reassembly of F-Actin that gives rise to plasma membrane blebs.

“These results may indicate that abnormal bundling of microtubules specifically induced by Tau promotes a reassembly of the F-Actin that gives rise to the formation of PM blebs.”

PM blebbing in Tau-expressing cells depends on F-Actin cytoskeleton remodeling.

“PM blebbing in Tau-expressing cells depends on F-Actin cytoskeleton remodeling”

PM blebbing in Tau-expressing C6 cells was regulated by RhoA-GTPase and ROCK signaling, because the addition of their specific inhibitors, C3-transferase and Y-27632, respectively, avoided the formation of these structures.

“In our study, we corroborated that PM blebbing in Tau-expressing C6 cells was also regulated by RhoA-GTPase and ROCK signaling, because the addition of their specific inhibitors, C3-transferase and Y-27632, respectively, avoided the formation of these structures (Figs. 7C, D).”

Plasma membrane blebs do not originate as apoptotic consequences in Tau-expressing cells.

“These results indicate that PM blebs do not originate as apoptotic consequences in Tau-expressing cells.”

Because glial cells contribute to neuronal maintenance and survival, Tau-induced alterations in these cells may also account for the neurodegenerative process observed in the brains of individuals suffering from tauopathies.

“Because glial cells contribute to the neuronal maintenance and survival, Tau-induced alterations in these cells may also account for the neurodegenerative process observed in the brains of individuals suffering from tauopathies.”

• AssumptionRests on an assumption: Glial cells contribute to neuronal maintenance and survival.

Tau-induced microtubule bundling may release microtubule-bound GEF-H1 into the cytoplasm to activate RhoA-GTPase.

“All of these effects were initiated upstream by Tau-induced microtubule bundling, which may release microtubule-bound Guanine nucleotide exchange factor-H1 (GEF-H1) into the cytoplasm in order to activate its major effector, RhoA-GTPase.”

• AssumptionRests on an assumption: C6-glial cells are a valid model for glial cells in Alzheimer's disease and other tauopathies.
• AssumptionRests on an assumption: Transient expression of Tau441 or Tau421 in C6-glial cells reproduces the pathological accumulation of Tau in glial cells.

These results may represent a new mechanism of Tau toxicity that contributes to impairment of glial activity and neuronal dysfunction in AD and other tauopathies.

“Taken together, these results may represent a new mechanism of Tau toxicity that contributes to the impairment of glial activity and neuronal dysfunction reported in AD and other tauopathies.”

• AssumptionRests on an assumption: C6-glial cells are a valid model for glial cells in Alzheimer's disease and other tauopathies.
• AssumptionRests on an assumption: Transient expression of Tau441 or Tau421 in C6-glial cells reproduces the pathological accumulation of Tau in glial cells.

F-Actin remodeling is produced by indirect activation of the GEF-H1-RhoA-ROCK pathway.

“We focused on some intracellular mechanisms leading to F-Actin remodeling, and found that it is produced by indirect activation of the GEF-H1-RhoA-ROCK pathway.”

Tau-induced remodeling of the Actin cytoskeleton is involved in plasma membrane blebbing.

“It appears that Tau-induced remodeling of the Actin cytoskeleton is involved in PM blebbing.”