PubMed: 27594586

MMP-9 and MMP-2 Contribute to Neuronal Cell Death in iPSC Models of Frontotemporal Dementia with MAPT Mutations.
Stem cell reports
Karydas A | Futai K | Lopez-Gonzalez R | Miller BL | Mao W | Biswas MHU | Almeida S | Gao FB | Geschwind MD | Zhang Z | Biernat J | Mandelkow EM

Evidence c5ad644c6e

ERK phosphorylation in MAPT mutant neurons was blocked by treatment with PD98059, a specific inhibitor of MEK-mediated activation of ERK (Li et al., 2001) (Figures 4B and 4C)

Evidence 368b73c15b

Correspondingly, the level and activity of secreted MMP-9 was greatly suppressed in both tau-A152T and MAPT IVS10+16 patient neurons (Figures 4B and 4C), suggesting that MMP-9 expression is regulated by the ERK signaling pathway in cortical neurons

Evidence c71bf60195

Interestingly, the level and activity of MMP-2 was not affected after the PD98059 treatment in tau-A152T neurons (Figure 4B) and MAPT IVS10+16 neurons (Figure 4C)

Evidence c793ba0246

We treated these neurons with staurosporine, a broad-spectrum kinase inhibitor that induces apoptosis (Budd et al., 2000); rotenone, a complex inhibitor that induces mitochondrial dysfunction (Sherer et al., 2003); and rapamycin, an inhibitor of the mammalian target of the rapamycin (mTOR) signaling pathway with many downstream effects (Li et al., 2014)

Evidence db72c96425

MMP-9/MMP-2 mRNA levels in tau-A152T and MAPT IVS10+16 neurons were significantly higher than that in control neurons (Figure S2J), and rapamycin increased these levels (p < 0.01) (Figure 3C)

Evidence 851d6f66c8

When measuring cell viability using the MTT assay, we found that patient-specific neurons are more sensitive than control neurons to cell death induced by rapamycin (Figure 3B) but not by staurosporine or rotenone (Figures S3B and S3C), suggesting compromised function in the mTOR pathway or related molecular pathways

Evidence b73844fc4f

Four weeks after terminal differentiation, 80% of cells were positive for the neuron-specific marker MAP2, and there was no significant difference between control and patient neurons (Figure S2A)

Evidence 5d39c34f14

The percentages of VGLUT1-positive excitatory and GABA-positive inhibitory neurons were similar in neuronal cultures differentiated from control and patient iPSCs (Figures S2B and S2C)

Evidence 285cf3f279

Moreover, the total expression levels of tau and PSD-95 were the same in 1-month-old neurons derived from control and patient iPSCs (Figure 1B)

Evidence c1b66358a1

The resting membrane potential did not differ among neurons differentiated from all iPSC lines (Figure S2D)

Evidence 8ebb0ac36f

Thus, both molecular and electrophysiological analyses suggest MAPT mutations do not affect early neuronal differentiation

Evidence e96d8f5473

Tau-A152T neurons had >10-fold higher level and activity of secreted MMP-9 than control neurons (Figures 2A and 2B), as did MAPT IVS10+16 neurons to a lesser extent (Figures 2C and 2D)

Evidence 7e13deb6c0

The level of secreted MMP-9 and MMP-2 was also increased in cortical neurons derived from a published iPSC line with the tau-A152T mutation (Fong et al., 2013) (Figures 2E and 2F)

Evidence f4ac108ff7

It is worth noting that the level and activity of secreted MMP-9 in tau- A152T neurons is also substantially higher than that in MAPT IVS10+16 neurons (Figure 2)

Evidence b70b83a701

This increase is likely in part due to transcriptional regulation, since the mRNA levels of MMP-9 and MMP-2 in tau-A152T and MAPT IVS10+16 neurons were higher than in control neurons (Figure S2J)

Evidence 9e83c30177

We found that, indeed, TUJ1+ neurons showed reduced survival as measured by TUNEL analysis (Figure 3A)

Evidence 3d24568e7b

The level of phosphorylated ERK (p-ERK) was significantly increased in tau-A152T neurons and to a lesser extent in MAPT IVS10+16 patient neurons (Figure 4A)

Evidence 204b51d746

Interestingly, this ratio is also increased, but to a lesser extent, in tau-A152T neurons (Figure S3F)

Evidence a573b45019

As expected, the 4R/3R tau ratio is greatly increased in MAPT IVS10+16 neurons (Figure S3F) but not in GRN mutant neurons (Figure S3G)

Evidence 493a258a08

It is known that MMP-9 expression is regulated by ERK (Wang et al., 2006)

Evidence a3052f89cb

We found the level and activity of secreted MMP-9 to correlate well with the extent of ERK phosphorylation (Figure S4A)

Evidence b6812f0664

Interestingly, the level and activity of secreted MMP-9 in HEK293 cells expressing WT 3R tau and 3R tau-A152T are similar (Figures S4B and S4C)

Evidence c0b14909b1

ERK phosphorylation is also not affected by A152T mutation in 3R tau (Figure S4D)

Evidence a7e0e27798

However, the secreted MMP-9 level and activity in conditioned medium was significantly higher in cultures of cells expressing tau-A152T than WT tau (Figure 4E)

Evidence 95d3a27b5b

Taken together, these findings strongly suggest that 4R tau-A152T activates the ERK pathway more strongly, which in turn increases the level and activity of secreted MMP-9. 4R tau-A152T expression (Figures 4E and 4F) but not inhibition of ERK activity (Figures 4B and 4C) also increases the total activity of secreted MMP-2, suggesting that tau-A152T increases the level and activity of secreted MMP-2, likely through an ERK-independent pathway

Evidence 5a355741ce

The MMP-9 mRNA level was also higher in cells expressing tau-A152T (Figure 4F)

Evidence 5f63702704

Correspondingly, the level of p-ERK was also elevated in HEK293 cells expressing tau-A152T (Figure 4E)

Evidence 4ab634b851

To examine the contributions of increased MMP-9 and MMP-2 to neuronal cell death, we used two compounds that are known to inhibit MMP-9 and MMP-2 activity (catalog #444278 and #444244, Calbiochem). We found that this treatment partially rescued the increased sensitivity to rapamycin and improved survival (Figure 3B)

Evidence e83e3d2797

MMP-2 and MMP-9 seem to induce cell death directly, since this treatment also increased the TUNEL signal in iPSC-derived neurons (Figures S3D and S3E)

Evidence 6054f27e78

MMP-9 directly cleaves Huntingtin protein in Huntington’s disease, a progressive neurodegenerative disorder (Miller et al., 2010)

Evidence 23f1cf8c8e

Moreover, dysregulation of MMP-9 in the CNS is a cause of neuronal developmental disorders (Reinhard et al., 2015)


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