Transcriptomic analysis of these isolated astrocytes revealed an increase in TFEB tran- scripts, as well as transcripts of several of its well-known target genes in the rTg4510 relative to wild-type controls
In addition, flow cytometry analysis demonstrated that heparin reduces dye-conjugated pff uptake in both TFEB and EGFP transduced astrocytes, suggesting that macropino- cytosis is responsible for astroglial pff uptake
Inhibiting mTORC1 blocks the phosphor- ylation of TFEB, allowing unphosphorylated TFEB to translocate to the nucleus and become transcriptionally active
TFEB activation has been linked to lysosomal stress and the ac- cumulation of aberrant protein aggregates, indicated by TFEB’s nuclear localization in lysosomal storage disorders
as expected, the expression of several TFEB lysosomal target genes was signifi- cantly up-regulated in TFEB-transduced astrocytes, including lysosomal marker LAMP1 and lysosomal proteases, cathepsins A and B
In sum, these results demonstrate that TFEB regulates the lysosomal pathway in primary astrocytes.
TFEB not only increased the proportion of cells taking up beads, but also increased the pro- portion of cells taking up multiple beads, suggesting an enhanced phagocytic capacity
Our results demonstrate a statistically significant increase of 12% in the uptake of pffs in TFEB transduced astrocytes relative to control at the 1-h time point, with a 22% relative increase in up- take in the TFEB transduced cells at 4 h
Flow cytometry revealed that Torin1 treatment of TFEB transduced astrocytes increased dye-conjugated pff uptake 63% relative to EGFP transduced controls as shown by median fluorescence in- tensity, while under basal conditions, the TFEB overexpressing astrocytes increased uptake just 18% relative to EGFP expressing controls (Fig. 2 K). Thus, TFEB enhances phagocytic pathways in astrocytes, in particular increasing the uptake of pffs.
. Recapitulating the uptake assay with dye-conjugated pffs in TFEB KO astrocytes, we observed a modest reduction pff uptake compared with primary astrocytes from littermate controls
In sum, these data suggest that TFEB stimulates uptake and trafficking of pffs to the lysosome for degradation.
qRT-PCR demonstrated undetectable transcript levels of TFEB as well as a reduction in LAMP1 mRNA in TFEB KO astrocytes compared with littermate controls
Flow cytometry analysis of primary astrocytes incubated with DQ-BSA revealed an ∼60% increase in median fluorescence for TFEB-transduced astro- cytes, indicating that TFEB enhances uptake and proteolysis of DQ-BSA
Immunoblot- ting for total tau and phospho-tau antibodies, PHF1 and CP13, demonstrated no statistical difference in hippocampal brain ly- sate from TFEB- versus EGFP-injected rTg4510 male or female mice
Western blot a reduction in phospho-tau species recognized by AT8 (trending), AT180, and CP13 antibodies in the TFEB-injected mice
In the insoluble fraction, phospho- and total tau species were all reduced in the TFEB mice, as demonstrated by Western blot
Quantifying the area fluorescence of MC1 staining for both hippocampi from sections representing the entire volume of hippocampus showed that astroglial TFEB had no impact on MC1 staining on the ipsi- lateral side, but significantly reduced the area of MC1 staining on the contralateral side
Overall, these results show that astroglial TFEB overexpression reduces tau pathology and gliosis in the hippocampus of PS19 tauopathy mice.
Transcriptional levels of TFEB and several of its well-known lysosomal targets were stratified by no demen- tia, mild cognitive impairment, and dementia,these transcript levels positively correlate with cognitive decline
. In addition, human brains diagnosed with frontotemporal demen- tia (FTD) demonstrated an increase in transcriptional levels of TFEB and TFEB lysosomal target gene LAMP1
On the protein level, TFEB lysosomal target genes LAMP1 and Cathep- sin D (CTSD) are also increased in the FTD frontal cortex relative to normal subjects, and TFEB protein levels trended increased with statistical significance in the CBD group
In the rTg4510 mouse model of tauopathy (Ramsden et al., 2005; SantaCruz et al., 2005), gene set enrichment analysis (GSEA) of microarray data revealed a similar enrichment of TFEB’s tran- scriptional profile when comparing 4-mo-old transgenic mice with widespread NFTs to wild-type mice, indicating TFEB’s activation with tau pathology (Fig. 1 F).
Examining the protein levels in another tauopathy mouse model, hippocampal lysates from PS19 mice (Yoshiyama et al., 2007) at 10 mo of age showed increases in TFEB and lysosomal proteins LAMP1 and CTSD com- pared with wild-type mice (Fig. S1, G and H).
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