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Transcription Factor EB Is Selectively Reduced in the Nuclear Fractions of Alzheimer's and Amyotrophic Lateral Sclerosis Brains.

Wang H, Wang R, Xu S, Lakshmana MK - Neurosci J (2016)

Bottom Line: Most importantly, normalized (to lamin) TFEB levels in the nuclear fractions were consistently reduced starting from Braak stage IV (52%, p < 0.01), stage V (67%, p < 0.01), and stage VI (85%, p < 0.01) when compared to normal control (NC) brains.These data suggest that nuclear TFEB is selectively lost in ALS as well as AD brains, in which TFEB reduction was Braak-stage-dependent.Taken together, the observed reductions in TFEB protein levels may be responsible for the widely reported autophagy defects in these disorders.

View Article: PubMed Central - PubMed

Affiliation: Section of Neurobiology, Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, FL 34987, USA.

ABSTRACT
Multiple studies suggest that autophagy is strongly dysregulated in Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), as evidenced by accumulation of numerous autophagosomes, lysosomes with discontinuous membranes, and aggregated proteins in the patients' brains. Transcription factor EB (TFEB) was recently discovered to be a master regulator of lysosome biogenesis and autophagy. To examine whether aberrant autophagy in AD and ALS is due to alterations in TFEB expression, we systematically quantified the levels of TFEB in these brains by immunoblotting. Interestingly, cytoplasmic fractions of AD brains showed increased levels of normalized (to tubulin) TFEB only at Braak stage IV (61%, p < 0.01). Most importantly, normalized (to lamin) TFEB levels in the nuclear fractions were consistently reduced starting from Braak stage IV (52%, p < 0.01), stage V (67%, p < 0.01), and stage VI (85%, p < 0.01) when compared to normal control (NC) brains. In the ALS brains also, nuclear TFEB levels were reduced by 62% (p < 0.001). These data suggest that nuclear TFEB is selectively lost in ALS as well as AD brains, in which TFEB reduction was Braak-stage-dependent. Taken together, the observed reductions in TFEB protein levels may be responsible for the widely reported autophagy defects in these disorders.

No MeSH data available.


Related in: MedlinePlus

Braak-stage-dependent loss of nuclear expression of the transcription factor TFEB in Alzheimer's brains. Homogenates were prepared from the hippocampus of AD brains classified as Braak stages II to VI or age-matched normal controls (NC) and nuclear fractions were separated and subjected to SDS-PAGE. Nuclear fractions showed robustly decreased normalized (to lamin) TFEB levels starting from Braak stage IV (52%), stage V (67%), and stage VI (85%). Nuclear lamin was detected as a marker of nuclear fractions. ∗∗p < 0.01 by ANOVA followed by Dunnett multiple comparisons test. Data are mean ± SEM, and “n” are indicated on the figure.
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fig2: Braak-stage-dependent loss of nuclear expression of the transcription factor TFEB in Alzheimer's brains. Homogenates were prepared from the hippocampus of AD brains classified as Braak stages II to VI or age-matched normal controls (NC) and nuclear fractions were separated and subjected to SDS-PAGE. Nuclear fractions showed robustly decreased normalized (to lamin) TFEB levels starting from Braak stage IV (52%), stage V (67%), and stage VI (85%). Nuclear lamin was detected as a marker of nuclear fractions. ∗∗p < 0.01 by ANOVA followed by Dunnett multiple comparisons test. Data are mean ± SEM, and “n” are indicated on the figure.

Mentions: Although a majority of endogenous TFEB is found in the cytoplasm under basal conditions, lower levels of TFEB can also be found in the nucleus and a fraction on the lysosomes as well [37]. However, under conditions of stress such as starvation, majority of cytoplasmic TFEB translocate to the nucleus and regulate transcription [37], resulting in biogenesis of new lysosomes [28, 29]. Therefore, we quantified TFEB protein levels in the nuclear and cytosolic fractions of AD brains and compared them with those of NC brains. Positive detection of tubulin in the cytosolic fractions (Figure 1) and lamin A and lamin C in the nuclear fractions (Figure 2) ensured noncontamination of the cytosolic and nuclear preparations. In the cytosolic fractions, normalized TFEB protein levels (to tubulin) were significantly increased by 61% (p < 0.01) at Braak stage IV AD brains compared to NC (Figure 1). Though Braak stage II also showed increased trend, it was not significantly different from NC brains (Figure 1). On the contrary, nuclear levels of normalized (to lamin) TFEB protein were consistently reduced starting from Braak stage IV (Figure 2). The reduction was 52% (p < 0.01) at Braak stage IV, 67% (p < 0.01) at stage V, and 85% (p < 0.01) at stage VI (Figure 2). Thus, nuclear TFEB is almost completely lost at Braak stage VI. This also suggests that expression levels of nuclear TFEB are inversely proportional to the extent of tau pathology in AD brains, since Braak staging is based on the extent of tau pathology [38]. Also, alterations in TFEB levels were independent of postmortem interval, sex, or age. Since TFEB protein levels were normalized to that of lamin or tubulin levels, neuronal loss occurring in AD was controlled. Therefore, it is unlikely that reduction in TFEB protein levels in AD brains is due to neuronal cell loss.


Transcription Factor EB Is Selectively Reduced in the Nuclear Fractions of Alzheimer's and Amyotrophic Lateral Sclerosis Brains.

Wang H, Wang R, Xu S, Lakshmana MK - Neurosci J (2016)

Braak-stage-dependent loss of nuclear expression of the transcription factor TFEB in Alzheimer's brains. Homogenates were prepared from the hippocampus of AD brains classified as Braak stages II to VI or age-matched normal controls (NC) and nuclear fractions were separated and subjected to SDS-PAGE. Nuclear fractions showed robustly decreased normalized (to lamin) TFEB levels starting from Braak stage IV (52%), stage V (67%), and stage VI (85%). Nuclear lamin was detected as a marker of nuclear fractions. ∗∗p < 0.01 by ANOVA followed by Dunnett multiple comparisons test. Data are mean ± SEM, and “n” are indicated on the figure.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940567&req=5

fig2: Braak-stage-dependent loss of nuclear expression of the transcription factor TFEB in Alzheimer's brains. Homogenates were prepared from the hippocampus of AD brains classified as Braak stages II to VI or age-matched normal controls (NC) and nuclear fractions were separated and subjected to SDS-PAGE. Nuclear fractions showed robustly decreased normalized (to lamin) TFEB levels starting from Braak stage IV (52%), stage V (67%), and stage VI (85%). Nuclear lamin was detected as a marker of nuclear fractions. ∗∗p < 0.01 by ANOVA followed by Dunnett multiple comparisons test. Data are mean ± SEM, and “n” are indicated on the figure.
Mentions: Although a majority of endogenous TFEB is found in the cytoplasm under basal conditions, lower levels of TFEB can also be found in the nucleus and a fraction on the lysosomes as well [37]. However, under conditions of stress such as starvation, majority of cytoplasmic TFEB translocate to the nucleus and regulate transcription [37], resulting in biogenesis of new lysosomes [28, 29]. Therefore, we quantified TFEB protein levels in the nuclear and cytosolic fractions of AD brains and compared them with those of NC brains. Positive detection of tubulin in the cytosolic fractions (Figure 1) and lamin A and lamin C in the nuclear fractions (Figure 2) ensured noncontamination of the cytosolic and nuclear preparations. In the cytosolic fractions, normalized TFEB protein levels (to tubulin) were significantly increased by 61% (p < 0.01) at Braak stage IV AD brains compared to NC (Figure 1). Though Braak stage II also showed increased trend, it was not significantly different from NC brains (Figure 1). On the contrary, nuclear levels of normalized (to lamin) TFEB protein were consistently reduced starting from Braak stage IV (Figure 2). The reduction was 52% (p < 0.01) at Braak stage IV, 67% (p < 0.01) at stage V, and 85% (p < 0.01) at stage VI (Figure 2). Thus, nuclear TFEB is almost completely lost at Braak stage VI. This also suggests that expression levels of nuclear TFEB are inversely proportional to the extent of tau pathology in AD brains, since Braak staging is based on the extent of tau pathology [38]. Also, alterations in TFEB levels were independent of postmortem interval, sex, or age. Since TFEB protein levels were normalized to that of lamin or tubulin levels, neuronal loss occurring in AD was controlled. Therefore, it is unlikely that reduction in TFEB protein levels in AD brains is due to neuronal cell loss.

Bottom Line: Most importantly, normalized (to lamin) TFEB levels in the nuclear fractions were consistently reduced starting from Braak stage IV (52%, p < 0.01), stage V (67%, p < 0.01), and stage VI (85%, p < 0.01) when compared to normal control (NC) brains.These data suggest that nuclear TFEB is selectively lost in ALS as well as AD brains, in which TFEB reduction was Braak-stage-dependent.Taken together, the observed reductions in TFEB protein levels may be responsible for the widely reported autophagy defects in these disorders.

View Article: PubMed Central - PubMed

Affiliation: Section of Neurobiology, Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, FL 34987, USA.

ABSTRACT
Multiple studies suggest that autophagy is strongly dysregulated in Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), as evidenced by accumulation of numerous autophagosomes, lysosomes with discontinuous membranes, and aggregated proteins in the patients' brains. Transcription factor EB (TFEB) was recently discovered to be a master regulator of lysosome biogenesis and autophagy. To examine whether aberrant autophagy in AD and ALS is due to alterations in TFEB expression, we systematically quantified the levels of TFEB in these brains by immunoblotting. Interestingly, cytoplasmic fractions of AD brains showed increased levels of normalized (to tubulin) TFEB only at Braak stage IV (61%, p < 0.01). Most importantly, normalized (to lamin) TFEB levels in the nuclear fractions were consistently reduced starting from Braak stage IV (52%, p < 0.01), stage V (67%, p < 0.01), and stage VI (85%, p < 0.01) when compared to normal control (NC) brains. In the ALS brains also, nuclear TFEB levels were reduced by 62% (p < 0.001). These data suggest that nuclear TFEB is selectively lost in ALS as well as AD brains, in which TFEB reduction was Braak-stage-dependent. Taken together, the observed reductions in TFEB protein levels may be responsible for the widely reported autophagy defects in these disorders.

No MeSH data available.


Related in: MedlinePlus