In Situ Detection of Starvation-induced Autophagy

Abstract

Autophagy is a regulated bulk degradation process involved in many different human pathologies. Transmission electron microscopy (TEM) is currently the only reliable method for monitoring autophagy in situ. Because TEM is labor intensive, we questioned whether useful marker proteins can be found for unambiguous detection of autophagy in tissue via routinely used colorimetric, immunohistochemical, or fluorescent techniques. Starved HepG2 hepatocytes and nutrient deprived liver tissue were used as a model for the initiation of autophagy. Our findings indicate that starvation-induced autophagy in HepG2 cells was associated neither with differential mRNA gene expression nor with changes in the expression level of known autophagy-related proteins. On the contrary, both transcription and translation were inhibited, suggesting that the identification of autophagy-specific biomarkers for tissue is highly compromised. Light chain 3 (LC3) protein, which is an attractive marker of autophagosomes, revealed a relatively low expression level in tissue and cultured cells, but could be detected via immunohistochemistry in liver from GFP-LC3 transgenic mice. The number of LC3 immunopositive dot-like structures was significantly upregulated in liver tissue from nutrient-deprived GFP-LC3 mice as compared with nonstarved control tissue. Our results suggest that LC3 immunostaining can be used as an alternative detection method for autophagy in situ, but only when this protein is overexpressed.

Immunohistochemical analysis of macroautophagy

“…[3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] Detection guidelines have also been established for the correct use and interpretation of such methods. [19][20][21] Still demonstration of autophagy in tissue has not received extensive evaluation, particularly in clinical specimens for which genetic techniques (e.g., GFP-LC3 overexpression), autophagic flux measurements and injection of specific dyes are inconceivable.…”

“…Because mammalian LC3 is one of the most frequently used biomarkers for autophagy both in vitro 3,4,19,21 and in situ, [11][12][13][14][15][16] starved and nonstarved livers Nutrient deprivation induces autophagy in liver. To validate immunocytochemical methods for the detection of autophagy, nutrient deprivation was used as a trigger.…”

“…11,12 A western blot analysis of different mouse tissue lysates revealed that the expression of LC3A and LC3B may vary considerably. Expression of LC3A and LC3B is relatively weak in liver as well as in many other organs such as heart, spleen and lung.…”

“…[9][10][11][12][13][14][15][16][17][18] Advantages of immunohistochemistry include a fast turnaround time (typically 2 d vs 5-7 d for TEM), low cost, ease of performance and widespread familiarity. However, most immunohistochemical staining protocols for autophagy-associated proteins have not been validated (e.g., by using autophagy-deficient tissue as negative control) or optimized to obtain high sensitivity.…”

Sequestration Revisited: Integrating Traditional Electron Microscopy, De Novo Assembly and New Results

“…1 Since the first picture of a macroautophagic structure was published in 1957, 2 electron microscopy (EM) remains an indispensable method and ultimate reference base to study this process [3][4][5] even though the discovery of autophagy genes 6,7 makes it possible to construct fluorescence-tagged autophagy proteins and produce antibodies against them. In traditional transmission EM preparations the cellular structures are made visible by the differential incorporation of heavy metals into chemically fixed cellular material.…”