Beth Levine's Legacy: From the Discovery of BECN1 to Therapies. A Mentees' Perspective.

With great sadness, the scientific community received the news of the loss of Beth Levine on 15 June 2020. Dr. Levine was a pioneer in the autophagy field and work in her lab led not only to a better understanding of the molecular mechanisms regulating the pathway, but also its implications in multiple physiological and pathological conditions, including its role in development, host defense, tumorigenesis, aging or metabolism. This review does not aim to provide a comprehensive view of autophagy, but rather an outline of some of the discoveries made by the group of Beth Levine, from the perspective of some of her own mentees, hoping to honor her legacy in science.

TLR9 and beclin 1 crosstalk regulates muscle AMPK activation in exercise.

The activation of adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle coordinates systemic metabolic responses to exercise1. Autophagy-a lysosomal degradation pathway that maintains cellular homeostasis2-is upregulated during exercise, and a core autophagy protein, beclin 1, is required for AMPK activation in skeletal muscle3. Here we describe a role for the innate immune-sensing molecule Toll-like receptor 9 (TLR9)4, and its interaction with beclin 1, in exercise-induced activation of AMPK in skeletal muscle. Mice that lack TLR9 are deficient in both exercise-induced activation of AMPK and plasma membrane localization of the GLUT4 glucose transporter in skeletal muscle, but are not deficient in autophagy. TLR9 binds beclin 1, and this interaction is increased by energy stress (glucose starvation and endurance exercise) and decreased by a BCL2 mutation3,5 that blocks the disruption of BCL2-beclin 1 binding. TLR9 regulates the assembly of the endolysosomal phosphatidylinositol 3-kinase complex (PI3KC3-C2)-which contains beclin 1 and UVRAG-in skeletal muscle during exercise, and knockout of beclin 1 or UVRAG inhibits the cellular AMPK activation induced by glucose starvation. Moreover, TLR9 functions in a muscle-autonomous fashion in ex vivo contraction-induced AMPK activation, glucose uptake and beclin 1-UVRAG complex assembly. These findings reveal a heretofore undescribed role for a Toll-like receptor in skeletal-muscle AMPK activation and glucose metabolism during exercise, as well as unexpected crosstalk between this innate immune sensor and autophagy proteins.

Autosis is a Na+,K+-ATPase-regulated form of cell death triggered by autophagy-inducing peptides, starvation, and hypoxia-ischemia.

A long-standing controversy is whether autophagy is a bona fide cause of mammalian cell death. We used a cell-penetrating autophagy-inducing peptide, Tat-Beclin 1, derived from the autophagy protein Beclin 1, to investigate whether high levels of autophagy result in cell death by autophagy. Here we show that Tat-Beclin 1 induces dose-dependent death that is blocked by pharmacological or genetic inhibition of autophagy, but not of apoptosis or necroptosis. This death, termed "autosis," has unique morphological features, including increased autophagosomes/autolysosomes and nuclear convolution at early stages, and focal swelling of the perinuclear space at late stages. We also observed autotic death in cells during stress conditions, including in a subpopulation of nutrient-starved cells in vitro and in hippocampal neurons of neonatal rats subjected to cerebral hypoxia-ischemia in vivo. A chemical screen of ~5,000 known bioactive compounds revealed that cardiac glycosides, antagonists of Na(+),K(+)-ATPase, inhibit autotic cell death in vitro and in vivo. Furthermore, genetic knockdown of the Na(+),K(+)-ATPase α1 subunit blocks peptide and starvation-induced autosis in vitro. Thus, we have identified a unique form of autophagy-dependent cell death, a Food and Drug Administration-approved class of compounds that inhibit such death, and a crucial role for Na(+),K(+)-ATPase in its regulation. These findings have implications for understanding how cells die during certain stress conditions and how such cell death might be prevented.

Identification of a candidate therapeutic autophagy-inducing peptide.

The lysosomal degradation pathway of autophagy has a crucial role in defence against infection, neurodegenerative disorders, cancer and ageing. Accordingly, agents that induce autophagy may have broad therapeutic applications. One approach to developing such agents is to exploit autophagy manipulation strategies used by microbial virulence factors. Here we show that a peptide, Tat-beclin 1-derived from a region of the autophagy protein, beclin 1, which binds human immunodeficiency virus (HIV)-1 Nef-is a potent inducer of autophagy, and interacts with a newly identified negative regulator of autophagy, GAPR-1 (also called GLIPR2). Tat-beclin 1 decreases the accumulation of polyglutamine expansion protein aggregates and the replication of several pathogens (including HIV-1) in vitro, and reduces mortality in mice infected with chikungunya or West Nile virus. Thus, through the characterization of a domain of beclin 1 that interacts with HIV-1 Nef, we have developed an autophagy-inducing peptide that has potential efficacy in the treatment of human diseases.

Autophagy, antiviral immunity, and viral countermeasures.

The autophagy pathway likely evolved not only to maintain cellular and tissue homeostasis but also to protect cells against microbial attack. This conserved mechanism by which cytoplasmic cargo is delivered to the endolysosomal system is now recognized as a central player in coordinating the host response to diverse intracellular pathogens, including viruses. As an endolysosomal delivery system, autophagy functions in the transfer of viruses from the cytoplasm to the lysosome where they are degraded, in the transfer of viral nucleic acids to endosomal sensors for the activation of innate immunity, and in the transfer of endogenous viral antigens to MHC class II compartments for the activation of adaptive immunity. Viruses have, in turn, evolved different strategies to antagonize, and potentially, to exploit the host autophagic machinery. Moreover, through mechanisms not yet well understood, autophagy may dampen host innate immune and inflammatory responses to viral infection. This review highlights the roles of autophagy in antiviral immunity, viral strategies to evade autophagy, and potential negative feedback functions of autophagy in the host antiviral response.

Inhibitory function of adapter-related protein complex 2 alpha 1 subunit in the process of nuclear translocation of human immunodeficiency virus type 1 genome.

The transfection of human cells with siRNA against adapter-related protein complex 2 alpha 1 subunit (AP2alpha) was revealed to significantly up-regulate the replication of human immunodeficiency virus type 1 (HIV-1). This effect was confirmed by cell infection with vesicular stomatitis virus G protein-pseudotyped HIV-1 as well as CXCR4-tropic and CCR5-tropic HIV-1. Viral adsorption, viral entry and reverse transcription processes were not affected by cell transfection with siRNA against AP2alpha. In contrast, viral nuclear translocation as well as the integration process was significantly up-regulated in cells transfected with siRNA against AP2alpha. Confocal fluorescence microscopy revealed that a subpopulation of AP2alpha was not only localized in the cytoplasm but was also partly co-localized with lamin B, importin beta and Nup153, implying that AP2alpha negatively regulates HIV-1 replication in the process of nuclear translocation of viral DNA in the cytoplasm or the perinuclear region. We propose that AP2alpha may be a novel target for disrupting HIV-1 replication in the early stage of the viral life cycle.

The RING finger ubiquitin ligase RNF125/TRAC-1 down-modulates HIV-1 replication in primary human peripheral blood mononuclear cells.

CXCR4-using HIV-1 was previously shown to replicate more efficiently in a healthy donor-derived CD4(+) CD38(+) than in a CD4(+) CD38(-) T-cell subset after stimulation with interleukin (IL)-4. Here, we identified 3 cellular genes, which were expressed to a higher level in an IL-4-stimulated CD38(-) subset. One of the 3 genes, RNF125/TRAC-1, was involved in the down-regulation of HIV-1 replication not only in cell lines, but also in peripheral blood mononuclear cells. RNF125/TRAC-1 bears the RING finger domain, important for E3 ubiquitin protein ligase. Mutations in this domain of RNF125/TRAC-1 led to the loss of HIV-1 down-modulatory activity, suggesting that E3 ligase activity is necessary. In addition, the results of Northern blotting and reporter gene analysis indicated that RNF125/TRAC-1 function occurs at the viral transcription step. These results suggest that RNF125/TRAC-1 could function to recruit host factor(s) controlling HIV-1 transcription to the ubiquitin-proteasome pathway.

p21WAF1 modulates NF-kappaB signaling and induces anti-apoptotic protein Bcl-2 in Tax-expressing rat fibroblast.

Of the cell cycle-associated genes regulated by human T-cell leukemia virus type-1 (HTLV-1) Tax, cyclin-dependent kinase (CDK) inhibitor p21WAF1 is upregulated in HTLV-1-infected cells. Previously, we reported that p21WAF1 stimulated Tax-dependent NF-kappaB activation which influences a variety of cellular processes, including proliferation, differentiation, and apoptosis. In HTLV-1-infected cells, Tax is primarily involved in the constitutive activation of NF-kappaB signaling. Here, we demonstrate that p21WAF1 affects Tax-dependent NF-kappaB signaling by inducing p100/52, an NF-kappaB-related protein. W4, a Tax-transformed rat fibroblast cell line, exhibits the constitutive activation of NF-kappaB signaling, potentially mediated by overexpression of RelB. Ectopic expression of p21WAF1 in W4 cells, which lack endogenous expression due to methylation of the p21WAF1 promoter, induces the expression of p100/52. Bcl-2 expression was also upregulated by ectopic p21WAF1 in this cell line, suggesting that p21WAF1 plays an important role in the regulation of apoptosis by modulating NF-kappaB signaling in Tax-expressing rat fibroblasts. We also address the expression of NF-kappaB-related proteins in HTLV-1-infected cells.

Studies on the conditions required for structural and functional maturation of rabies virus glycoprotein (G) in G cDNA-transfected cells.

When the rabies virus G cDNA was expressed with the help of T7 RNA polymerase provided by a recombinant vaccinia virus (RVV-T7), functional G proteins were produced in terms of their ability to induce low pH-dependent syncytium formation and the formation of conformational epitopes, including the acid-sensitive epitope recognized by mAb #1-30-44. Such an ability and the 1-30-44 epitope formation, however, were not associated with the G gene products when G cDNA was expressed without the help of RVV-T7 using a tetracycline-regulated expression vector (pTet-G), although they were normally transported to the surface of established G protein-producing BHK-21 (G-BHK) cells. But, when the G-BHK cells were treated with 2.5 m M sodium butyrate (NaB) after the removal of tetracycline, we could observe not only a much increased frequency of G protein-producing cells, but also the greatly enhanced maturation of the protein. Another short acylate, sodium propionate (NaP), similarly induced increased G protein synthesis at a concentration of 2.5 m M as NaB; however, such proteins were mostly not endowed with the fusion activity nor the 1-30-44 epitope, while NaP at a higher concentration as 5.0 m M did induce similarly the increased production and enhanced maturation of G protein, including the 1-30-44 epitope formation. From these results, we conclude that functional maturation of G protein to acquire fusogenic activity is correlated with 1-30-44 epitope formation, and 2.5 m M NaB not only stimulates G protein production, but also provides such cellular conditions as are required for the structural and functional maturation of the protein.

p21(Waf1/Cip1/Sdi1) prevents apoptosis as well as stimulates growth in cells transformed or immortalized by human T-cell leukemia virus type 1-encoded tax.

Human T-cell leukemia virus type 1 (HTLV-1) Tax regulates the expression of virally encoded genes, as well as various endogenous host genes in trans. Tax-mediated regulation of gene expression is important for the immortalization of normal human T lymphocytes and the transformation of fibroblast cells, such as Rat-1 cells. Tax has the ability to transactivate p21(Waf1/Cip1/Sdi1), resulting in high expression levels in HTLV-1-immortalized cells. Since p21 expression is suppressed due to methylation of the promoter region in Rat-l cell line, p21 may not be critical for the transformation of this cell line by Tax. To further understand the role of p21 for the proliferation of Tax-transformed Rat-1 cells, we examined the effect of ectopic expression of p21 in these cells. Here, we observed that p21 expression enhanced the transformation of this cell line via at least two mechanisms: (i) the enhancement of NF-kappaB activation and/or CREB signaling and (ii) the excitation of antiapoptotic machinery. To analyze the role of p21 that is overexpressed in HTLV-1-immortalized lymphocytes, p21 expression was suppressed by using an antisense oligonucleotide specific for p21 mRNA; these cells then became sensitive to apoptotic induction. These results suggest that p21 plays an important role in the proliferation of Tax-expressing cells through the regulation of at least two independent mechanisms.