Impairment of the autophagy pathway has been observed during the pathogenesis of Alzheimer’s disease (AD), a neurodegenerative disorder characterized by abnormal deposition of extracellular and intracellular amyloid β (Aβ) peptides. Yet the role of autophagy in Aβ production and AD progression is complex. To study whether increased basal autophagy plays a beneficial role in Aβ clearance and cognitive improvement, we developed a novel genetic model to hyperactivate autophagy in vivo. We found that knock-in of a point mutation F121A in the essential autophagy gene Beclin 1/Becn1 in mice significantly reduces the interaction of BECN1 with its inhibitor BCL2, and thus leads to constitutively active autophagy even under non-autophagy-inducing conditions in multiple tissues, including brain. Becn1F121A-mediated autophagy hyperactivation significantly decreases amyloid accumulation, prevents cognitive decline, and restores survival in AD mouse models. Using an immunoisolation method, we found biochemically that Aβ oligomers are autophagic substrates and sequestered inside autophagosomes in the brain of autophagy-hyperactive AD mice. In addition to genetic activation of autophagy by Becn1 gain-of-function, we also found that ML246, a small-molecule autophagy inducer, as well as voluntary exercise, a physiological autophagy inducer, exert similar Becn1-dependent protective effects on Aβ removal and memory in AD mice. Taken together, these results demonstrate that genetically disrupting BECN1-BCL2 binding hyperactivates autophagy in vivo, which sequestrates amyloid oligomers and prevents AD progression. The study establishes new approaches to activate autophagy in the brain, and reveals the important function of Becn1-mediated autophagy hyperactivation in the prevention of AD.

SUMMARY Autophagy, a stress-induced lysosomal degradative pathway, has been assumed to exert similar metabolic effects in different organs. Here, we establish a model where autophagy plays different roles in insulin-producing β cells versus insulin-responsive cells, utilizing knockin (Becn1F121A) mice manifesting constitutively active autophagy. With a high-fat-diet challenge, the autophagy-hyperactive mice unexpectedly show impaired glucose tolerance, but improved insulin sensitivity, compared to mice with normal autophagy. Autophagy hyperactivation enhances insulin signaling, via suppressing ER stress in insulin-responsive cells, but decreases insulin secretion by selectively sequestrating and degrading insulin granule vesicles in β cells, a process we term “vesicophagy.” The reduction in insulin storage, insulin secretion, and glucose tolerance is reversed by transient treatment of autophagy inhibitors. Thus, β cells and insulin-responsive tissues require different autophagy levels for optimal function. To improve insulin sensitivity without hampering secretion, acute or intermittent, rather than chronic, activation of autophagy should be considered in diabetic therapy development.

The herpes simplex virus 1 ORF UL41 encodes a protein (virion host shutoff or vhs) associated with selective degradation of mRNA early in infection. Some mRNAs, exemplified by GAPDH or ␤-actin mRNAs, are degraded rapidly. Others, for example IEX-1 mRNA, are degraded in two stages: whereas the 3 domain disappears rapidly, a large 5 domain fragment of the mRNA lingers for several hours. Still a third, exemplified by tristetraprolin mRNA, is not degraded, allowing its protein product to accumulate in infected cells. Here we report the following: (i) a GST-vhs protein produced in Escherichia coli, solubilized and purified to homogeneity acts as bona fide endoribonuclease when tested on in vitro transcribed IEX-1 probes. A GST-vhs protein in which three key vhs amino acids were replaced with alanines, solubilized and purified by the same protocol, had no enzymatic activity. (ii) The number of fragments generated by cleavage of a truncated IEX-1 RNA by vhs appears to be small; the cleavage sites are centered at or near the AU-rich elements located at the 3 untranslated region of the mRNA. A truncated RNA containing only the IEX-1 coding domain was cleaved numerous times. (iii) In cells infected at high multiplicity and exposed to a large number of particles per cell, the vhs protein accumulated within 3 h after infection, in small uniform cytoplasmic granules raising the possibility that vhs colocalizes with tristerapolin, a protein induced after infection, in structures involved in degradation of RNA.endoribonuclease ͉ mRNA ͉ virion host shutoff O ne of the key early events in the replication of herpes simplex virus 1 (HSV-1) is the shutoff of host macromolecular metabolism (1). The shutoff of protein synthesis was mapped to a gene designated U L 41, and the protein product was designated virion host shutoff or vhs (2, 3). During productive infection, copies of vhs protein, which enter the cell as components of the virion tegument, cause the degradation of preexisting and newly transcribed mRNAs during the first few hours after infection (2-4). After the onset of viral transcription, vhs also accelerates the turnover of viral mRNAs, facilitating the sequential expression of different classes of viral genes (3, 5, 6). At later times after infection, newly made vhs is sequestered and rendered inactive by another viral protein, VP16 also known as ␣-trans inducing factor, the product of U L 48 (7). The vast literature of the past decade, reviewed in ref. 8, speaks eloquently of the importance of vhs in the biology of HSV. Some of the key studies, relevant to this report, are as follows: (i) vhs degrades mRNA in the absence of other viral proteins, as shown by inhibition of reporter gene expression in mammalian cells transiently cotransfected with a vhs expression vector (9, 10). (ii) vhs, along with homologues from other alphaherpesviruses, shares amino acid sequence similarity with a larger family of human, yeast, bacterial and phage nucleases and mutations of highly conserved residues, known to be essential for the cat...

The accumulation of cellular transcripts from cells infected with herpes simplex virus 1 (HSV-1) as measured with the aid of Affymetrix microchips has been reported elsewhere. Among these transcripts were genes that respond to stress and that could have a noxious effect on viral replication. We have selected the stress-inducible cellular gene encoding the immediate-early response protein IEX-1 to verify and determine the significance of the accumulation of these transcripts in infected cells. We report that we verified the increase in accumulation of IEX-1 transcripts after infection by Northern analyses and real-time PCR. These transcripts reach peak levels between 3 and 7 h after infection and decrease thereafter. However, IEX-1 protein was detected in cells 1 h after infection but not at later intervals. Studies designed to elucidate the failure of IEX-1 protein to be synthesized revealed the following points.

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