Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disorder resulting from motor neuron death. Approximately 10% of cases are familial (FALS), typically with a dominant inheritance mode. Despite numerous advances in recent years1-9, nearly 50% of FALS cases have unknown genetic etiology. Here we show that mutations within the profilin 1 (PFN1) gene can cause FALS. PFN1 is critical for monomeric (G)-actin conversion to filamentous (F)-actin. Exome sequencing of two large ALS families revealed different mutations within the PFN1 gene. Additional sequence analysis identified 4 mutations in 7 out of 274 FALS cases. Cells expressing PFN1 mutants contain ubiquitinated, insoluble aggregates that in many cases contain the ALS-associated protein TDP-43. PFN1 mutants also display decreased bound actin levels and can inhibit axon outgrowth. Furthermore, primary motor neurons expressing mutant PFN1 display smaller growth cones with a reduced F-/G-actin ratio. These observations further document that cytoskeletal pathway alterations contribute to ALS pathogenesis.

Ethanol exposure diminishes intestinal abundance in both mice and humans and can be recovered in experimental ALD by oral supplementation. promotes intestinal barrier integrity and ameliorates experimental ALD. Our data suggest that patients with ALD might benefit from supplementation.

Objective Several studies have suggested an increased frequency of variants in the gene encoding angiogenin (ANG) in patients with amyotrophic lateral sclerosis (ALS). Interestingly, a few ALS patients carrying ANG variants also showed signs of Parkinson disease (PD). Furthermore, relatives of ALS patients have an increased risk to develop PD, and the prevalence of concomitant motor neuron disease in PD is higher than expected based on chance occurrence. We therefore investigated whether ANG variants could predispose to both ALS and PD. Methods We reviewed all previous studies on ANG in ALS and performed sequence experiments on additional samples, which allowed us to analyze data from 6,471 ALS patients and 7,668 controls from 15 centers (13 from Europe and 2 from the USA). We sequenced DNA samples from 3,146 PD patients from 6 centers (5 from Europe and 1 from the USA). Statistical analysis was performed using the variable threshold test, and the Mantel-Haenszel procedure was used to estimate odds ratios. Results Analysis of sequence data from 17,258 individuals demonstrated a significantly higher frequency of ANG variants in both ALS and PD patients compared to control subjects (p = 9.3 × 10−6 for ALS and p = 4.3 × 10−5 for PD). The odds ratio for any ANG variant in patients versus controls was 9.2 for ALS and 6.7 for PD. Interpretation The data from this multicenter study demonstrate that there is a strong association between PD, ALS, and ANG variants. ANG is a genetic link between ALS and PD.

Cellular homeostais, that is normally maintained through autophagy, is disrupted in alcoholic liver disease (ALD). Because autophagy and exosome biogenesis share common elements, we hypothesized that increased exosome production in ALD may be linked to disruption of autophagic function. We found impaired autophagy both in ALD and alcoholic hepatitis (AH) mouse models and human livers with ALD as indicated by increased hepatic p62 and LC3‐II levels. Alcohol reduced autophagy flux in vivo in chloroquine‐treated mice as well as in vitro in hepatocytes and macrophages treated with bafilomycin A. Our results revealed that alcohol targets multiple steps in the autophagy pathway. Alcohol‐related decrease in mechanistic target of rapamycin (mTOR) and Ras homolog enriched in brain (Rheb), that initiate autophagy, correlated with increased Beclin1 and autophagy‐related protein 7 (Atg7), proteins involved in phagophore‐autophagosome formation, in ALD. We found that alcohol disrupted autophagy function at the lysosomal level through decreased lysosomal‐associated membrane protein 1 (LAMP1) and lysosomal‐associated membrane protein 2 (LAMP2) in livers with ALD. We identified that micro‐RNA 155 (miR‐155), that is increased by alcohol, targets mTOR, Rheb, LAMP1, and LAMP2 in the authophagy pathway. Consistent with this, miR‐155‐deficient mice were protected from alcohol‐induced disruption of autophagy and showed attenuated exosome production. Mechanistically, down‐regulation of LAMP1 or LAMP2 increased exosome release in hepatocytes and macrophages in the presence and absence of alcohol. These results suggested that the alcohol‐induced increase in exosome production was linked to disruption of autophagy and impaired autophagosome and lysosome function. Conclusion: Alcohol affects multiple genes in the autophagy pathway and impairs autophagic flux at the lysosome level in ALD. Inhibition of LAMP1 and LAMP2 promotes exosome release in ALD. We identified miR‐155 as a mediator of alcohol‐related regulation of autophagy and exosome production in hepatocytes and macrophages.

Edited by Jeffrey PessinFibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon regulatory factor 3 (IRF3) regulates hepatocyte apoptosis and production of type I IFNs. In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the endoplasmic reticulum (ER) adapter, stimulator of interferon genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically induced liver fibrogenesis. To test this, we performed acute or chronic CCl 4 administration to WT and IRF3-, Toll/ Interleukin-1R (TIR) domain-containing adapter-inducing interferon-␤ (TRIF)-, TRIF-related adaptor molecule (TRAM)-, and STING-deficient mice. We report that acute CCl 4 administration to WT mice resulted in early ER stress, activation of IRF3, and type I IFNs, followed by hepatocyte apoptosis and liver injury, accompanied by liver fibrosis upon repeated administration of CCl 4 . Deficiency of IRF3 or STING prevented hepatocyte death and fibrosis both in acute or chronic CCl 4 . In contrast, mice deficient in type I IFN receptors or in TLR4 signaling adaptors, TRAM or TRIF, upstream of IRF3, were not protected from hepatocyte death and/or fibrosis, suggesting that the proapoptotic role of IRF3 is independent of TLR signaling in fibrosis. Hepatocyte death is required for liver fibrosis with causal involvement of STING and IRF3. Thus, our results identify that IRF3, by its association with STING in the presence of ER stress, couples hepatocyte apoptosis with liver fibrosis and indicate that innate immune signaling regulates outcomes of liver fibrosis via modulation of hepatocyte death in the liver.

Kupffer cell (KC) and macrophage (MØ) activation contribute to steatosis, inflammation and fibrosis in alcoholic liver disease (ALD). We found increased frequency of MØ, T cells and expression of Ccr2 and Ccr5 in the livers of patients with ALD and increased circulating chemokines, CCL2 and CCL5 in alcoholic hepatitis patients. We hypothesized that inhibition of CCL2 signaling with the dual CCR2/5 inhibitor, cenicriviroc (CVC), would attenuate ALD. In a mouse model of ALD, liver injury (ALT) and steatosis were prevented by CVC whether administered as "prevention" throughout the alcohol feeding or as "treatment" started after the development of ALD. Alcohol-induced increases in early liver fibrosis markers (Sirius-red, hydroxyproline and collagen-1) were normalized by both modes of CVC administration. We found that "prevention" and "treatment" with CVC reversed alcohol-related increases in liver mRNA and protein expression of TNFα, IL-1β, IL-6 and CCL2. CVC administration regimens prevented the increase in infiltrating MØ (F4/80 CD11b ) and reduced proinflammatory Ly6C MØ in livers of alcohol-fed mice. CVC increased liver T cell numbers and attenuated Il-2 expression without an effect on CD69 or CD25 T cell expression. In vitro, CVC inhibited CCL2-induced increases in hepatocyte Fasn and Adrp while it augmented Acox-1, Pgc1α and Ucp-2 expression, suggesting mechanisms for attenuated hepatocyte steatosis. We found that CCL2 and CCL5 sensitized hepatocytes to LPS-induced liver injury (TNFα, ALT and LDH release). Alcohol feeding induced apoptosis (PARP and caspase-3 cleavage) and pyroptosis (gasdermin D cleavage) in livers and CVC prevented both these forms of cell death. Together, our data demonstrate preclinical evidence for CCR2/CCR5 inhibition with CVC as a potent intervention to ameliorate alcohol-induced steatohepatitis and liver damage. This article is protected by copyright. All rights reserved.

Background & Aims Chronic, excessive alcohol consumption leads to alcoholic liver disease (ALD) characterized by steatosis, inflammation, and eventually cirrhosis. The hepatocyte specific microRNA 122 (MIR122) regulates hepatocyte differentiation and metabolism. We investigated whether an alcohol-induced decrease in level of MIR122 contributes to development of ALD. Methods We obtained liver samples from 12 patients with ALD and cirrhosis and 9 healthy individuals (controls) and analyzed them by histology and immunohistochemistry. C57Bl/6 mice were placed on a Lieber-DeCarli liquid diet, in which they were fed ethanol for 8 weeks, as a model of ALD, or a control diet. These mice were also given injections of CCl4, to increase liver fibrosis, for 8 weeks. On day 28, mice with ethanol-induced liver disease and advanced fibrosis, and controls, were given injections of recombinant adeno-associated virus 8 vector that expressed the primary miR-122 transcript (pri-MIR122, to overexpress MIR122 in hepatocytes) or vector (control). Two weeks before ethanol feeding, some mice were given injections of a vector that expressed an anti-MIR122, to knock down its expression. Serum and liver tissues were collected; hepatocytes and liver mononuclear cells were analyzed by histology, immunoblots, and confocal microscopy. We performed in silico analyses to identify targets of MIR122 and chromatin immunoprecipitation quantitative PCR analyses in Huh-7 cells. Results Levels of MIR122 were decreased in liver samples from patients with ALD and mice on the Lieber-DeCarli diet, compared with controls. Transgenic expression of MIR122 in hepatocytes of mice with ethanol-induced liver disease and advanced fibrosis significantly reduced serum levels of alanine aminotransferase (ALT) and liver steatosis and fibrosis, compared to mice given injections of the control vector. Ethanol feeding reduced expression of pri-MIR122 by increasing expression of the spliced form of the transcription factor grainyhead like transcription factor 2 (GRHL2) in livers tissues from mice. Levels of GRHL2 were also increased in liver tissues from patients with ALD, compared with controls; increases correlated with decreases in levels of MIR122 in human liver. Mice given injections of the anti-MIR122 before ethanol feeding had increased steatosis, inflammation, and serum levels of ALT compared to mice given a control vector. Levels of hypoxia inducible factor 1 alpha (HIF1A) mRNA, a target of MIR122, were increased in liver tissues from patients and mice with ALD, compared with controls. Mice with hepatocyte-specific disruption of Hif1a developed less-severe liver injury following administration of ethanol, injection of anti-MIR122, or both. Conclusions Levels of MIR122 decrease in livers from patients with ALD and mice with ethanol-induced liver disease, compared with controls. Transcription of MIR122 is inhibited by GRHL2, which is increased in livers of mice and patients with ALD. Expression of an anti-MIR122 worsened the severity of liver damage followin...

Disease severity in alcoholic liver disease (ALD) is associated with a significant presence of neutrophils (a type of immune cell) in the liver. It remains unknown how alcohol affects the capacity of neutrophils to control infection, a major hallmark of ALD. We found that binge alcohol drinking impaired important strategies used by neutrophils to contain and resolve infection, resulting in increased liver injury during ALD.