Michael Bouchard

In the context of neurodegeneration, activated microglia facilitate inflammation via secretion of TNF-α, IL-1α, and C1q. Astrocytes exposed to this signaling array polarize to a reactive inflammatory phenotype, termed A1 or A1-like. Astrocytes are essential for neuronal survival, synaptic support, and blood–brain barrier (BBB) function, but A1-like astrocytes upregulate inflammatory gene expression, downregulate neurotrophic factors, and secrete neurotoxic signals. The consequences of A1-like polarization on BBB function are unknown but may have etiological implications for some diseases. Frequently identified by upregulation of complement component 3 (C3), A1-like astrocytes have been characterized in neurodegenerative disorders like Alzheimer’s disease, with polarization correlated with disease progression and severity. However, the role of A1-like astrocytes in neurodegeneration associated with chronic viral infections, like HIV-1-associated neurocognitive disorder (HAND), remains unclear. An in vitro system using primary human astrocytes, as well as a BBB model featuring primary human brain microvascular endothelial cells (BMECs) co-cultured with astrocytes, was used to elucidate cellular and molecular consequences of chronic astrocyte activation. As measured by whole transcriptome analysis and protein expression assays, repeated treatment with TNF-α, IL-1α, and C1q induced A1-like polarization of astrocytes both in monoculture and in a BBB model, resulting in increased secretion of pro-inflammatory signals. No substantial change to BBB permeability was observed. In contrast, exposure to HIV-1 viral protein Tat did not independently induce A1-like polarization. Ongoing investigations into the effect of astrocyte polarization on BBB integrity and treatment with pathogenic proteins may provide insights into the role of neurotoxic astrocytes in neurovirologic pathologies.

Primary liver cancer represents a significant global health burden, with limited therapeutic options for advanced disease. Long non-coding RNAs (lncRNAs) are increasingly found to play crucial roles in hepatic biology and disease progression. Here, we identify FAM99A as a highly liver-specific lncRNA that is systematically downregulated across liver malignancies, with reduced expression correlating with poor clinical outcomes. FAM99A exhibits remarkable tissue specificity with minimal expression outside the liver, and its levels rapidly decline during primary hepatocyte dedifferentiation in culture. Through isoform analysis, we establish FAM99A-203 as the predominant transcript in normal liver tissue and observe altered isoform distribution in liver cancers. Functionally, FAM99A overexpression inhibits anchorage-independent growth in liver cancer cell lines. Transcriptomic analysis reveals that FAM99A negatively regulates translation-related pathways in both liver cancer cells and primary hepatocytes. This is corroborated by protein synthesis assays showing that FAM99A overexpression substantially reduces global translation rates. Targeted RNase H-mediated extraction coupled with mass spectrometry identifies multiple components of the translation machinery as direct FAM99A binding partners, including eukaryotic translation initiation factors and RNA helicases involved in ribosome biogenesis. Clinical data analysis demonstrates significant inverse correlations between FAM99A expression and ribosomal protein genes in liver cancer patients. Additionally, hepatitis B virus appears to downregulate FAM99A expression, potentially contributing to its oncogenic properties. Our findings establish FAM99A as a liver-specific translational regulator that exerts tumor-suppressive effects by restraining protein synthesis rates, offering insights into hepatocarcinogenesis and the potential of FAM99A as both a biomarker and agent in new therapeutic avenues.

Liver cancer research demands robust cellular models and molecular biomarkers that accurately reflect disease biology. We present a comprehensive dual investigation addressing both challenges through transcriptomic analyses of 541 samples spanning multiple liver cancer subtypes and cell lines. Using a signature of 2,523 highly variable genes, we identified distinct molecular profiles distinguishing cancer subtypes and their corresponding cell lines, determining optimal models for each disease type. We identified potential mischaracterizations, notably demonstrating HepG2 cells most closely resemble hepatoblastoma rather than their commonly reported hepatocellular carcinoma (HCC) origin, and revealed that physioxic primary hepatocyte culture conditions better preserve liver-specific expression programs. Building on these findings, we next conducted a systematic biomarker screening that identified FAM99A as a liver-specific long non-coding RNA significantly downregulated in HCC and other liver cancers, with progressive loss during hepatocyte de-differentiation. Through clinical data analyses, isoform characterization, colony formation assays, and RNA-seq, we established FAM99A's potential tumor suppressor function. GSEA revealed translation and ribosome biogenesis as the primary pathways affected by FAM99A overexpression, which we validated through protein synthesis assays. Analysis of TREX mass spectrometry data identified FAM99A's interaction with key translation initiation factors and RNA helicases, suggesting a liver-specific role in regulating translation to balance the organ's regenerative capacity and protein synthesis demands. Ultimately, this work identifies FAM99A as a strong candidate biomarker and translation regulator in hepatic biology while characterizing its fundamental roles. Understanding FAM99A's mechanisms carries significant clinical implications, establishing it as an attractive therapeutic target for novel liver cancer treatment strategies.

Globally, a chronic hepatitis B virus (HBV) infection is the leading cause of hepatocellular carcinoma (HCC). The increased risk of HCC persists in patients treated with antiviral therapy, despite suppressing viral replication. Co-infection with human immunodeficiency virus (HIV) increases the risk of developing HCC by 18-fold relative to an HBV mono-infection. Despite the clear clinical burden posed by an HIV/HBV co-infection, the mechanisms underlying the increased risk of HCC are understudied due to a lack of relevant model systems. We developed an in vitro model of HIV/HBV co-infection using Jurkat cells with inducible expression of HIV-1 Tat co-cultured with HBV-infected primary rat hepatocytes. The co-culture model was used to investigate the effect of HIV-1 Tat on HBV replication and cell survival in HBV-infected hepatocytes. Results from Jurkat-secreted Tat were complemented with purified HIV-1 Tat to determine the effect of Tat alone. Exposure to Tat significantly upregulated ERK1/2 and NF-[kappa]B signaling and cell survival in HBV-infected and uninfected hepatocytes. The activation of ERK1/2 and NF-[kappa]B signaling by Tat also inhibited HBV replication, which could be rescued by inhibition of ERK1/2 with temuterkib. Tat-dependent activation of NF-[kappa]B signaling and cell survival was also reversed by treatment with temuterkib. Tat-mediated activation of NF-[kappa]B signaling and cell survival is initiated by the TLR-4 cell surface receptor and recruitment of PKC-delta to the plasma membrane. Overall, the results of these studies indicate that Tat-mediated upregulation of ERK1/2 may promote oncogenesis and contribute to the development of HCC during an HIV/HBV co-infection by promoting cell survival.

Reovirus (ReoV) is an enteric dsRNA virus which can infect a wide range of mammals and induce intestinal and autoimmune diseases due to the cell death elicited during infection. The mechanisms by which ReoV induces such cell death are unclear. For decades, ReoV was thought to cause mainly apoptosis in infected cells. Recent studies have shown that ReoV can also activate necroptosis, a caspase-independent form of inflammatory programmed cell death reliant on the kinase RIPK3 and its substrate MLKL. The mechanisms by which both necroptosis and apoptosis are activated during ReoV infection, and the importance of these cell death pathways to ReoV pathogenesis, are largely unknown. Here, we show that Z-DNA Binding Protein 1 (ZBP1) initiates both apoptosis and necroptosis during ReoV infections. We have also discovered that ReoV generates Z-RNA, the left-handed conformation of double-helical (ds)RNA, which are sensed by ZBP1 in the cytoplasm of infected cells. These ReoV Z-RNAs originate primarily from the positive-sense viral RNAs, which fold-back to produce dsRNA. ZBP1 then initiates RIPK3-driven pathways of apoptosis and necroptosis, as well as RIPK3- independent apoptosis driven by RIPK1. In vivo, ZBP1 is essential for limiting virus dissemination to cardiac tissues following oral and intracranial infection, and for preventing lethality in mice. These results demonstrate that ZBP1-driven Z-RNA sensing and cell death play an integral role in controlling ReoV infection.

Hepatocellular carcinoma (HCC), a primary liver malignancy, is responsible for approximately 90% of liver cancers and is a leading cause of cancer-related deaths globally. Despite its prevalence and impact, the molecular mechanisms driving the development, maintenance, and progression of HCC remain poorly understood, resulting in limited treatment options and a poor prognosis that is dependent on the stage of the tumor. Recent research has highlighted the important role of long non-coding RNAs (lncRNAs), which are RNA transcripts longer than 200 nucleotides that have no protein-coding potential, in regulating cellular signaling pathways and disease progression. To investigate the potential involvement of lncRNAs in HCC, we performed a comparative analysis of HCC tissue and adjacent nontumor tissue transcriptome data from The Cancer Genome Atlas (TCGA). We identified FAM99A, a liver-specific lncRNA, as significantly downregulated in HCC. Moreover, Kaplan-Meier analysis revealed a correlation between low FAM99A expression and poor overall survival in HCC patients. To further explore the functional role of FAM99A in HCC, we assessed its expression levels in liver cancer cell lines (HepG2, Huh7, and Hep3B) and primary human hepatocytes using quantitative PCR analysis. The results confirmed significantly lower FAM99A expression in liver cancer cell lines compared to primary human hepatocytes. Additionally, TREX and mass spectrometry assays were conducted to identify FAM99A's interacting partners. Accumulating evidence suggests that certain lncRNAs are dysregulated in HBV-related HCC and may influence the HBV lifecycle. Characterizing the functions of these lncRNAs can provide a more comprehensive understanding of HBV replication and oncogenesis. Intriguingly, we observed a significant decrease in FAM99A expression in cells transfected with a replication-competent cDNA of HBV, indicating that HBV downregulates FAM99A expression. In conclusion, our findings suggest that FAM99A is a potential tumor suppressor in HCC, and its downregulation by HBV may contribute to the development of HBV-associated HCC. These insights underscore the importance of further research into the role of FAM99A in HCC and its potential as a therapeutic target or prognostic marker.

Approximately 350 million individuals are chronically infected with the hepatitis B virus (HBV). Chronic HBV infections are associated with the development of hepatocellular carcinoma (HCC). The HBV X protein (HBx) is thought to play an important role in the development of HBV-associated HCC. One fundamental function of HBx is the elevation of cytosolic calcium signals; this HBx activity has been linked to HBx stimulation of cell proliferation and transcription pathways as well as HBV replication. Exactly how HBx elevates cytosolic calcium signals is not clear. The studies described here show that HBx stimulates calcium entry into cells, resulting in an increased plateau level of IP3-linked calcium signals. This increased calcium plateau can be inhibited by blocking mitochondrial calcium uptake and store-operated calcium entry (SOCE). Blocking SOCE also reduces HBV replication. Finally, these studies also demonstrate that there is increased mitochondrial calcium uptake in HBx-expressing cells. Cumulatively, these studies suggest that HBx can increase mitochondrial calcium uptake and promote increased SOCE to sustain higher cytosolic calcium and stimulate HBV replication.

Chronic infection with the hepatitis B virus (HBV) is a major risk factor for developing severe liver diseases such as hepatocellular carcinoma (HCC). Despite the availability of a vaccine, HBV-associated liver diseases remain a major worldwide health problem. HBx is a multifunctional protein encoded by the HBV genome; HBx stimulates HBV replication and is thought to play an important role in the development of HBV-associated HCC. HBx can activate the phosphatidylinositol 3 kinase (PI3K)/AKT signaling pathway in some cell lines; however, whether HBx regulates the PI3K/AKT signaling in normal hepatocytes has not been evaluated. In studies described here, we assessed HBx activation of PI3K/AKT signaling in an ex vivo model of cultured primary rat and human hepatocytes and determined how this HBx activity affected HBV replication. We report that HBx activates AKT in primary rat and human hepatocytes and that activation of AKT decreases HBV replication in these cells. HBx activation of AKT in primary hepatocytes decreased HBV mRNA and core protein levels, suggesting that AKT regulates HBV replication by affecting transcription of HBV mRNAs. In agreement with this, we show that the transcription factor hepatocyte nuclear factor (HNP) 4[alpha] is a target of HBx-regulated AKT, and we directly link HNF4[alpha] to HBx-regulated AKT modulation of HBV transcription and replication. Although 14Bx activates AKT to diminish HBV replication, we also show that an important effect of HBx activation of AKT is inhibition of apoptosis. HBx also activates the transcription factor NF-[kappa]B to inhibit apoptosis in cultured primary rat hepatocytes. The molecular mechanisms that underlie HBx activation of NF-[kappa]B in normal hepatocytes are undefined. We now report that HBx increases the levels of reactive oxygen species (ROS) in primary rat hepatocytes and that this elevation of ROS is responsible for HBx activation of NF-[kappa]B. Activation of NF-[kappa]B by HBx decreases HBV replication in primary rat hepatocytes. Consequently, our studies suggest that HBx activates cellular signaling pathways that simultaneously regulate HBV replication and cell survival. We propose that HBx functions as a rheostat that controls the level of HBV replication and the cellular environment to ensure non-cytopathic HBV replication.