Biology subjects

Lian, E.

Publications and source records attributed to Lian, E..

6 recordsLinked to original sources

bioRxiv2022

Genetic and pharmacological reduction of CDK14 mitigates synucleinopathy

Parkinsonas disease (PD) is a debilitating neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons (DaNs) and the abnormal accumulation of -Synuclein (-Syn) protein. Currently, no treatment can slow nor halt the progression of PD. Multiplications and mutations of the -Syn gene (SNCA) cause PD-associated syndromes and animal models that overexpress -Syn replicate several features of PD. Decreasing total -Syn levels, therefore, is an attractive approach to slow down neurodegeneration in patients with synucleinopathy. We previously performed a genetic screen for modifiers of -Syn levels and identified CDK14, a kinase of largely unknown function as a regulator of -Syn. To test the potential therapeutic effects of CDK14 reduction in PD, we ablated Cdk14 in the -Syn preformed fibrils (PFF)-induced PD mouse model. We found that loss of Cdk14 mitigates the grip strength deficit of PFF-treated mice and ameliorates PFF-induced cortical -Syn pathology, indicated by reduced numbers of pS129 -Syn-containing cells. In primary neurons, we found that Cdk14 depletion protects against the propagation of toxic -Syn species. We further validated these findings on pS129 -Syn levels in PD patient neurons. Finally, we leveraged the recent discovery of a covalent inhibitor of CDK14 to determine whether this target is pharmacologically tractable in vitro and in vivo. We found that CDK14 inhibition decreases total and pathologically aggregated -Syn in human neurons, in PFF- challenged rat neurons and in the brains of -Syn-humanized mice. In summary, we suggest that CDK14 represents a novel therapeutic target for PD-associated synucleinopathy.

neuroscience↗

bioRxiv2022

Shifting a Cellular Metabolic Landscape Identifies a Refractory Environment for Flavivirus Replication

Host-targeted therapeutics to control viral infection are gaining prominence given the vulnerability of viral replication at select host-interaction points and the limited possibility of developing drug resistant mutants. Nevertheless, the chemical and biological impact of many host-targeted therapeutics on both the cell and virus has not been elucidated and remains a key complication. Previously, it has been demonstrated that inhibition of fatty acid metabolism has significant antiviral potential. Here, we use a multidisciplinary approach to demonstrate how inhibition of fatty acid biosynthesis creates a metabolically refractory environment that drives viral dependence on alternate metabolic pathways for survival. By profiling the global metabolic landscape following inhibition of fatty acid biosynthesis, we identified additional biochemical pathways that, when inhibited in combination with fatty acid biosynthesis, displayed increased antiviral potential. Our studies also demonstrated that there was a direct link between changes in cellular chemical composition and the ultrastructural membrane architecture induced by viral gene products. Utilizing inhibitors to change these metabolic environments significantly impacted early viral replication and disrupted the membrane architecture critical for the viral life cycle. Here, we have defined at a molecular level how shifting metabolic landscapes can be exploited to identify combinations of therapeutics that have a greater antiviral effect. Author SummaryDengue viruses are transmitted by Aedes aegypti mosquitoes which are prevalent in the tropical and subtropical regions of the world. These viruses cause over 350 million infections annually. There are no antivirals to combat infection and the only vaccine available is suboptimal. Since these viruses are obligate pathogens, they hijack lipid metabolic pathways in host cells to drive new lipid synthesis critically required for their replication. Mechanisms of how lipid synthesis impacts viral replication is unknown. These viruses also rearrange cellular membranes to form platforms for assembly of viral replication complexes. Here, for the first time, we show that virus-hijacking of de novo fatty acid biosynthesis pathways is required for the formation of membranous replication platforms and if inhibited disrupted synthesis of replicative form viral RNA. Importantly, these inhibitors drastically rearranged the metabolic landscape of the cell resulting in an activation of compensatory nucleotide synthesis pathways that allowed the virus to survive at a low level through the inhibition. However, if both pathways were inhibited in combination, infectious virus release was reduced to below detection limits. The study demonstrates how understanding the metabolic landscape altered by specific inhibitors can lead to the discovery of compensatory metabolic pathways and targets that in combination can enhance intervention efficacy.

microbiology↗

bioRxiv2022

Expression of fatty acid synthase genes and their role in development and arboviral infection of Aedes aegypti

BackgroundFatty acids are the building blocks of complex lipids essential for living organisms. In mosquitoes, fatty acids are involved in cell membrane production, energy conservation and expenditure, innate immunity, development, and reproduction. Fatty acids are synthesized by a multifunctional enzyme complex called fatty acid synthase (FAS). Several paralogues of FAS were found in the Aedes aegypti (Ae. aegypti) mosquito. However, the molecular characteristics and the expression of some of these paralogues have not been investigated. MethodsGenome assemblies of Ae. aegypti were analyzed and orthologues of human FAS were identified. Phylogenetic analysis and in silico molecular characterization were performed to identify the functional domains of the Ae. aegypti FAS (AaFAS). Quantitative analysis and loss-of-function experiments were performed to determine the significance of different AaFAS transcripts in various stages of development, expression following different diets and the impact of AaFAS on dengue virus, serotype 2 (DENV2) infection and transmission. ResultsWe identified seven putative FAS genes in the Ae. aegypti genome assembly, based on nucleotide similarity to the FAS proteins (tBLASTn) of humans, other mosquitoes and invertebrates. Bioinformatics and molecular analyses suggested that only five of the FAS genes produce mRNA and therefore represent complete gene models. Expression levels of AaFAS varied among developmental stages and between male and female Ae. aegypti. Quantitative analyses revealed that expression of AaFAS1, the putative orthologue of the human FAS, was highest in adult females. Transient knockdown (KD) of AaFAS1 did not induce a complete compensation by other AaFAS genes but limited DENV2 infection of Aag2 cells in culture and the midgut of the mosquito. ConclusionAaFAS1 is the predominant AaFAS in the adult mosquitoes. It has the highest amino acid similarity to human FAS and contains all enzymatic domains typical of human FAS. AaFAS1 also facilitated DENV2 replication in both cell culture and in mosquito midguts. Our data suggest that AaFAS1 may play a role in transmission of dengue viruses, and could represent a target for intervention strategies.

microbiology↗

bioRxiv2021

Acyl-CoA Thioesterases; a rheostat that controls activated fatty acids modulates dengue virus serotype 2 replication

During infection with dengue viruses (DENVs), the lipid landscape within host cells is significantly altered to assemble membrane platforms that support viral replication and particle assembly. Fatty acyl-CoAs are key intermediates in the biosynthesis of complex lipids that form these membranes. They also function as key signaling lipids in the cell. Here, we carried out loss of function studies on acyl-CoA thioesterases (ACOTs), a family of enzymes that hydrolyze fatty acyl-CoAs to free fatty acids and coenzyme A, to understand their influence on the lifecycle of DENVs. Loss of function of the type I ACOTs 1 (cytoplasmic) and 2 (mitochondrial) together significantly increased DENV serotype 2 (DENV2) viral replication and infectious particle release. However, isolated knockdown of mitochondrial ACOT2 significantly decreased DENV2 protein translation, genome replication, and infectious virus release. Furthermore, loss of ACOT7 function, a mitochondrial type II ACOT, similarly suppressed DENV2. As ACOT1 and ACOT2 are splice variants, these data suggest that location (cytosol and mitochondria, respectively) rather than function of these proteins may account for the differences in DENV2 infection phenotype. Additionally, loss of mitochondrial ACOT2 and ACOT7 expression also altered the expression of several ACOTs located in multiple organelle compartments within the cell highlighting a complex relationship between ACOTs in the DENV2 virus lifecycle.

microbiology↗

bioRxiv2021

Three-dimensional drug screen identifies HDAC inhibitors as therapeutic agents in mTORC1-driven lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a rare disease involving cystic lung destruction by invasive LAM cells. These cells harbor loss-of-function mutations in TSC2, conferring constitutive mTORC1 signaling. Rapamycin is the only clinically approved disease-modifying treatment, but its action is cytostatic and disease progresses upon its withdrawal. There is a critical need to identify novel agents that prevent the invasive phenotype and/or eradicate the neoplastic LAM cells. Here, we employed novel cellular and extracellular models to screen for candidate therapeutics in a physiologically relevant setting. We observed that lung-mimetic hydrogel culture of pluripotent stem cell-derived diseased cells more faithfully recapitulates human LAM biology compared to conventional culture on two-dimensional tissue culture plastic. Leveraging our culture system, we conducted a three-dimensional drug screen using a custom 800-compound library, tracking cytotoxicity and invasion modulation phenotypes at the single cell level. We identified histone deacetylase (HDAC) inhibitors as a group of anti-invasive agents that are also selectively cytotoxic towards TSC2-/- cells. Unexpectedly, we observed that next generation ATP-competitive mTORC1/2 inhibitors potentiate invasion. We determined anti-invasive effects of HDAC inhibitors to be independent of genotype, while selective cell death is mTORC1-dependent and mediated by apoptosis. Drug performance was subsequently evaluated at the single cell level in zebrafish xenografts. We observed consistent therapeutic efficacy in vivo at equivalent concentrations to those used in vitro, substantiating HDAC inhibitors as potential therapeutic candidates for pursuit in patients with LAM. One Sentence SummaryWe performed a drug screen in 3D and discovered HDAC inhibitors exhibit therapeutic efficacy in models of the lung disease lymphangioleiomyomatosis.

cell biology↗

bioRxiv2020

Triple Combination Nitazoxanide, Ribavirin, and Hydroxychloroquine results in the multiplicative reduction of in vitro SARS-CoV-2 viral replication

BackgroundAn immediate unmet medical need exists to test and develop existing approved drugs against SARS-COV-2. Despite many efforts, very little progress has been made regarding finding low-cost oral medicines that can be made widely available worldwide to address the global pandemic. MethodsWe sought to examine if a triple combination of nitazoxanide (using its active metabolite tizoxanide), ribavirin, and hydroxychloroquine would lead to a multiplicative effects on viral replication of SARS-COV-2 resulting in a significant reduction of virus yield using VERO E6 cells as a model of viral replication. ResultsVirus yield measured in PFU/ml was ~ 2 logs lower with triple combination versus either drug alone, resulting in the prolongation of time to peak cytopathic effects (CPE). The time to produce 50% CPE increased from 2.8 days for viral controls versus 5.3 days for triple combination therapy. Finally, for each 1-log reduction in virus yield 24 hours post-infection, there was an additional 0.7-day delay in onset of CPE. ConclusionsA triple combination of tizoxanide, ribavirin, and hydroxychloroquine produced a reduction in SARS-COV-2 viral replication in Vero E6 cells, warranting exploration in additional cell lines as well as human clinical trials.

microbiology↗