Mills, S. A. | Search bioRxiv

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

Cortical hyperexcitability drives dying forward ALS symptoms and pathology in mice

Amyotrophic lateral sclerosis (ALS) is a progressive fatal disorder caused by degeneration of motor neurons in the cortex and spinal cord. The origin of ALS in the central nervous system is unclear, however cortical hyperexcitability appears as an early and intrinsic feature of ALS and has been linked to degeneration of spinal motor neurons via a dying-forward mechanism. Here, we implement chemogenetics to validate the dying forward hypothesis of ALS in mice. We show that chronic hyperexcitability of corticomotoneurons induced by excitatory chemogenetics results in motor symptoms and core neuropathological hallmarks of ALS, including corticomotoneuron loss, corticospinal tract degeneration and reactive gliosis. Importantly, corticomotoneuron loss was sufficient to drive degeneration of spinal motor neurons and neuromuscular junctions (NMJs), associated with cytoplasmic TAR DNA binding protein 43 (TDP-43) pathology. These findings establish a cortical origin of ALS mediated by neuronal hyperexcitability, consistent with a dying forward mechanism of neurodegeneration.

neuroscience↗

bioRxiv2020

Fractalkine-induced microglial vasoregulation occurs within the retina and is altered early in diabetic retinopathy

Local blood flow control within the CNS is critical to proper function and is dependent on coordination between neurons, glia and blood vessels. Macroglia such as astrocytes and Muller cells, contribute to this neurovascular unit within the brain and retina, respectively. This study explored the role of microglia, the innate immune cell of the CNS, in retinal vasoregulation and highlights changes during early diabetes. Structurally, microglia were found to contact retinal capillaries and neuronal synapses. In the brain and retinal explants, the addition of fractalkine, the sole ligand for monocyte receptor Cx3cr1, resulted in capillary constriction at regions of microglial contact. This vascular regulation was dependent on microglial involvement, since mice lacking Cx3cr1, exhibited no fractalkine-induced constriction. Analysis of the microglial transcriptome identified several vasoactive genes, including angiotensinogen, a constituent of the renin-angiotensin system (RAS). Subsequent functional analysis showed that RAS blockade via candesartan, abolished microglial-induced capillary constriction. Microglial regulation was explored in a rat streptozotocin (STZ) model of diabetic retinopathy. Retinal blood flow was reduced after 4 weeks due to reduced capillary diameter and this was coincident with increased microglial association. Functional assessment showed loss of microglial-capillary response in STZ-treated animals and transcriptome analysis showed evidence of RAS pathway dysregulation in microglia. While candesartan treatment reversed capillary constriction in STZ-treated animals, blood flow remained decreased likely due to dilation of larger vessels. This work shows microglia actively participate in the neurovascular unit, with aberrant microglial-vascular function possibly contributing to the early vascular compromise during diabetic retinopathy. Significance StatementThis work identifies a novel role for microglia, the innate immune cells of the CNS, in the local control of the retinal vasculature and identifies deficits early in diabetes. Microglia contact neurons and vasculature and express several vasoactive agents. Activation of microglial fractalkine-Cx3cr1 signalling leads to capillary constriction and blocking the renin-angiotensin system (RAS) with candesartan abolishes microglial-mediated vasoconstriction in the retina. In early diabetes, reduced retinal blood flow is coincident with capillary constriction, increased microglial-vessel association, loss of microglial-capillary regulation and altered microglial expression of the RAS pathway. While candesartan restores retinal capillary diameter early in diabetes, targeting of microglial-vascular regulation is required to prevent coincident dilation of large retinal vessels and reduced retinal blood flow.

cell biology↗