Anatomy and Neuroscience - Research Publications
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ItemNo Preview AvailableFractalkine-induced microglial vasoregulation occurs within the retina and is altered early in diabetic retinopathy( 2020)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 Müller 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 Statement
This 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. -
ItemNanopore direct RNA sequencing detects differential expression between human cell populations( 2020)Accurately quantifying gene and isoform expression changes is essential to understanding cell functions, differentiation and disease. Therefore, a crucial requirement of RNA sequencing is identifying differential expression. The recent development of long-read direct RNA (dRNA) sequencing has the potential to overcome many limitations of short and long-read sequencing methods that require RNA fragmentation, cDNA synthesis or PCR. dRNA sequences native RNA and can encompass an entire RNA in a single read. However, its ability to identify differential gene and isoform expression in complex organisms is poorly characterised. Using a mixture of synthetic controls and human SH-SY5Y cell differentiation into neuron-like cells, we show that dRNA sequencing accurately quantifies RNA expression and identifies differential expression of genes and isoforms. We generated ∼4 million dRNA reads with a median length of 991 nt. On average, reads covered 74% of SH-SY5Y transcripts and 29% were full-length. Measurement of expression and fold changes between synthetic control RNAs confirmed accurate quantification of genes and isoforms. Differential expression of 231 genes, 291 isoforms, plus 27 isoform switches were detected between undifferentiated and differentiated SH-SY5Y cells and samples clustered by differentiation state at the gene and isoform level. Genes upregulated in neuron-like cells were associated with neurogenesis. We further identified >30,000 expressed transcripts including thousands of novel splice isoforms and transcriptional units. Our results establish the ability of dRNA sequencing to identify biologically relevant differences in gene and isoform expression and perform the key capabilities of expression profiling methodologies.
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ItemNo Preview AvailableTranscriptional and epi-transcriptional dynamics of SARS-CoV-2 during cellular infection.( 2020-12-22)SARS-CoV-2 uses subgenomic (sg)RNA to produce viral proteins for replication and immune evasion. We applied long-read RNA and cDNA sequencing to in vitro human and primate infection models to study transcriptional dynamics. Transcription-regulating sequence (TRS)-dependent sgRNA was upregulated earlier in infection than TRS-independent sgRNA. An abundant class of TRS-independent sgRNA consisting of a portion of ORF1ab containing nsp1 joined to ORF10 and 3’UTR was upregulated at 48 hours post infection in human cell lines. We identified double-junction sgRNA containing both TRS-dependent and independent junctions. We found multiple sites at which the SARS-CoV-2 genome is consistently more modified than sgRNA, and that sgRNA modifications are stable across transcript clusters, host cells and time since infection. Our work highlights the dynamic nature of the SARS-CoV-2 transcriptome during its replication cycle. Our results are available via an interactive web-app at http://coinlab.mdhs.unimelb.edu.au/ .