Wednesday, May 18, 2022
5/18/2022
Abstract Spinal cord injury (SCI) is debilitating. Astrocytes are the predominant component of the spinal scar. Defining the complex molecular regulation during astrogliosis is fundamentally important for developing effective therapeutic strategies. We were the first to systematically analyze the expression profiles of long non- coding RNA (lncRNA) (>200 bp), a type of regulatory RNA that plays important roles, in purified astrocytes from adult spinal cords across SCI stages. There is a large knowledge gap in lncRNAs' roles and mechanisms in astrogliosis after SCI. As <2% of the genome is associated with protein-coding genes, innovative targeting of non-coding RNAs can greatly enlarge the number of druggable targets and facilitate new classes of mechanistic discoveries. Towards the overarching goal of defining the mechanisms of astrogliosis in SCI and identifying novel molecular targets for treating chronic SCI, we discovered that Zeb2os, a highly conserved lncRNA, plays an important functional role in astrogliosis. Data showed that Zeb2os expression has high correlation over the injury time course with its antisense protein coding gene Zeb2 and the essential astrogliosis factor Stat3. Significantly, Zeb2os shRNA knockdown (KD) in vivo by Adeno Associated Virus (AAV) injection led to attenuated astrogliosis, shown by astrocyte morphology, reduced GFAP and pSTAT3 expression, and reduced lesion size. Additionally, Zeb2os KD in primary astrocytes significantly decreased expression of many genes, including Gfap, Zeb2, Stat3, neurocan, phosphacan and integrins that are involved in scar development. Our RNAscope data demonstrated Zeb2os-FZeb2 and Zeb2os- pSTAT3 colocalization in GFAP+ cells. Furthermore, ChIP-Seq experiments revealed STAT3 bound to the Zeb2 promoter region. Notably, our data and studies by others provided evidence that, after injury, there is a change of astrocytes from an initial beneficial state (reactive astrocytes, RA) to a maladaptive state (scar-forming astrocytes, SA) expressing molecules that inhibit neuroregeneration. Importantly, attenuating astrocytic scar progression with anti-integrin neutralizing antibodies promoted functional improvement after SCI. Based on our exciting findings and others', we propose to: a) test the hypothesis that lncRNA Zeb2os regulates astrogliosis through a Zeb2os/Zeb2/Stat3 axis, b) decipher additional mechanisms by which Zeb2os regulates astrocytes by comprehensively identifying its target genes and pathways, and c) define the effects of manipulating Zeb2os and target genes on anatomical and functional outcomes in vivo by AAV injection. Our goal is not to remove the scar; it is to attenuate later stages of scar progression and the scar's inhibitory effects on neuroregeneration without interrupting neuroprotective effects at early phases. These team of investigators with unique and strong expertise in SCI, astrocyte biology and genomics. Overall, studies will be led by these a studieswill significantly advance our understanding oflncRNA function and mechanisms in astrogliosis, and thereby reveal new molecular targets for treating chronic SCI.
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