Background: Aicardi–Goutières syndrome (AGS) is a severe neurodegenerative disease with clinical features of early-onset encephalopathy and progressive loss of intellectual abilities and motor control. Gene mutations in seven protein-coding genes have been found to be associated with AGS. However, the causative role of these mutations in the early-onset neuropathogenesis has not been demonstrated in animal models, and the mechanism of neurodegeneration of AGS remains ambiguous. Methods: Via CRISPR/Cas-9 technology, we established a mutant mouse model in which a genetic mutation found in AGS patients at the ADAR1 coding gene (Adar) loci was introduced into the mouse genome. A mouse model carrying double gene mutations encoding ADAR1 and MDA-5 was prepared using a breeding strategy. Phenotype, gene expression, RNA sequencing, innate immune pathway activation, and pathologic studies including RNA in situ hybridization (ISH) and immunohistochemistry were used for characterization of the mouse models to determine potential disease mechanisms. Results: We established a mouse model bearing a mutation in the catalytic domain of ADAR1, the D1113H mutation found in AGS patients. With this mouse model, we demonstrated a causative role of this mutation for the early-onset brain injuries in AGS and determined the signaling pathway underlying the neuropathogenesis. First, this mutation altered the RNA editing profile in neural transcripts and led to robust IFN-stimulated gene (ISG) expression in the brain. By ISH, the brains of mutant mice showed an unusual, multifocal increased expression of ISGs that was cell-type dependent. Early-onset astrocytosis and microgliosis and later stage calcification in the deep white matter areas were observed in the mutant mice. Brain ISG activation and neuroglial reaction were completely prevented in the Adar D1113H mutant mice by blocking RNA sensing through deletion of the cytosolic RNA receptor MDA-5. Conclusions: The Adar D1113H mutation in the ADAR1 catalytic domain results in early-onset and MDA5-dependent encephalopathy with IFN pathway activation in the mouse brain.

Background: Astrogliosis and white matter lesions (WML) are key characteristics of vascular contributions to cognitive impairment and dementia (VCID). However, the molecular mechanisms underlying VCID remain poorly understood. Stimulation of Na‐K‐Cl cotransport 1 (NKCC1) and its upstream kinases WNK (with no lysine) and SPAK (the STE20/SPS1‐related proline/alanine‐rich kinase) play a role in astrocytic intracellular Na⁺ overload, hypertrophy, and swelling. Therefore, in this study, we assessed the effect of SPAK inhibitor ZT‐1a on pathogenesis and cognitive function in a mouse model of VCID induced by bilateral carotid artery stenosis (BCAS).Methods: Following sham or BCAS surgery, mice were randomly assigned to receive either vehicle (DMSO) or SPAK inhibitor ZT‐1a treatment regimen (days 14–35 post‐surgery). Mice were then evaluated for cognitive functions by Morris water maze, WML by ex vivo MRI‐DTI analysis, and astrogliosis/demyelination by immunofluorescence and immunoblotting.Results: Compared to sham control mice, BCAS‐Veh mice exhibited chronic cerebral hypoperfusion and memory impairments, accompanied by significant MRI DTI‐detected WML and oligodendrocyte (OL) death. Increased activation of WNK‐SPAK‐NKCC1‐signaling proteins was detected in white matter tissues and in C3d⁺GFAP⁺ cytotoxic astrocytes but not in S100A10⁺GFAP⁺ homeostatic astrocytes in BCAS‐Veh mice. In contrast, ZT‐1a‐treated BCAS mice displayed reduced expression and phosphorylation of NKCC1, decreased astrogliosis, OL death, and WML, along with improved memory functions.Conclusion: BCAS‐induced upregulation of WNK‐SPAK‐NKCC1 signaling contributes to white matter‐reactive astrogliosis, OL death, and memory impairment. Pharmacological inhibition of the SPAK activity has therapeutic potential for alleviating pathogenesis and memory impairment in VCID.

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID's high incidence, which is expected to continue rising as the population ages, it is critical to establish a robust animal model for the disease. This paper presents a novel method of creating a mouse model of VCID that is based on asymmetric bilateral common carotid artery stenosis, which mimics human chronic cerebral hypoperfusion caused by carotid atherosclerosis. Briefly, common carotid arteries (CCAs) are ligated to different gauge needles (32 G for the right CCA and 34 G for the left CCA) using 7-0 silk sutures followed by immediate needle removal. The remaining suture rings cause persistent blood flow reduction and long-term cognitive impairment associated with white matter injury, microinfarcts, and reactive gliosis, thus closely mimicking the pathogenesis of VCID. Importantly, in this needle model, the clinical representations do not revert with time, providing reliable long-term cognitive impairment. Moreover, the survival rate 24 weeks post surgery was 81.6%, which is higher compared to the other established models of VCID with a similar level of blood flow reduction. Additional advantages include low material cost and compatibility with MRI to monitor brain injury in live animals since no metal is implanted. The main challenge in employing the needle model of VCID is the requirement for developing advanced surgical skills since mouse CCAs are less than 0.6 mm in diameter and are very fragile. High-quality visual representation of the surgery will thus help researchers to master this technique and advance our understanding of VCID, potentially leading to the development of novel therapeutic modalities to decrease the devastating cognitive decline associated with VCID.

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID's high incidence, which is expected to continue rising as the population ages, it is critical to establish a robust animal model for the disease. This paper presents a novel method of creating a mouse model of VCID that is based on asymmetric bilateral common carotid artery stenosis, which mimics human chronic cerebral hypoperfusion caused by carotid atherosclerosis. Briefly, common carotid arteries (CCAs) are ligated to different gauge needles (32 G for the right CCA and 34 G for the left CCA) using 7-0 silk sutures followed by immediate needle removal. The remaining suture rings cause persistent blood flow reduction and long-term cognitive impairment associated with white matter injury, microinfarcts, and reactive gliosis, thus closely mimicking the pathogenesis of VCID. Importantly, in this needle model, the clinical representations do not revert with time, providing reliable long-term cognitive impairment. Moreover, the survival rate 24 weeks post surgery was 81.6%, which is higher compared to the other established models of VCID with a similar level of blood flow reduction. Additional advantages include low material cost and compatibility with MRI to monitor brain injury in live animals since no metal is implanted. The main challenge in employing the needle model of VCID is the requirement for developing advanced surgical skills since mouse CCAs are less than 0.6 mm in diameter and are very fragile. High-quality visual representation of the surgery will thus help researchers to master this technique and advance our understanding of VCID, potentially leading to the development of novel therapeutic modalities to decrease the devastating cognitive decline associated with VCID.