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The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune modulation and a delivery system capable of crossing the blood–brain barrier. The recent identification and characterization of a small population of regulatory T (Treg) cells resident in the brain presents one such potential therapeutic target. Here, we identified brain interleukin 2 (IL-2) levels as a limiting factor for brain-resident Treg cells. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially direct delivery to inflammatory sites. Mice with brain-specific IL-2 delivery were protected in traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL-2 gene delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients.Liston and colleagues design a gene-delivery system to specifically target astrocytes in the central nervous system to express IL-2 and thereby expand/maintain Treg cells to suppress neuroinflammation.

Charcot–Marie–Tooth disease (CMT) is the most common inherited peripheral neuropathy, with currently no effective treatment or cure. CMT1A is caused by a duplication of the PMP22 gene, which leads to Schwann cell differentiation defects and dysmyelination of the peripheral nerves. The epigenetic regulator histone deacetylase 3 (HDAC3) has been shown to negatively regulate myelination as well as its associated signaling pathways, PI3K-AKT and MAPK-ERK. We showed that these signaling pathways are indeed downregulated in the C3-PMP22 mouse model, similar to what has been shown in the CMT1A rat model. We confirmed that early postnatal defects are present in the peripheral nerves of the C3-PMP22 mouse model, which led to a progressive reduction in axon caliber size and myelination. The aim of this study was to investigate whether pharmacological HDAC3 inhibition could be a valuable therapeutic approach for this CMT1A mouse model. We demonstrated that early treatment of CMT1A mice with the selective HDAC3 inhibitor RGFP966 increased myelination and myelin g-ratios, which was associated with improved electrophysiological recordings. However, a high dose of RGFP966 caused a decline in rotarod performance and a decline in overall grip strength. Additionally, macrophage presence in peripheral nerves was increased in RGFP966 treated CMT1A mice. We conclude that HDAC3 does not only play a role in regulating myelination but is also important in the neuroimmune modulation. Overall, our results indicate that correct dosing of HDAC3 inhibitors is of crucial importance if translated to a clinical setting for demyelinating forms of CMT or other neurological disorders.

The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is an intronic G 4 C 2 repeat expansion in C9orf72 . The repeats undergo bidirectional transcription to produce sense and antisense repeat RNA species, which are translated into dipeptide repeat proteins (DPRs). As toxicity has been associated with both sense and antisense repeat-derived RNA and DPRs, targeting both strands may provide the most effective therapeutic strategy. CRISPR-Cas13 systems mature their own guide arrays, allowing targeting of multiple RNA species from a single construct. We show CRISPR-Cas13d variant CasRx effectively reduces overexpressed C9orf72 sense and antisense repeat transcripts and DPRs in HEK cells. In C9orf72 patient-derived iPSC-neuron lines, CRISPR-CasRx reduces endogenous sense and antisense repeat RNAs and DPRs and protects against glutamate-induced excitotoxicity. AAV delivery of CRISPR-CasRx to two distinct C9orf72 repeat mouse models significantly reduced both sense and antisense repeat-containing transcripts. This highlights the potential of RNA-targeting CRISPR systems as therapeutics for C9orf72 ALS/FTD. CRISPR-CasRx effectively reduces ALS- and FTD-causing C9orf72 sense and antisense repeat derived RNAs and proteins in cell lines, patient iPSC-neurons and two independent mouse models of C9orf72 repeat expansion.

The most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a G4C2 repeat expansion in intron 1 of the C9orf72 gene. This repeat expansion undergoes bidirectional transcription to produce sense and antisense repeat RNA species. Both sense and antisense-derived repeat RNAs undergo repeat-associated non-AUG translation in all reading frames to generate five distinct dipeptide repeat proteins (DPRs). Importantly, toxicity has been associated with both sense and antisense repeat-derived RNA and DPRs. This suggests targeting both sense and antisense repeat RNA may provide the most effective therapeutic strategy. The RNA-targeting CRISPR-Cas13 systems offer a promising avenue for simultaneous targeting of multiple RNA transcripts, as they mature their own guide arrays, thus allowing targeting of more than one RNA species from a single construct. We show that CRISPR-Cas13d originating from Ruminococcus flavefaciens (CasRx) can successfully reduce C9orf72 sense and antisense repeat transcripts and DPRs to background levels in HEK cells overexpressing C9orf72 repeats. CRISPR-CasRx also markedly reduced the endogenous sense and antisense repeat RNAs and DPRs in three independent C9orf72 patient-derived iPSC-neuron lines, without detectable off-target effects. To determine whether CRISPR-CasRx is effective in vivo, we treated two distinct C9orf72 repeat mouse models using AAV delivery and observed a significant reduction in both sense and antisense repeat-containing transcripts. Taken together this work highlights the potential for RNA-targeting CRISPR systems as therapeutics for C9orf72 ALS/FTD.Competing Interest StatementLiam Kempthorne and Adrian Isaacs are co-inventors listed on UK Patent Application No. 2105455.6, International application No. PCT/EP2022/060296 which are related to the presented data. All other authors declare they have no competing interests.

Duplication of PMP22 causes Charcot-Marie-Tooth disease type 1A (CMT1A) and is known to disrupt the lipid metabolism in myelinating Schwann cells by unknown mechanisms. By using two CMT1A mouse models overexpressing human PMP22, we discovered that PMP22 dose-dependently downregulates genes that are involved in lipid and cholesterol metabolism. Lipidomic analysis on CMT1A mouse sciatic nerves confirmed lipid metabolic abnormalities primarily associated with cholesterol and sphingolipids. We observed similar lipidomic profiles and downregulation of genes associated with lipid metabolism in human CMT1A patient induced pluripotent stem cell-derived Schwann cell precursors (iPSC-SCPs). We confirmed these findings by demonstrating altered lipid raft dynamics and plasma membrane fluidity in CMT1A iPSC-SCPs. Additionally, we identified impaired cholesterol incorporation in the plasma membrane due to altered lipid storage homeostasis in CMT1A iPSC-SCPs, which could be modulated by changing the lipid composition of the cell culture medium. These findings suggest that PMP22 plays a role in regulating the lipid composition of the plasma membrane and lipid storage homeostasis. Targeting lipid metabolism may hold promise as a potential treatment for CMT1A patients. PMP22 copy number causes a dose-dependent suppression of cholesterol and lipid biosynthesis in peripheral nerves of CMT1A mice Lipid composition is altered in the sciatic nerves of CMT1A mice and in the membranes of patient derived iPSC-SCPs, with a significant reduction in sphingolipids CMT1A iPSC-SCPs show decreased plasma membrane lipids required for regulating lipid raft dynamics, membrane fluidity, and membrane order Lipid storage misregulation is key in the pathogenesis of CMT1A

The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune-modulation and a delivery system capable of crossing the blood-brain-barrier. The recent identification and characterization of a small population of regulatory T cells (Tregs) resident in the brain presents one such potential therapeutic target. Here we identified brain IL2 levels as a limiting factor for brain-resident Tregs. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially-direct delivery to inflammatory sites. Mice with brain-specific IL2 delivery were protected from traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL2 gene-delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients. Competing Interest Statement The VIB and Babraham Institute are owners of a patent based on work included in the manuscript, with L.Y., E.P., J.D., M.G.H. and A.L. potential financial beneficiaries of commercialization.

The presence of vegetation in stream ecosystems is highly dynamic in both space and time. A digital photography technique is developed to map aquatic vegetation cover at species level, which has a very high spatial and a flexible temporal resolution. A digital single-lens reflex (DSLR) camera mounted on a handheld telescopic pole is used. The low-altitude (5 m) orthogonal aerial images have a low spectral resolution (red-green-blue), high spatial resolution (∼1.9 pixels cm −2 , ∼1.3 cm length) and flexible temporal resolution (monthly). The method is successfully applied in two lowland rivers to quantify four key properties of vegetated rivers: vegetation cover, patch size distribution, biomass and hydraulic resistance. The main advantages are that the method is (i) suitable for continuous and discontinuous vegetation covers, (ii) of very high spatial and flexible temporal resolution, (iii) relatively fast compared to conventional ground survey methods, (iv) non-destructive and (v) relatively cheap and easy to use, and (vi) the software is widely available and similar open source alternatives exist. The study area should be less than 10 m wide, and the prevailing light conditions and water turbidity levels should be sufficient to look into the water. Further improvements of the image processing are expected in the automatic delineation and classification of the vegetation patches.

In-stream submerged macrophytes have a complex morphology and several species are not rigid, but are flexible and reconfigure along with the major flow direction to avoid potential damage at high stream velocities. However, in numerical hydrodynamic models, they are often simplified to rigid sticks. In this study hydraulic resistance of vegetation is represented by an adapted bottom friction coefficient and is calculated using an existing two layer formulation for which the input parameters were adjusted to account for (i) the temporary reconfiguration based on an empirical relationship between deflected vegetation height and upstream depth-averaged velocity, and (ii) the complex morphology of natural, flexible, submerged macrophytes. The main advantage of this approach is that it removes the need for calibration of the vegetation resistance coefficient. The calculated hydraulic roughness is an input of the hydrodynamic model Telemac 2D, this model simulates depth-averaged stream velocities in and around individual vegetation patches. Firstly, the model was successfully validated against observed data of a laboratory flume experiment with three macrophyte species at three discharges. Secondly, the effect of reconfiguration was tested by modelling an in situ field flume experiment with, and without, the inclusion of macrophyte reconfiguration. The inclusion of reconfiguration decreased the calculated hydraulic roughness which resulted in smaller spatial variations of simulated stream velocities, as compared to the model scenario without macrophyte reconfiguration. We discuss that including macrophyte reconfiguration in numerical models input, can have significant and extensive effects on the model results of hydrodynamic variables and associated ecological and geomorphological parameters.