The most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide repeat expansion (HRE) in C9ORF72 that contributes to neurodegeneration by both loss-of-function (decreased C9ORF72 protein levels) and gain-offunction (e.g. dipeptide repeat protein production) mechanisms. Although therapeutics targeting the gain-of-function mechanisms are in clinical development, it is unclear if these will be efficacious given the contribution of C9ORF72 loss-of-function processes to neurodegeneration. Moreover, there is a lack of therapeutic strategies for C9ORF72 ALS/FTD with demonstrated efficacy in vivo. Here, we show that small molecule inhibition of PIKFYVE kinase rescues both loss-and gain-of-function C9ORF72 disease mechanisms in vivo. We find that the reduction of C9ORF72 in mouse motor neurons leads to a decrease in early endosomes. In contrast, treatment with the PIKFYVE inhibitor apilimod increases the number of endosomes and lysosomes. We show that reduced C9ORF72 levels increases glutamate receptor levels in hippocampal neurons in mice, and that apilimod treatment rescues this excitotoxicity-related phenotype in vivo. Finally, we show that apilimod also alleviates the gainof-function pathology induced by the C9ORF72 HRE by decreasing levels of dipeptide repeat proteins derived from both sense and antisense C9ORF72 transcripts in hippocampal neurons in vivo. Our data demonstrate the neuroprotective effect of PIKFYVE kinase inhibition in both gain-and loss-of-function murine models of C9ORF72 ALS/FTD. Keywordsamyotrophic lateral sclerosis, frontotemporal dementia, apilimod, C9ORF72, NMDA-induced injury, endosomal trafficking, dipeptide repeat proteins, lysosomes, endosomes, glutamate receptors A. Images of EEA1+ vesicles in the hippocampus of C9-BAC mice treated by direct injection with DMSO or apilimod. Scale bar = 10 µm. B. Number of EEA1+ vesicles per cytosolic area in the hippocampus of C9-BAC mice treated by direct injection with apilimod (n=186 cells from 8 mice) or DMSO (vehicle, n=185 cells from 8 mice) for 48 hours. Mean +/-standard deviation, unpaired t-test. C. Number of EEA1+ vesicles per cytosolic area in the hippocampus of C9-BAC mice treated by direct injection with apilimod (n=8 mice) or DMSO (vehicle, n= 8 mice) for 48 hours. Median +/-interquartile range of cells for each mouse, unpaired t-test comparing mean values per mouse. Horizontal grey dotted line indicates the median number of EEA1+ vesicles per cytosolic area in the hippocampus of C9-BAC mice treated by direct injection with DMSO. D. Images of poly(GP)+ punctae in the hippocampus of C9-BAC mice 48 hours after being treated by direct injection with DMSO or apilimod. Scale bar = 10 µm.E. Number of poly(GP)+ punctae per neuronal area in the hippocampus of C9-BAC mice treated by direct injection with apilimod (n=150 cells from 9 mice) or DMSO (vehicle, n=150 cells from 9 mice) for 48 hours. Control group, included for reference, are untreated wild-type mice (n=125 cells from 6 mice). Median...

30RNA therapies such as oligonucleotides (OGNs) offer precision treatments for a variety of 31 neurological diseases, including epilepsy but their deployment is hampered by the blood brain 32 barrier (BBB). Here we used brain imaging and assays of serum proteins and tracer 33 extravasation, to determine that BBB disruption occurring after status epilepticus in mice was 34 sufficient to permit passage of systemically-injected antisense OGNs targeting microRNA-134 35 (Ant-134) into the brain parenchyma. A single intraperitoneal injection of Ant-134 two hours 36 after status epilepticus in mice resulted in potent suppression of spontaneous recurrent 37 seizures, reaching a 99.5% reduction during recordings at three months. The duration of 38 spontaneous seizures, when they occurred, was also reduced in Ant-134-treated mice. These 39 studies indicate that systemic delivery of Ant-134 reaches the brain and produces disease-40 modifying effects after systemic injection in mice when timed with BBB disruption and may be 41 a clinically-viable approach for this and other disease-modifying microRNA therapies. 42 43 44 45