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Huntington's disease (HD) is characterised by progressive symptoms including cognitive deficits and sleep/wake disturbances reflected in an abnormal electroencephalography (EEG). Modafinil, a wake-promoting and cognitive-enhancing drug, has been considered as a treatment for HD. We used HD (R6/2) mice to investigate the potential for using modafinil to treat sleep-wake disturbance in HD. R6/2 mice show sleep-wake and EEG changes similar to those seen in HD patients, with increased rapid eye movement sleep (REMS), decreased wakefulness/increased non-REMS (NREMS), and pathological changes in EEG spectra, particularly an increase in gamma power. We recorded EEG from R6/2 and wild-type mice treated with modafinil acutely (with single doses between 25 and 100 mg/kg; at 12 and 16 weeks of age), or chronically (64 mg/kg modafinil/day from 6 to 15 weeks). Acutely, modafinil increased wakefulness in R6/2 mice and restored NREMS to wild-type levels at 12 weeks. It also suppressed the pathologically increased REMS. This was accompanied by decreased delta power, increased peak frequency of theta, and increased gamma power. At 16 weeks, acute modafinil also restored wakefulness and NREMS to wild-type levels. However, whilst REMS decreased, it did not return to normal levels. By contrast, in the chronic treatment group, modafinil-induced wakefulness was maintained at 15 weeks (after 9 weeks of treatment). Interestingly, chronic modafinil also caused widespread suppression of power across the EEG spectra, including a reduction in gamma that increases pathologically in R6/2 mice. The complex EEG effects of modafinil in R6/2 mice should provide a baseline for further studies to investigate the translatability of these result to clinical practice.

Background Mutations in microtubule‐associated protein tau (MAPT) gene are causative of Frontotemporal Dementia (FTD). Many of the features associated with the development of tau pathology, e.g high levels of tau phosphorylation, are also present in early development. iPSC‐neurons have gene expression signatures similar to fetal neurons, and iPSC‐neurons with MAPT mutations do not develop tau aggregates. We hypothesise that iPSC‐neurons are resistant to developing tau aggregates due to high activity levels of the proteostasis network during early development. To test this hypothesis, we investigated the levels of proteasome subunits in developing iPSC derived neurons and measured the proteasome activity of iPSC‐neurons throughout development. To test whether manipulation of proteostasis in iPSC‐neurons could lead to tau pathology we treated cells with a proteasome inhibitor, and measured changes in total and phosphorylated tau, proteasome subunits and autophagy regulators. Methods Human cortical neurons were derived from isogenic iPSCs with the following MAPT genotypes: WT, 10+16 monoallelic, 10+16 biallelic and 10+16/P301S biallelic. RNA and protein analysis of proteasome subunits was performed by qPCR and Western blot at during neuronal development (DIV 0, 10, 30 and 100) and after proteasome inhibition treatments. Results We show that proteasome activity decreases during the differentiation of iPSCs to cortical neurons, accompanied by a reduction in levels of the proteasome regulatory subunits Rpt6 and Rpn6. Proteasome inhibition in WT neurons does not lead to a significant change in total or phosphorylated tau levels but resulted in an increase in the autophagy associated protein BAG3, together with an induction of tau cleaved by caspase‐3 at Asp421. Conclusion Proteasome activity decreases during the differentiation of iPSCs into neurons, which may be due to a reduction in in proteasome regulators. Our results suggest that proteasome inhibition causes an increase in tau cleavage which may be preferentially cleared through the autophagy pathway. The ability of these ‘fetal‐like’ neurons to adapt and upregulate their protein clearance system may be enhanced compared to FTD brain tissue. In ongoing work we are investigating proteasome expression and activity in FTD post‐mortem tissue as well as the localisation and interactions of tau and the proteasome after proteasome inhibition treatments.

Background GRN mutations, resulting in haploinsufficiency of progranulin, cause frontotemporal dementia (FTD) with TDP‐43 pathology. Impairments in mitophagy, the selective autophagy of damaged mitochondria, have been identified in several neurodegenerative diseases, and multiple neurodegenerative disease genes, including PINK1, Parkin, VCP and TBK1, have been shown to play a role in this pathway. A role for progranulin in the regulation of neuronal mitophagy has not been explored, however progranulin deficient mice exhibit reduced xenophagy (selective clearance of bacteria). We therefore hypothesised that loss of progranulin could lead to defective mitophagy. Method We used two in vitro models to investigate PINK1/Parkin mitophagy: SHSY5Y cells overexpressing Parkin (PoE‐SHSY5Y) +/‐ siRNA against GRN, and induced pluripotent stem cell (iPSC) derived cortical neurons from two patients with FTD‐associated GRN mutations (R493X and C31fs). PINK1/Parkin mitophagy was induced using a combination of Antimycin A (respiratory complex III inhibitor) and oligomycin (ATP synthase inhibitor), and PINK1 accumulation and levels of S65 phosphorylated ubiquitin (the substrate of PINK1 and a marker of its activity) were examined using western blot and immunofluorescence. Subsequent mitophagy was assessed by examining Mitofusin‐2 ubiquitination and degradation, and TIM23 levels by western blot. Result A reduction in progranulin levels resulted in reduced accumulation of PINK1 and lower levels of mitochondrial phospho‐ubiquitin following oligomycin/antimycin treatment in both PoE‐SHSY5Y and iPSC‐neurons with GRN mutations. Ubiquitination and degradation of mitofusin‐2 was also decreased in cells with reduced progranulin. Conclusion These results suggest that progranulin plays a role in mitophagy by regulating stability and/or activity of PINK1. Ongoing work aims to understand the mechanisms by which progranulin and/or individual granulins contribute to this process and to dissect cell‐type specific contributions of progranulin to mitophagy in iPSC‐ derived neurons, astrocytes and microglia.

Background GRN mutations that result in progranulin haploinsufficiency cause frontotemporal dementia (FTD). Mitophagy, the selective autophagy of damaged mitochondria, is impaired in several neurodegenerative diseases. A number of genes linked to FTD (e.g. OPTN, SQSTM, VCP and TBK1) are also known to play a role in mitophagy. Xenophagy, the selective autophagy of non‐host pathogens, relies on some of the same proteins as mitophagy (Parkin and TBK1) and is reduced in GRN knockout mice. Progranulin has also been found to play a role in mitophagy in mouse kidney cells. We therefore hypothesised that loss of progranulin could lead to defective mitophagy in neurons, astrocytes and microglia. Methods We investigated PINK1/Parkin mitophagy in astrocytic‐like H4 cells and neuron‐like Parkin overexpressing SHSY5Y cells (PoE‐5Ys) +/‐ siRNA against GRN. We also examined induced pluripotent stem cells (iPSCs) from four controls, three patients with FTD‐associated GRN mutations (R493X and C31fs) and a CRISPR series from the human iPSC Neurodegenerative Disease Initiative1,2,3 (iNDI) with an isogenic control, heterozygous and homozygous R493X mutation lines. The iPSCs were differentiated to cortical neurons, astrocytes and microglia. PINK1/Parkin mitophagy was induced using Antimycin A (respiratory complex III inhibitor) and oligomycin (ATP synthase inhibitor). PINK1 accumulation, levels of S65 phosphorylated ubiquitin (pUb), other mitophagy markers as well as proteins hypothesised to play a role in the mechanism were examined using western blotting and immunofluorescence (ICC). Results Lower levels of pUb were detected in PoE‐5Ys treated with oligomycin/antimycin following GRN knockdown. There was a significant reduction in mitophagy in GRN siRNA treated H4 cells, detected by ICC and western blotting of mitophagy markers. Initial results from iPSC neurons found no significant difference in mitophagy between control and patient lines. Work is ongoing in a larger set of iPSC neurons, astrocytes and microglia. Preliminary results suggest that reducing progranulin affects mitophagy in astrocytes but has a limited effect on mitophagy in neurons. Conclusions The results suggest that progranulin has a cell specific role in mitophagy with progranulin regulating stability and/or activity of PINK1. Current work aims to understand the mechanisms of this process and to dissect cell‐type specific contributions of progranulin to mitophagy.