Longitudinal modeling of human neuronal aging identifies RCAN1-TFEB pathway contributing to neurodegeneration of Huntington’s disease

Aging is a common risk factor in neurodegenerative disorders and the ability to investigate aging of neurons in an isogenic background would facilitate discovering the interplay between neuronal aging and onset of neurodegeneration. Here, we perform direct neuronal reprogramming of longitudinally collected human fibroblasts to reveal genetic pathways altered at different ages. Comparative transcriptome analysis of longitudinally aged striatal medium spiny neurons (MSNs), a primary neuronal subtype affected in Huntington’s disease (HD), identified pathways associated with RCAN1, a negative regulator of calcineurin. Notably, RCAN1 undergoes age-dependent increase at the protein level detected in reprogrammed MSNs as well as in human postmortem striatum. In patient-derived MSNs of adult-onset HD (HD-MSNs), counteracting RCAN1 by gene knockdown (KD) rescued HD-MSNs from degeneration. The protective effect of RCAN1 KD was associated with enhanced chromatin accessibility of genes involved in longevity and autophagy, mediated through enhanced calcineurin activity, which in turn dephosphorylates and promotes nuclear localization of TFEB transcription factor. Furthermore, we reveal that G2-115 compound, an analog of glibenclamide with autophagy-enhancing activities, reduces the RCAN1-Calcineurin interaction, phenocopying the effect of RCAN1 KD. Our results demonstrate that RCAN1 is a potential genetic or pharmacological target whose reduction-of-function increases neuronal resilience to neurodegeneration in HD through chromatin reconfiguration.

our study highlights RCAN1 as an effective genetic or pharmacological target that can confer 74 neuronal resilience against the age-associated neurodegeneration of HD.

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Neuronal conversion of longitudinally collected human adult fibroblasts and 78 transcriptome analysis We investigated age-related differences in reprogrammed MSNs from 79 longitudinally collected fibroblasts from three independent healthy individuals and carried out 80 comparative transcriptome analysis between the age groups (Fig. 1a). We designate fibroblasts 81 initially collected during middle age as "young" and samples subsequently collected approximately 82 20 years later from three independent individuals as "old" groups (Coriell NINDS and NIGMS   Fig. 2d). Moreover, RCAN1 expression was significantly increased in 110 HD-MSNs from older symptomatic patients compared to HD-MSNs from younger pre-111 symptomatic patients (pre-HD-MSNs) (~35 years age difference) (Fig. 1i), suggesting the global 112 age-associated upregulation of RCAN1 in MSNs. Altogether, our results indicate that RCAN1 is 113 an age-associated factor whose protein expression undergoes upregulation in aged-MSNs.

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Validation of RCAN1 as a disease modifier gene in HD Genome-wide association studies 116 have identified Genetic Modifiers of HD (GeM-HD) comprised of polymorphic gene variants 117 associated with accelerated or delayed onset of HD 29 . These genes were discovered as modifiers 118 that can affect the age of symptomatic onset. Interestingly, RCAN1 is also included in the list of 119 candidate HD-modifier genes. Thus, as a parallel investigation, we knocked down 246 candidate 120 genes to identify genes whose reduction-of-function would protect HD-MSNs from degeneration, 121 thereby genes that contribute to HD-MSN degeneration (Extended Data Fig. 3a

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well plates to be assayed for neuronal death using Sytox-Green as previously described (approximately 3,000 cells counted per well) (Extended Data Fig. 3c) 9,10 . For this assay, we first 125 used HD-MSN from the GM04194 line (CAG repeat size 46; HD.46) which showed a two-fold 126 increase in cell death at around 50% compared to Control (Ctrl)-MSNs from the healthy individual 127 (GM02171) (Extended Data Fig. 3d). We then added lentivirus carrying gene-specific shRNAs to 128 HD-MSN and the average cell death level for each gene KD was compared to the average level 129 with scrambled control shRNA (shCtrl) (Extended Data Fig. 3e). Interestingly, in this unbiased 130 testing, we also identified RCAN1 whose KD led to the most significant reduction in neuronal 131 death compared to other identified genes, RTCA and UBE2D4 (pink zone = plus or minus 10% 132 of healthy control level). This protective effect was further validated in HD-MSNs from other 133 independent HD patients (Extended Data Fig. 3f). We also tested if KD of the identified genes 134 would lower mHTT aggregation and found that among the genes tested, only RCAN1 KD 135 significantly decreased the amount of mHTT inclusion bodies (Extended Data Fig. 3g).

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To confirm the specificity of shRCAN1 for HD survival, we prolonged RCAN1 expression 137 by overexpressing RCAN1 cDNA in HD-MSNs in the presence of shRCAN1 (Extended Data Fig.   138 4a). Continuous RCAN1 expression reversed the neuroprotective effect of shRCAN1 in HD-MSNs 139 from multiple patients (Fig. 2a). Furthermore, since caspase activation signals have been 140 detected in HD patient brains 30-36 , we also assessed Caspase 3/7 activation and Annexin V signal 141 (an apoptotic marker via its ability to bind to phosphatidylserine on the extracellular surface) in 142 HD-MSNs as previously described 10 . RCAN1 KD significantly reduced Caspase activation and 143 Annexin V signals while the rescuing effect of shRCAN1 was abolished by RCAN1 cDNA (Fig.   144 2b-c). Moreover, the clearance of HTT aggregation following RCAN1 KD was also reversed by 145 overexpressing RCAN1 in the presence of shRCAN1 (Fig. 2d). Therefore, our results overall 146 indicate that RCAN1 is an age-associated disease modifier whose KD leads to clearance of mHTT 147 aggregation and neuroprotection of HD-MSNs.

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Neurodegeneration via RCAN1 KD is associated with changes in chromatin accessibility.

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RCAN1 primarily functions to inhibit its interacting partner, CaN, calcium-and calmodulin-151 dependent protein serine/threonine phosphatase, which in turn regulates phosphorylation of 152 target transcription factors (TFs) [14][15][16] . Due to this potential link between RCAN1 KD and changes 153 in TF activities, we investigated whether RCAN1 KD would lead to changes in chromatin 154 accessibilities in HD-MSNs by performing comparative Omni-ATAC-seq 37 analyses between 155 shCtrl-and shRCAN1-expressing HD-MSNs from multiple HD samples (

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KEGG pathway enrichment analysis revealed that genes associated with DARs opened 166 by shRCAN1 in HD-MSNs were enriched with longevity-regulating pathway, PI3K-AKT/ MAPK/ 167 AMPK signaling pathway, autophagy, and endocytosis, suggesting that RCAN1 KD led to 168 chromatin changes proximal to genes associated with aging in HD-MSNs (Fig. 2f top).

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Interestingly, the role of autophagy in clearing mHTT aggregates and neuroprotection was 170 previously shown by the discovery of miR-29b-3p-STAT3 axis, Beclin1, and autophagy-related 171 FYVE protein (ALFY) that modifies the amount of mHTT aggregation 10,38,39 . Genes associated 172 with closed DARs in shRCAN1-HD-MSNs were, however, enriched in other pathways including 173 long-term depression, circadian entrainment, axon guidance, protein digestion and absorption, 174 focal adhesion, and phospholipase D signaling pathway (Fig. 2f bottom). Altogether, these results demonstrate that the protective effect of RCNA1 KD is accompanied by increased chromatin 176 accessibility to genes involved in longevity and autophagy.   binding sites were enriched with longevity and autophagy pathways ( Fig. 4b top). Some of these 218 genes include RB1CC1, an autophagy inducer 48,49 , and MAPK1, whose function has been shown 219 to decline with brain aging 50,51 (Fig. 4b bottom).

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TFEB is known as a regulator of lysosomal biogenesis and autophagy 25-27 which may also 221 regulate longevity 52-57 . Phosphorylation keeps TFEB localization in the cytoplasm whereas its 222 dephosphorylation allows TFEB shuttling into the nucleus 26,58,59 . Since RCAN1 inhibits CaN 223 function and CaN has been shown to dephosphorylate TFEB 41 , we tested whether RCAN1 KD 224 would lead to TFEB dephosphorylation and nuclear localization in HD-MSNs. RCAN1 KD reduced 225 the level of phosphorylated TFEB, which was reversed by overexpressing RCAN1 as assessed by immunoblots in HD-MSNs from multiple HD patients (Fig. 4c). Also, while expressing 227 exogenous TFEB led to the localization of TFEB in both cytoplasm and nucleus, RCAN1 KD 228 significantly increased nuclear localization of TFEB, which was mimicked by phosphor-mutant 229 (S142/211A) TFEB containing mutations at the serine residue S142 and S211 (CaN's 230 dephosphorylation sites in TFEB) to alanine 26,41,60 ( Fig. 4d and Extended Data Fig. 5a). Our results 231 thus indicate that RCAN1 KD enhances TFEB activity by promoting its nuclear localization.       Wyss-Coray, T. Ageing, neurodegeneration and brain rejuvenation.