Absence of Sac2/INPP5F enhances the phenotype of a Parkinson’s disease mutation of synaptojanin 1 Mian Caoa,b,c,d,1,2, Daehun Parka,b,c,d,1, Yumei Wua,b,c,d, and Pietro De Camillia,b,c,d,e,3 aDepartment of Neuroscience, Yale University School of Medicine, New Haven, CT 06510; bDepartment of Cell Biology, Yale University School of Medicine, New Haven, CT 06510; cHoward Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510; dProgram in Cellular Neuroscience, Neurodegeneration, and Repair, Yale University School of Medicine, New Haven, CT 06510; and eKavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT 06510 Contributed by Pietro De Camilli, April 9, 2020 (sent for review March 9, 2020; reviewed by Volker Haucke and David Sulzer) Numerous genes whose mutations cause, or increase the risk of, analysis of cultured neurons and brain tissue of these mice Parkinson’s disease (PD) have been identified. An inactivating mu- demonstrated defects in synaptic vesicle recycling and dystrophic tation (R258Q) in the Sac inositol phosphatase domain of synapto- changes selectively in nerve terminals of dopaminergic neurons janin 1 (SJ1/PARK20), a phosphoinositide phosphatase implicated in the striatum (17). Levels of auxilin (PARK19) and other in synaptic vesicle recycling, results in PD. The gene encoding Sac2/ endocytic factors were abnormally increased in the brains of such INPP5F, another Sac domain-containing protein, is located within a mice (17). In addition, levels of the PD gene parkin (PARK2) PD risk locus identified by genome-wide association studies. were strikingly elevated (17), strengthening the evidence for Knock-In mice carrying the SJ1 patient mutation (SJ1RQKI) exhibit a functional partnership among endophilin, SJ1, and parkin PD features, while Sac2 knockout mice (Sac2KO) do not have ob- reported by previous studies (18–20). vious neurologic defects. We report a “synthetic” effect of the SJ1 Most interestingly, the intronic SNP rs117896735 located mutation and the KO of Sac2 in mice. Most mice with both muta- within the gene encoding Sac2/INPP5F, another protein con- tions died perinatally. The occasional survivors had stunted ttaining a Sac domain that functions primarily as a PI4P 4- growth, died within 3 wk, and showed abnormalities of striatal phosphatase (Fig. 1A) (21, 22), was identified as a risk locus in RQ dopaminergic nerve terminals at an earlier stage than SJ1 KI PD by genome-wide association studies (23, 24). Sac2 has been mice. The abnormal accumulation of endocytic factors observed implicated in the endocytic pathway via its interaction with Rab5 RQ at synapses of cultured SJ1 KI neurons was more severe in and, like SJ1, is preferentially expressed in the nervous system double-mutant neurons. Our results suggest that SJ1 and Sac2 (21, 22). These findings raise the hypothesis that the Sac domains have partially overlapping functions and are consistent with a of SJ1 and Sac2 may have some overlapping functions in the potential role of Sac2 as a PD risk gene. down-regulation of a same phosphoinositide pool on membranes of the endocytic pathway at synapses. Here we used mouse ge- endocytosis | Sac2 | synaptojanin 1 | INPP5F | PI4P netics to explore this possibility. We report the occurrence of a strong synthetic effect at both the organismal level and the ynaptojanin 1 (SJ1), also known as PARK20, is a phosphoi- synaptic level of the Sac2 null mutation and of the EOP mutation Snositide phosphatase concentrated at synapses implicated in endocytic membrane traffic. SJ1 dephosphorylates PI(4,5)P2 on Significance endocytic membranes via the sequential action of two tandemly arranged inositol phosphatase modules: a central 5-phosphatase Extensive genetic studies have identified numerous genes whose A domain and an N-terminal Sac phosphatase domain (Fig. 1 ). mutations results on Parkinson’s disease (PD), including syn- The latter functions primarily as a 4-phosphatase domain, al- aptojanin 1 (SJ1/Park20), a nerve terminal enriched protein that though it can also dephosphorylate PI3P and PI(3,5)P2 in addi- includes an inositol 4-phosphatase domain (Sac domain). In tion to PI4P in vitro (1). addition, many PD candidate genes have been identified by Full loss-of-function mutations of the SJ1 gene result in early genome-wide association studies, but for most of these genes, postnatal lethality in both mice and humans (2–4). In contrast, a the link to PD remains hypothetical. One such gene is Sac2/INPP5F, homozygous missense R > Q mutation at amino acid position which, interestingly, also includes an inositol 4-phosphatase do- 258 within the Sac domain of SJ1 (a highly conserved amino acid main. While Sac2KO mice do not show obvious defects, we show position) is responsible for early-onset Parkinsonism (EOP) with a striking synthetic effect in mice of the KO of Sac2 and the Sac epilepsy, leading to the naming of SJ1 as PARK20 (5–7). domain mutation of SJ1 found in PD patients. These findings The well-established main function of SJ1 is to participate in support a synergistic role of SJ1 and Sac2 on a PI4P pool whose clathrin uncoating during the endocytic recycling of synaptic dysfunction results in PD. vesicles. SJ1 recruitment to endocytic pits by endophilin is re- Author contributions: M.C., D.P., Y.W., and P.D.C. designed research; M.C., D.P., and Y.W. quired for dissociation of the PI(4,5)P2-dependent interaction of – performed research; M.C., D.P., Y.W., and P.D.C. analyzed data; and M.C., D.P., and P.D.C. the clathrin adaptors with the membrane (8 11). Such action wrote the paper. cooperates in uncoating with the disassembly of the clathrin Reviewers: V.H., Leibniz-Forschungsinstitut für Molekulare Pharmakologie and Freie Uni- lattice mediated by the AAA ATPase HSC70 and its synaptically versität Berlin; and D.S., Columbia University. enriched cofactor auxilin (12, 13). Interestingly, loss-of-function The authors declare no competing interest. mutations in auxilin (PARK19) also result in EOP with epilepsy This open access article is distributed under Creative Commons Attribution-NonCommercial- (14–16). These observations support a link between dysfunction NoDerivatives License 4.0 (CC BY-NC-ND). in clathrin-mediated budding and EOP. 1M.C. and D.P. contributed equally to this work. We previously showed that the R258Q mutation abolishes the 2Present address: Program in Neuroscience and Behavioral Disorders, Duke-NUS Medical activity of the Sac phosphatase domain of SJ1 without affecting School, Singapore, 169857. the activity of its 5-phosphatase domain (5). We also generated 3To whom correspondence may be addressed. Email: [email protected]. RQ homozygous knock-in (KI) mice (SJ1 KI mice) that carry the This article contains supporting information online at https://www.pnas.org/lookup/suppl/ patient mutation and have neurologic manifestations reminiscent doi:10.1073/pnas.2004335117/-/DCSupplemental. of those of human patients (17). Functional and microscopic First published May 18, 2020. 12428–12434 | PNAS | June 2, 2020 | vol. 117 | no. 22 www.pnas.org/cgi/doi/10.1073/pnas.2004335117 Downloaded by guest on September 27, 2021 Sac2 was not abolished by expression of mCherry-Rab3. These results suggest the plausibility of overlapping roles of Sac2 and SJ1 in nerve terminals. Accumulation of Endocytic Factors in Axon Terminals of SJRQKI Neurons Is Enhanced by the Absence of Sac2. We previously reported the occurrence of a very robust exaggerated accumulation of endocytic factors, including clathrin coat components and their endocytic accessory factors, such as amphiphysin and auxilin, in nerve ter- minals of SJ1RQKI neurons both in situ (in all of several brain regions examined) and in neuronal cultures (17). Most likely, this abnormal accumulation of endocytic factors at synapses reflects their impaired shedding from membranes due to the lack of PI4P dephosphorylation by the Sac domain of SJ1, with a resulting accumulation of PI4P. Fig. 1. Genetic interaction between SJ1 and Sac2/INPP5F. (A) Domain We performed a similar analysis on neuronal cortical cultures structures of SJ1 and Sac2. Sac/4-Ptase, 4-phosphatase domain; 5-Ptase, of WT, Sac2KO, SJ1RQKI, and Sac2KO/SJ1RQKI mice. No such 5-phosphatase domain; RRM, RNA recognition motif; PRD, proline-rich do- accumulation/clustering was observed in nerve terminals of Sac2KO main; PH, pleckstrin homology. (B) Survival curves of mice with indicated RQ genotypes. Numbers of animals are indicated in parentheses. (C) Littermate neurons relative to controls (Fig. 3); however, in Sac2KO/SJ1 KI mice at P15. The Sac2KO/SJ1RQKI shown is one of the few survivors at neurons, accumulation of amphiphysin 2, clathrin light chain this age. (CLC), auxilin, and SJ1 itself was strongly more pronounced than in SJ1RQKI neurons, corroborating evidence for a synthetic effect of the two mutations (Fig. 3). As in the case of SJ1RQKI neurons in the Sac domain of SJ1. Our results support the identification (17), the enhanced presynaptic accumulation of endocytic factors of Sac2/INPP5F as the gene product responsible for the PD risk observed in double-mutant cultured cortical neurons was more at SNP rs117896735 and corroborate evidence of a link between pronounced at inhibitory than at excitatory presynaptic terminals, impaired endocytic flow at synapses and EOP. as detected by immunolabeling for vGAT and vGlut1, respectively SI Appendix ( , Fig. S3). NEUROSCIENCE Results Since previous studies of Sac2 had revealed that pools of this Synthetic Effect of SJ1 and Sac2 Mutations on Mice Survival. Sac2KO protein are localized on several endocytic compartments (21, mice do not have an obvious pathological neurologic phenotype 22), we also analyzed the distribution of EEA1 (early endosomal (25). Although SJ1RQKI mice are neurologically impaired and ex- marker) and LAMP1 (lysosome marker) in cultured cortical RQ RQ perience seizures, approximately 60% of them can reach adulthood neurons of WT, Sac2KO, SJ1 KI, and Sac2KO/SJ1 KI mice.