Parkinsonism Sac domain mutation in Synaptojanin-1 affects ciliary properties in iPSC-derived dopaminergic neurons

Synaptojanin-1 (SJ1) is a major neuronal-enriched PI(4,5)P2 4- and 5-phosphatase implicated in the shedding of endocytic factors during endocytosis. A mutation (R258Q) that impairs selectively its 4-phosphatase activity causes Parkinsonism in humans and neurological defects in mice (SJ1RQKI mice). Studies of these mice showed, besides an abnormal assembly state of endocytic factors at synapses, the presence of dystrophic nerve terminals selectively in a subset of nigro-striatal dopamine (DA)-ergic axons, suggesting a special lability of DA neurons to the impairment of SJ1 function. Here we have further investigated the impact of SJ1 on DA neurons using iPSC-derived SJ1 KO and SJ1RQKI DA neurons and their isogenic controls. In addition to the expected enhanced clustering of endocytic factors in nerve terminals, we observed in both SJ1 mutant neuronal lines increased cilia length. Further analysis of cilia of SJ1RQDA neurons revealed abnormal accumulation of the Ca2+ channel Cav1.3 and of ubiquitin chains, suggesting an impaired clearing of proteins from cilia which may result from an endocytic defect at the ciliary base, where a focal concentration of SJ1 was observed. We suggest that SJ1 may contribute to the control of ciliary protein dynamics in DA neurons, with implications on cilia-mediated signaling.


ABSTRACT
Synaptojanin-1 (SJ1) is a major neuronal-enriched PI(4,5)P2 4-and 5-phosphatase implicated in the shedding of endocytic factors during endocytosis.A mutation (R258Q) that impairs selectively its 4-phosphatase activity causes Parkinsonism in humans and neurological defects in mice (SJ1 RQ KI mice).Studies of these mice showed, besides an abnormal assembly state of endocytic factors at synapses, the presence of dystrophic nerve terminals selectively in a subset of nigro-striatal dopamine (DA)-ergic axons, suggesting a special lability of DA neurons to the impairment of SJ1 function.Here we have further investigated the impact of SJ1 on DA neurons using iPSC-derived SJ1 KO and SJ1 RQ KI DA neurons and their isogenic controls.In addition to the expected enhanced clustering of endocytic factors in nerve terminals, we observed in both SJ1 mutant neuronal lines increased cilia length.Further analysis of cilia of SJ1 RQ DA neurons revealed abnormal accumulation of the Ca 2+ channel Cav1.3 and of ubiquitin chains, suggesting an impaired clearing of proteins from cilia which may result from an endocytic defect at the ciliary base, where a focal concentration of SJ1 was observed.We suggest that SJ1 may contribute to the control of ciliary protein dynamics in DA neurons, with implications on ciliamediated signaling.

INTRODUCTION
While the cause of most Parkinson's disease (PD) is not known, mutations in a selected list of genes are responsible for the development of familial forms of the disease, often Early-Onset Parkinsonism (EOP) (Blauwendraat et al. 2020).One such gene is SYNJ1, which encodes the protein synaptojanin-1 (SJ1), a polyphosphoinositide phosphatase highly expressed in neurons and enriched at synapses (McPherson et al. 1996, Krebs et al. 2013, Quadri et al. 2013).SJ1 dephophorylates PI(4,5)P2 via the sequential action of two tandemly arranged inositol phosphatase modules: a central 5-phosphatase domain and an N-terminal Sac1 domain which functions primarily as a 4-phosphatase (McPherson et al. 1996, Guo et al. 1999, Nemoto et al. 2000).These catalytic modules are followed by a proline-rich region which is responsible for its subcellular targeting and undergoes alternative splicing to generate a shorter (145 kD, the predominant neuronal form) and a longer (170 kD) isoform (McPherson et al. 1996, Ramjaun and McPherson 1996, Rosivatz 2006).One of the main known roles of SJ1 is to participate in the shedding from endocytic vesicles of clathrin coats and other endocytic factors, including actin regulatory proteins, which bind PI(4,5)P2 at the plasma membrane to initiate the endocytic reaction (Cremona et al. 1999, Di Paolo andDe Camilli 2006).While absence of SJ1 leads to early postnatal lethality in mice (Cremona et al. 1999) and humans (Dyment et al. 2015, Hardies et al. 2016), a patient R258Q missense mutation (SJ1 RQ ) (accession number: NM_003895) also known as R219Q (accession number: NM_001160302) is responsible for EOP with epilepsy.This mutation selectively abolishes the catalytic action of its Sac1 domain (SJ1 RQ ) (Krebs et al. 2013).
We previously showed that knock-in mice with this mutation (SJ1 RQ KI) display neurologic manifestations reminiscent of those of human patients (Cao et al. 2017).These manifestations are accompanied at the cellular level not only by endocytic defects and an accumulation of clathrin-coated vesicles at synapses, but also by degenerative changes selectively of a subset of dopaminergic nerve terminals in the dorsal striatum (Cao et al. 2017, Ng et al. 2023).
One cell compartment which is regulated by PI4P and PI(4,5)P2 dynamics is the primary cilium (Chavez et al. 2015, Garcia-Gonzalo et al. 2015, Demmel et al. 2016, Ojeda Naharros and Nachury 2022).PI(4,5)P2 in the plasma membrane of the ciliary pocket at the base of the cilium, which is a site of intense exo-endocytosis, helps regulate the turnover of cilia-related signaling proteins (Garcia-Gonzalo et al. 2015, Demmel et al. 2016, Nachury and Mick 2019).Moreover PI(4,5)P2 is the precursor of the pool of PI4P generated in the ciliary shaft through dephosphorylation of PI(4,5)P2 by INPP5E, a polyphosphoinositide 5-phosphatase concentrated in the shaft of primary cilia.This PI4P pool has a critical role in cilia biology (Bielas et al. 2009, Jacoby et al. 2009, Chavez et al. 2015, Garcia-Gonzalo et al. 2015, Phua et al. 2017, Klink et al. 2020).Primary cilia are key players in the hedgehog signaling pathway which has a crucial importance in the nigrostriatal system (Hynes et al. 1995, Nordstroma et al. 2015, Dhekne et al. 2018, Derderian et al. 2023).The importance of hedgehog signaling in the development of DA neurons is proven by the essential requirement of Sonic Hedgehog (Shh) for the differentiation of iPSCs into DA neurons (Kriks et al. 2011, Kim et al. 2021).Primary cilia of neurons are increasingly recognized as major signaling hub with a major impact on neuronal function.
Interestingly, disease causing mutations in another PD gene, LRRK2 (PARK8) (Paisan-Ruiz et al. 2004, Zimprich et al. 2004, Poewe et al. 2017, Alessi and Sammler 2018, Sobu et al. 2021) interfere with ciliogenesis (Dhekne et al. 2018, Khan et al. 2021, Sobu et al. 2021), suggesting a potential contribution of ciliary-related defects to PD pathology.While one effect of PD LRRK2 mutations is to impact DA neurons indirectly, via an impairment of cilia-dependent hedgehog signaling in striatal cholinergic neurons (Dhekne et al. 2018, Khan et al. 2021), additional directs effects of these mutations via an impairment of cilia in DA neurons cannot be excluded.These considerations raise the question of whether phenotypic manifestations of SJ1 impairment may include perturbations of ciliary functions and whether such perturbations may occur in DA neurons.
Here we have used iPSC-derived DA neurons as a model system to address this question.We report that DA neurons with impaired SJ1 function have abnormally long cilia which display an ectopic accumulation of ubiquitinated proteins within them.The Cav1.3, a voltage-gated calcium channel, which is important for the rhythmic pacemaking activity of DA neurons (Gregory et al. 2011, Felix and Weiss 2017, Liss and Striessnig 2019, Grimaldo et al. 2022), is also ectopically accumulated within them.Together, our results demonstrate a role of SJ1 in the cilia of DA neurons and implicates this protein in the control of their signaling properties.

Generation of WT and SJ1 mutant iPSC-derived DA neurons
Human iPSCs (WTC11 line) were gene edited in house by CRISPR/Cas9 to delete expression of SJ1 (SJ1 KO).Correct editing was validated by PCR and absence of SJ1 in KO cells was confirmed by western blotting (Supplementary Figure 1A and B).iPSCs (KOLF2.1 line) harboring the EOP RQ mutation at position 258 (accession number: NM_003895) were obtained from the iPSC Neurodegeneration Initiative (iNDI) (Ramos et al. 2021) and validated by polymerase chain reaction (PCR).SJ1 KO and SJ1 RQ KI iPSCs, as well as their corresponding isogenic controls were differentiated either into cortical-like i 3 neurons or into DA neurons (Supplementary Figure 1C-G).To generate cortical-like i 3 Neurons, we used the doxycycline-inducible neurogenin-2 (NGN2)-driven differentiation protocol (Wang et al. 2017) as described in Fernandopulle et al.
(2018) which results in mature neuronal cultures within 15-19 days.For the generation of DA neurons, we used the procedure described by Kriks et al. (2011) and Bressan et al. (2021).This differentiation process is slower than the NGN2-driven neuronal differentiation (Fernandopulle et al. 2018, Bressan et al. 2021, Hulme et al. 2022).However, 30 days from the beginning of differentiation, cells had acquired neuronal morphology with the formation of a complex network of processes (Supplementary Figure 1C and D).Moreover, western blotting and immunofluorescence of these cultures showed the expression of two key markers of DA neurons, tyrosine hydroxylase (TH) and the dopamine transporter (DAT), in both the control and the two SJ1 mutant lines (Supplementary Figure 1E-I).

iPSC-derived SJ1 mutant DA neurons display abnormal accumulation of endocytic factors in nerve terminals
A key and defining phenotype of SJ1 KO and SJ1 RQ KI neurons in situ and in primary cultures is a very robust and exaggerated accumulation in their nerve terminals of endocytic membrane intermediates and endocytic factors, including clathrin coat components and their accessory factors, with amphiphysin-2 being the most strikingly accumulated protein (Cao et al. 2017).To validate the use of iPSC-derived DA neurons as model systems to assess the impact of SJ1 mutations, we examined if this phenotype was recapitulated in these cells.
At day 50-55 from the beginning of differentiation, SJ1 KO neurons, SJ1 RQ KI DA neurons and their corresponding control neurons showed a similar and prominent punctate pattern of immunoreactivity for the synaptic vesicle marker synaptophysin, revealing abundant formation of synapses in all four conditions.However, a very strong and robust accumulation of puncta of amphiphysin-2 immunoreactivity, which overlapped with synaptophysin immunoreactivity (Figure 1A-F), was observed in SJ1 KO and SJ1 RQ KI DA neurons, but not in control neurons, demonstrating that the accumulation of endocytic factors typical of SJ1 KO neurons is replicated in these iPSC-derived neurons.These accumulations were also seen when SJ1 RQ KI DA neurons were cocultured for 7 days with iPSC-derived medium spiny neurons (MSNs) (Figure 1G-J) using a microfluidic compartmentalization device (eNuvio).In this device, DA neurons and MSNs are seeded in two distinct chambers connected by narrow channels through which axons can grow.Large abnormal puncta of amphiphysin-2 immunoreactivity, which overlapped with puncta positive for synapsin, a marker of presynaptic nerve terminals (De Camilli et al. 1983), were observed in both chambers, with the puncta found in the MSN-containing chamber likely reflecting DA synapses on MSNs.We conclude that iPSC-derived DA neurons are good models to study SJ1 mutant phenotypes.
Presence of primary cilia in iPSC-derived DA neurons and abnormal ciliary length in SJ1 KO and SJ1 RQ KI DA neurons Cilia brightly positive for the primary cilia marker Arl13b (Caspary et al. 2007) were clearly visible in undifferentiated iPSCs, but no longer detectable after differentiation to cortical-like i 3 Neurons (Figure 2A and Figure 2B).This is in agreement with the decrease of the levels of mRNAs encoding cilia-related proteins as detected by RNAseq during iPSC-differentiation in i 3 Neurons (Tian et al. 2019).In contrast, the great majority of iPSC-derived DA neurons retained Arl13b -positive cilia (89.45 ± 1.68%; mean ± S.E.M.), which were also positive for acetylated tubulin (a general cilia marker) and for adenylate cyclase type III (AC3), a marker specific of neuronal cilia (Sipos et al. 2018, Sterpka andChen 2018)(Figure 2C-E).
Cilia, as assessed by Arl13b, acetylated microtubules and AC3 immunolabeling, were almost two-fold longer in SJ1 KO neurons when compared to control neurons, while the percentage of cilia-forming cells was the same in both conditions (Figure 3A-F).Furthermore, abnormally shaped Arl13b-positive cilia were observed in SJ1 KO DA neurons with presence of multiple abnormal cilia in a small proportion of SJ1 KO DA neurons, but not in their controls (Supplementary Figure 2).
We next analyzed presence of cilia in two different iPSC-derived clones of SJ1 RQ KI DA neurons (Figure 4).While again there was no difference in the percentage of cilia-forming DA neurons relative to controIs, the length of cilia was significantly longer in both clones in comparison to control (Figure 4A-E).We conclude that lack of a functional SJ1 affects some properties of cilia in DA neurons.

Accumulation of Cav1.3 in cilia of SJ1 RQ KI DA neurons
A special property of DA neurons is an intrinsic pacemaker function, whose activity is highly dependent on the L-type Cav1.2 and Cav1.3 voltage-gated calcium channels (Gregory et al. 2011, Felix and Weiss 2017, Liss and Striessnig 2019, Grimaldo et al. 2022).Interestingly, these channels, which are broadly localized throughout the surface of the cell bodies and dendrites of neurons (Liss and Striessnig 2019) are also present in cilia or cilia derived structures, in several cell types, including cells of the retina and kidney (Kersten et al. 2010, Jin et al. 2014, Jin et al. 2014, Korkka et al. 2019, Sanchez et al. 2023).Prompted by this reported localization, we explored whether cilia of iPSC-derived DA neurons were labeled by anti-Cav1.3antibodies that had been validated in Cav1.3 knockout cells (Shi et al. 2017).We found that in control iPSCderived DA neurons Cav1.3 immunoreactivity displayed, as previously reported (Kersten et al. 2010, Korkka et al. 2019), an accumulation at the base of cilia, whose position was marked by g-tubulin (Figure 5A and B).Strikingly, in SJ1 RQ KI DA neurons bright Cav1.3 fluorescence intensity was observed throughout the Arl13b-positive ciliary shaft (Figure 5C-E).These findings suggest that in iPSC-derived DA neurons harboring the SJ1 PD mutation, cilia are not only abnormal in length but also in some functional properties.

Accumulation of ubiquitinated proteins in SJ1 RQ KI DA neurons
A major mechanism underlying turnover of membrane protein in cilia is their ubiquitination, primarily via lysine 63-linked Ub (UbK63) linkage, as this process controls their exit from cilia to allow their endocytosis and targeting for degradation (Desai et al. 2020, Shinde et al. 2020, Ojeda Naharros and Nachury 2022).Thus, we investigated whether presence of ubiquitin conjugates is higher in cilia using the well-characterized FK2 and FK1 monoclonal antibodies that label ubiquitin conjugates but not free ubiquitin (Fujimuro and Yokosawa 2005)(Figure 6).
While no detectable FK2 and FK1 signal was observed in the cilia of control cells, a strong signal was present in cilia of SJ1 RQ KI DA neurons (Figure 6A-F, Supplementary Figure 3A).This result reveals a link between SJ1 function and the clearance of proteins in DA neurons, possibly reflecting back-up of endocytic traffic.

Concentration of SJ1 at the base of primary cilia
The impact of SJ1 mutations on primary cilia could be explained by the indirect effect of an endocytic impairment throughout the neuronal surface, or to the loss of a specific function in proximity of cilia.To gain insight into this question, we assessed the localization of SJ1 by immunofluorescence in iPSCs before and after differentiation into DA neurons.We found that one or two closely apposed bright spots of SJ1 immunoreactivity colocalized with g-tubulin, a marker of centrioles, were present in undifferentiated iPSCs and control DA neurons (Figure 7A and B).This staining at the base of cilia, was lost in SJ1 KO iPSCs and SJ1 KO DA neurons (Figure 7C and D).The localization of SJ1 at centrioles supports a role of SJ1 in cilia as it could serve as a mechanism to generate a focal high concentration of the protein in their proximity.
A frequently used model for the analysis of cilia is the RPE1 cell line, in which serum starvation for 48 hours robustly induces ciliogenesis (Figure 8A) (Spalluto et al. 2013, Ganga et al. 2021).
Upon expression of either mCherry-SJ1-145 or GFP-tagged SJ1-170 (the short and long forms of SJ1, respectively, Figure 8B and C) in these cells, bright spots of mCherry and GFP fluorescence were observed at the base of primary cilia.Co-expression in these cells of mCherry-SJ1-145 with another phosphoinositide phosphatases, the 5-phosphatase INPP5E (GFP-INPP5E), a known component of the cilia shaft (Bielas et al. 2009, Jacoby et al. 2009) confirmed the specific and selective localization of SJ1 at the cilia base (Figure 8B).

DISCUSSION
Our study shows that impairment of SJ1 function in human iPSC-derived DA neurons has an impact on the properties of their primary cilia, in addition to the well-established disrupting effect on presynaptic vesicle traffic.Both the lack of SJ1 and the selective loss of its 4phosphatase activity due to the EOP patient mutation (SJ1 RQ ) leads to increased cilia length in these cells.Further analysis of cilia in SJ1 RQ KI iPSC-derived DA neurons revealed abnormal protein localization in them, as exemplified by the accumulation of the Cav1.3 channel and of ubiquitin chains throughout the ciliary shaft.Given the increasingly appreciated importance of primary cilia in neuronal signaling, it is plausible that a defect in ciliary function may contribute to the pathological manifestations resulting from the EOP SJ1 mutation.
Traffic of plasma membrane proteins and lipids in and out of cilia is controlled by a diffusion barrier in which PI4P (which is the predominant phosphoinositide in the ciliary shaft) and PI(4,5)P2 (which is the predominant phosphoinositide the ciliary pocket) play an important role (Chavez et al. 2015, Garcia-Gonzalo et al. 2015, Nguyen et al. 2022, Ojeda Naharros and Nachury 2022).Impairment of SJ1 function may disrupt the function of the diffusion barrier between the two compartments by perturbing the physiological concentration and relative ratio of PI4P and PI(4,5)P2.Alternatively, or in addition, SJ1 may help control membrane protein clearing from cilia indirectly via its function in the endocytic pathway (Cremona et al. 1999) as the fate of ubiquitinated proteins is to undergo endocytosis in the ciliary pocket for subsequent targeting to degradation (Nachury andMick 2019, Shinde et al. 2020).While SJ1 appears to have a primary role in the shedding of endocytic factors after endocytosis (Nguyen et al. 2022), its loss-of-function, as shown by studies of nerve terminals, also results in a back-up of endocytic traffic with a partial stranding in the plasma membrane of proteins and membrane that needs to be internalized.As we have now shown that a pool of SJ1 is concentrated at the ciliary base, a special role of this protein in the endocytosis that occurs at the ciliary pocket is plausible.
Protein ubiquitination plays an important role in controlling protein turn-over in cilia, as a key regulatory mechanism for the exit of proteins from cilia is their ubiquitination (Anvarian et al. 2019, Nachury and Mick 2019, Shinde et al. 2020).Thus, increased cilia length and abnormal accumulation of ubiquitinated proteins in cilia may be related and due to defective protein clearance from cilia.The BBSome, a protein complex localized at cilia (Nachury et al. 2007), is implicated in this clearance (Jin et al. 2010, Chamling et al. 2014, Prasai et al. 2020).
Interestingly, the BBSome components BBS7 and BBS9, as well as other proteins involved in centrosome/ciliary function, were hits in a proximity-labeling screen for SJ1 neighbors (Bartolome et al. 2022) suggesting a potential functional interplay between the BBsome and SJ1 in such clearing.
How SJ1 becomes concentrated at the base of cilia remain unclear.This localization is unlikely to be explained by its concentration on endocytic membranes in the ciliary pocket, since the localization of SJ1 closely overlaps with the localization of g-tubulin even when the two centrioles are clearly physically separated, pointing to a concentration around the two centrioles rather than on endocytic vesicles.As the pericentriolar material is enriched in actin and actin regulatory proteins (Farina et al. 2016, Kohli et al. 2017, Kiesel et al. 2020), SJ1 may be recruited to these sites by interactions of its C-terminal proline-rich domain with actinregulatory proteins (Rosivatz 2006).We suggest that low affinity binding to proteins that surround the centrioles may serve to create a high local concentration of SJ1, thus facilitating its action at endocytic events that takes place at these sites.We note that another inositol 5phosphatase implicated in endocytic traffic was shown to be concentrated on centrioles at the base of cilia and impact cilia length, although with conflicting results about cilia length (Coon et al. 2012, Luo et al. 2012, Rbaibi et al. 2012), with longer cilia in Rbaibi et al. (2012).
A role in primary cilia dynamics in PD pathogenesis has been previously suggested (Nordstroma et al. 2015, Dhekne et al. 2018, Schmidt et al. 2022).In particular, at least some effects of the PD gene LRRK2 have been attributed to a role of this protein in cilia, based on studies in cell lines and mouse brain tissue (Dhekne et al. 2018, Khan et al. 2021, Sobu et al. 2021).PD mutations in LRRK2 resulted in shorter rather than longer cilia as we have shown here for SJ1 mutations.However, it remains possible that some shared aspects of ciliary function may be disrupted by both PD LRRK2 mutations and the EOP SJ1 mutation, in spite of the different effect on cilia morphology.Importantly, studies of LRRK2 and cilia have focused on striatal cells, i.e. targets of dopaminergic innervation, while here we have focused on DA neurons.An additional role of LRRK2 mutations on cilia of DA neurons cannot be excluded.
Whether and how the abnormal features of cilia of SJ1 mutant DA neurons impact their function will require further investigations.Ca 2+ oscillations in primary cilia independent of somatic Ca 2+ levels have been detected in several cell types and attributed to ciliary calcium channel activation, suggesting that cilia could function as an autonomous Ca 2+ signaling hub in response to external stimuli (Delling et al. 2013, Yuan et al. 2015, Djenoune et al. 2023, Sanchez et al. 2023).In this context the striking accumulation of Cav1.3 proteins in the cilia of SJ1 RQ KI DA neurons are of special interest as it raises the possibility that Ca 2+ signaling in these cilia may be altered, with repercussion on cell physiology.SJ1 KO mice, which die perinatally, do not display obvious brain developmental defects at birth.Likewise, developmental defects are not observed in mice and humans with the EOP mutation.
Thus, the impact of SJ1 on cilia function must be more subtle than the one of other proteins whose mutations results in major such defects, collectively referred to as ciliopathies (Reiter and Leroux 2017).Similar considerations were made for LRRK2 mutations (Dhekne et al. 2018) and OCRL mutation (Rbaibi et al. 2012).
In conclusion, our study reveals a previously unknown role of SJ1 in primary cilia of DA neurons and raises the possibility that perturbation of such role by the EOP mutation may contribute to the pathological manifestations produced by this mutation.

Human iPSC culture, i 3 Neuron and DA differentiation
The following iPSC lines were obtained from the iPSC Neurodegeneration Initiative (iNDI) consortium and genome-edited by Jackson Laboratories (JAX): KOLF2.1,KOLF2.1 (with the NGN2 cassette at the AAVS locus, used for the i 3 Neurons experiments) and KOLF2.1 SJ1 RQ KI (R219Q): clones A09 and B02.The WTC11(with the NGN2 cassette at the AAVS locus) iPSC line, kind gift of M. Ward (NIH) was used to generate SJ1 KO cells.For the maintenance of iPSCs in culture, iPSCs were cultured on Geltrex (Life Technologies) coated dishes and maintained in Essential 8 Flex media (Thermo Fisher Scientific).The Rho-kinase (ROCK) inhibitor Y-27632 (EMD Millipore, 10 μM) was added to Essential 8 Flex media on the first day of plating and replaced with fresh media without ROCK inhibitor on the following day.
For long-term culture of DA neurons, cells were re-plated on 0.1 mg/ml poly-L-ornithine in PBS (Sigma-Aldrich) and 10 μg/ml laminin (Thermo Fisher Scientific) coated 35 mm glass-bottom dishes (MatTek) or 6-well plates (Corning) for imaging and immunoblotting, respectively.These neurons were cultured and maintained in complete NB/B27 medium followed by the addition of 0.1% anti-mitotic inhibitor (Supplement K, Brainxell) at day 25 to terminate division of nonneuronal cells.Fresh NB/B27 medium was added to the existing plates or dishes every 7 days and kept at 37°C with 5% CO2 in an enclosed incubator.

CRISPR-Cas9 mediated generation of SJ1 KO iPSCs
A CRISPR-based homologous recombination strategy was used to generate the SJ1 KO iPSC line.
Briefly, 1 × 10 5 WTC11-NGN2 iPSCs were plated on Geltrex-coated 6-well plate and transfected the following day using the Lipofectamine Stem transfection reagent (Invitrogen) and 3 µg of px458 plasmid (Addgene plasmid #48138) containing a small guide RNA with the following sense (5ʹC CACCGTGGTTATTACGTCTTATGTG3ʹ) and antisense (5'AAACCACATAAGACGTAATAACCAC3ʹ) sequences that was designed to selectively target the Exon 5 of SJ1.Pooled (GFP-positive) cells were enriched by fluorescence activated cell sorting (FACS) 2 days later.Sorted cells were expanded and then serially diluted to yield small clonal populations, screened using PCR amplification of genomic DNA flanking the sgRNA target site followed by sequencing of the amplicons using the following forward and reverse sequencing primers: 5'TCTCGTTTTATAGCCCTATCTTCTGATCC3', 5'AAGGCCCATAAGTAACCAAGAACAATC3', respectively.

Cell culture and transfections
hTERT-RPE1 cells were grown in DMEM/F12 (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific), 1% glutaMAX and 1% penicillin-streptomycin.Cells were kept at 37°C with 5% CO2 in an enclosed incubator.Cells were transfected with the relevant plasmids using 4 μls of Lipofectamine™ 2000 Transfection Reagent (Invitrogen).4-6 hours posttransfection the medium was changed to DMEM/F12 medium without FBS to induce ciliogenesis and examined at the microscope 48 hours later.For both i 3 Neuron and DA neuron transfections, plasmids were transfected with 4 μl of Lipofectamine™ Stem Transfection Reagent (Invitrogen) and visualized at least 48 hours later.

Immunofluorescence, live imaging and fluorescent microscopy
Cells were seeded on glass-bottom mat-tek dishes (MATtek corporation).For immunofluorescence, cells were fixed with 4% (v/v) paraformaldehyde (Electron Microscopy Sciences) in 1x phosphate-buffered saline (PBS) for 20 mins followed by three washes in PBS.
Cells were permeabilized with 0.25-0.5% (v/v) Triton X-100 in PBS for 5 mins followed by three washes in PBS.Cells were then incubated with fresh 1 mg/ml sodium borohydride (Sigma-Aldrich) in PBS for 7 mins to reduce autofluorescence, and then washed thrice in PBS.They were further blocked for 30 min in 5% bovine serum albumin (BSA, Sigma-Aldrich) in PBS and then incubated overnight at 4 °C with the primary antibodies listed in Table S1.Subsequently, cells were washed with PBS thrice the following day and incubated with Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific) for 1 h at room temperature, followed by three washes in PBS.DAPI (Thermo Fisher Scientific) was used for nuclear staining.
Transfections were carried out as described above.For live imaging, cells were maintained in Live Cell Imaging buffer (Life Technologies) for COS7 cells, while both i 3 Neurons and DA neurons were maintained in CM and NB/B27 media, respectively, in a caged incubator with humidified atmosphere (5% CO2) at 37°C.The Yokogawa spinning disk field scanning confocal system with microlensing (CSU-W1 SoRa, Nikon) controlled by NIS elements (Nikon) software was used for neuronal imaging.Excitation wavelengths between 405-640 nm, CFI SR Plan ApoIR 60XC WI objective lens and SoRa lens-switched light path at 1x, 2.8x or 4x were used.SoRa images were deconvolved using the Batch Deconvolution (Nikon) software.

Neuronal co-culture device
Control or SJ1RQKI DA neurons (day 30) were replated on one side of the two-chamber microfluidic compartmentalization device (OMEGA 4 , eNuvio), where only axonal processes can migrate through the microfluidic channels connected to the adjacent chamber.After an additional 25 days in the co-culture device, frozen iPSC-derived medium spiny neurons (MSN) from Brainxell were plated on the other half of the device (where only the axons of DA neurons are present).The DA-MSN co-cultures were then fixed 7-10 days later for immunofluorescence.

Immunoblotting
i 3 Neurons, DA neurons and MSNs were grown on six-well plates (3-5 × 10 5 cells/well).After differentiation in their respective maturation media, neurons were washed with ice-cold PBS and then lysed in 1xRIPA lysis buffer (10X RIPA lysis buffer, Sigma-Aldrich) supplemented with cOmplete™ EDTA-free protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche), followed by centrifugation at 13,000 × g for 6 min.The supernatant was collected and incubated at 95 °C for 5 min in SDS sample buffer containing 1% 2mercaptoethanol (Sigma).The extracted proteins were separated by SDS-PAGE in Mini-PROTEAN TGX precast polyacrylamide gels (Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad) at 100 V for 1 h or 75 V for 2 h (for high molecular weight proteins: >150 kDa).Subsequently, the nitrocellulose membranes were blocked for 1 h with 5% non-fat milk (AmericanBIO) in TBST (tris-buffered saline [TBS] + 0.1% tween 20), then incubated overnight at 4 °C with primary antibodies and then incubated with IRDye 680RD or 800CW (LI-COR) secondary antibodies (1:8000) for 1 h at room temperature in TBST.Finally, blots were imaged using the Gel Doc imaging system (Bio-Rad) using manufacturer's protocols.

Statistical analysis
Quantification of ciliary ubiquitination and Cav1.3 levels were carried out according to Shinde et al. (2020).Briefly, total fluorescence intensity of ubiquitin or Cav1.3 levels at individual Arl13bpositive cilium were substracted from background ubiquitin or Cav1.3 fluorescence measured in the adjacent area.The methods for statistical analysis and sizes of the samples (n) are specified in the results section or figure legends for all quantitative data.Student's t test or Mann-Whitney test was used when comparing two datasets.Differences were accepted as significant for P < 0.05.Prism version 9 (GraphPad Software) was used to plot, analyze and represent the data.

Figure 7 :Figure 8 :
Figure 7: Presence of a pool of SJ1 at the ciliary base of iPSCs and iPSC-derived DA neurons (A and B) Fluorescence images of control (A) iPSCs and (B) iPSC-derived DA neurons immunolabeled with antibodies directed against g-tubulin (green) and SJ1 (magenta) showing overlap of spots of SJ1 immunoreactivity in control but not in SJ1 KO cells.(C and D) Fluorescence image of SJ1 KO iPSCs and iPSC-derived SJ1 KO DA neurons (day 30) immunolabeled with antibodies against Arl13b (green) and SJ1 (magenta) showing lack of SJ1 staining at the base of cilia.High magnifications of boxed areas in (A -D) are shown at right.(Scale bars, 10 µm; cropped areas: 2 µm).

Table S1 .
List of antibodies/dyes used in this study.