Host-to-graft propagation of inoculated α-synuclein into transplanted human induced pluripotent stem cell-derived midbrain dopaminergic neurons

Introduction Cell therapeutic clinical trials using fetal mesencephalic tissue provided a proof-of-concept for regenerative therapy in patients with Parkinson's disease. Postmortem studies of patients with fetal grafts revealed that α-synuclein+ Lewy body (LB)-like inclusions emerged in long-term transplantation and might worsen clinical outcomes even if the grafts survived and innervated in the recipients. Various studies aimed at addressing whether host-derived α-synuclein could be transferred to the grafted neurons to assess α-synuclein+ inclusion appearance in the grafts. However, determining whether α-synuclein in the grafted neurons has been propagated from the host is difficult due to the intrinsic α-synuclein expression. Methods We induced midbrain dopaminergic (mDA) neurons from human induced pluripotent stem cells (hiPSCs) and transplanted them into the striatum of immunodeficient rats. The recombinant human α-synuclein preformed fibrils (PFFs) were inoculated into the cerebral cortex after transplantation of SNCA−/− hiPSC-derived mDA neural progenitors into the striatum of immunodeficient rats to evaluate the host-to-graft propagation of human α-synuclein PFFs. Additionally, we examined the incorporation of human α-synuclein PFFs into SNCA−/− hiPSC-derived mDA neurons using in vitro culture system. Results We detected human α-synuclein-immunoreactivity in SNCA−/− hiPSC-derived mDA neurons that lacked endogenous α-synuclein expression in vitro. Additionally, we observed host-to-graft α-synuclein propagation into the grafted SNCA−/− hiPSC-derived mDA neurons. Conclusion We have successfully proven that intracerebral inoculated α-synuclein PFFs are propagated and incorporated from the host into grafted SNCA−/− hiPSC-derived mDA neurons. Our results contribute toward the basic understanding of the molecular mechanisms related to LB-like α-synuclein deposit formation in grafted mDA neurons.


Introduction
Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by progressive loss of midbrain dopaminergic (mDA) neurons, leading to motor symptoms (e.g., resting tremor, rigidity, and bradykinesia) that are potentially treatable by symptomatic therapy in the early stage of disease.Pathological features from postmortem studies include accumulation of a-synuclein protein in Lewy bodies (LBs) that appear in the remaining mDA neurons of patients with PD [1].Moreover, a-synuclein þ aggregates and neurites widely propagated across the brain in a prion-like manner corresponding to the neuropathological stage of PD [2].
Former cell transplantation studies in patients with PD provide a proof-of-concept for regenerative therapeutic strategy using a fetal mesencephalic tissue.In successful cases, grafted fetal mesencephalic tissue has survived, got innervated in the host brain, and improved the quality of life of the recipients with long-term symptomatic relief [3,4].However, ethical issues and unstable fetal tissue supply make the standardized cell transplantation therapy difficult.Instead of fetal mesencephalic tissue, human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC)-derived mDA neurons have emerged as alternative sources of cell transplantation therapy.Recently, the stem cell technological advantages have allowed for scalable generation of highly purified hESC/hiPSC-derived mDA neurons, demonstrating survival of the long-term grafted mDA neurons and improvement of motor functions in nonhuman primate and rodent PD models [5e8].Furthermore, the standardized protocols provide hESC/ hiPSC-derived mDA progenitors under good manufacturing practice conditions for clinical trials of cell transplantation therapy for patients with PD [9,10].
However, a drawback was observed in the postmortem studies of patients with PD who received fetal transplants, demonstrating LB-like a-synuclein deposit appearance in long-term grafts [11e14].Additionally, a-synuclein in the grafts could be transferred from the host into grafts in a prion-like manner.Furthermore, a study of a patient with PD having received unilateral fetal transplantation in the putamen revealed that grafted mDA neurons survived in the host brain and recovered motor functions in the first decade.However, this patient gradually lost motor functions from 14 years post-transplantation.A postmortem study revealed LB-like a-synuclein þ inclusions in 24-year grafts [15].This clinical case suggested that even if grafted mDA neurons survived and got innervated in the host brain, their therapeutic effects might be lost due to synucleinopathy in the longterm follow-up.To reveal the underlying molecular mechanisms of a-synuclein þ inclusion in the grafted neurons, several studies aimed at assessing how host-derived a-synuclein is incorporated into the neuronal grafts derived from healthy rodent fetal mesencephalic tissue-and hESC-derived mDA neurons [16e18].
However, intrinsic a-synuclein expression in the grafted neurons might affect host-to-graft extrinsic a-synuclein transfer observations.Herein, we inoculated human a-synuclein preformed fibrils (PFFs) into the brain of rats with X-linked severe combined immunodeficiency (X-SCID) and demonstrated that a-synuclein PFFs got incorporated into SNCA À/À hiPSC-derived mDA neuronal grafts.Since SNCA À/À hiPSC-derived mDA neurons do not express endogenous a-synuclein protein, our results visibly indicated that host-derived human a-synuclein PFFs could be transmitted into grafted hiPSC-derived mDA neurons.

Animals
We purchased 8-week-old wild-type C57BL/6 mice from Japan SLC Inc. (Hamamatsu, Japan).F344-Il2rg em1Iexas X-SCID rats were supplied by the National BioResource Project -Rat, Kyoto University (Kyoto, Japan) [19].The animals were kept at 25 C under a 12-h light/dark cycle with ad libitum access to food and water in the Bioscience Research Center at Kyoto Pharmaceutical University.We performed the animal experiments in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.Our protocols were approved by the Committee for Animal Research, Kyoto Pharmaceutical University.

hiPSC culture
The 1231A3 hiPSC line derived from ePBMC®, purchased from Cellular Technology Limited (http://www.immunospot.com/),it was established by Kyoto University, and provided by the RIKEN Bioresource Research Center (Tsukuba, Japan) through the National BioResource Project of the Ministry of Education, Culture, Sports, Science, and Technology/Japan Agency for Medical Research and Development (MEXT/AMED), Japan [20].Herein, we used SNCA À/À hiPSCs generated from the 1231A3 hiPSC line [21].The hiPSCrelated experiments were approved by the Ethical Review Committee for Medical and Health Research Involving Human Subjects, Kyoto Pharmaceutical University.
For the neurosphere culture, hiPSCs were dissociated into single cells and reseeded at a density of 9000 cells/well on ultra-low attachment V-bottom 96-well plates (Thermo Fisher Scientific) in the above-described differentiation medium and maintained until Day 60.

Recombinant human wild-type a-synuclein PFF preparation
Recombinant human a-synuclein PFFs was prepared as previously described [24].Briefly, lyophilized a-synuclein was dissolved in 20 mM glycine buffer (pH 8.0) and solubilized by adding 2 M NaOH [25].The solution was centrifuged at 10,000Âg for 30 min at 4 C to remove the insoluble aggregates after dialysis against PBS overnight.The supernatant containing monomeric a-synuclein (1 mg/mL) was incubated at 37 C for 5 days with rotation for PFF production.

Thioflavin T (ThT) fluorescence assay
a-synuclein PFF formation was verified using an amyloidspecific fluorescent dye, ThT, which was added to a 10-times diluted a-synuclein solution up to a final concentration of 10 mM.
The ThT fluorescent spectra were measured using a Hitachi F-2500 fluorescence spectrometer at 25 C.The spectra were recorded between 450 and 600 nm at an excitation of 440 nm.

Far-ultraviolet (UV) circular dichroism (CD) spectroscopy
Far-UV CD spectra were recorded between 190 and 260 nm at 25 C using a Jasco J-1500 spectropolarimeter (JASCO, Tokyo, Japan).The results were corrected by subtracting the buffer values.

Atomic force microscopy (AFM)
The a-synuclein PFF solution was deposited on freshly cleaved mica (The Nilaco Corporation, Tokyo, Japan) and incubated for 10 min.After washing the mica with distilled water, sample imaging was performed under ambient conditions at room temperature using a NanoScope IIIa Tapping Mode AFM (Veeco, Plainview, NY, USA) and microcantilever OMCLAC160TS-R3 (Olympus, Tokyo, Japan).

Immunocytochemistry
We performed immunocytochemistry as previously described [26].Briefly, we fixed the cells and neurospheres in 4 % paraformaldehyde (PFA) for 30 min at 4 C. Next, we embedded the neurospheres in an optimal cutting temperature (OCT) compound (Sakura Finetek Japan Co., Ltd., Osaka, Japan) and performed sample sectioning at a thickness of 20 mm using a cryostat.We then blocked the fixed samples for 1 h at room temperature using a 5 % normal donkey serum in 0.1 M phosphate-buffered saline containing 0.1 % Triton-X (PBST), incubated at 4 C overnight them with primary antibodies (Table 1), followed by an incubation with Alexa Fluorlabeled secondary antibodies (1:500; ThermoFisher) at room temperature for 2 h in the dark.Finally, we counterstained the cell nuclei using Hoechst 33342 (Dojindo, Kumamoto, Japan).

Immunohistochemistry
We performed the DAB staining and immunofluorescence as described previously [27].Briefly, we post-fixed the brain samples in 4 % PFA for 2 days and then transferred the samples into a 30 % sucrose solution at 4 C. Subsequently, we embedded the brain samples in the OCT compound and cut them to 40-mm-think sections.

Histological analysis evaluation
We determined the graft volume through hNCAM-positive area identification in every sixth section using a BZ-X800 microscope (Keyence, Osaka, Japan) and totaling the volumes of whole tall cylinders according to Cavalieri's principle.We estimated the immunoreactive cell numbers in each graft by manual cell counting in every sixth section.

5-ethynyl-2 0 -deoxyuridine (EdU) labeling in vivo
We performed the EdU labeling using Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye (Thermo Fisher Scientific).X-SCID rats were administrated with EdU (50 mg/kg) through intraperitoneal injection 4 h before sacrifice.We performed signal detection according to the manufacturer's protocol.

Statistical analysis
The values are represented as the mean ± standard deviation or standard error of the mean.The means of the two groups were compared using independent samples Student's t-test.All statistical analyses were conducted using Prism 10 (GraphPad, San Diego, CA, USA).

Histological verification of grafted hiPSC-derived cells
Next, we transplanted the hiPSC-derived mDA neuronal progenitors after 16 days of differentiation into the striatum of X-SCID rats.The X-SCID rats were already validated for human cell-derived graft xeno-transplantation into the brain [19,28].Our immunohistochemical analysis using an anti-human neuronal cell adhesion molecule (hNCAM) antibody indicated that human neuronal cells survived in the striatum at weeks 2 and 8 post-transplantation (Fig. 2A, B, and B'), and the graft volume at week 8 significantly increased compared to that at week 2 (0.32 ± 0.28 mm 3 and 3.91 ± 1.28 mm 3 at weeks 2 and 8, respectively, n ¼ 3e5) (Fig. 2C).FOXA2 þ cells could be dominantly observed in the grafts at both weeks 2 and 8 post-transplantation (Fig. 2D and E).The proliferative ability of the grafted cells was assessed by Ki67 þ cells and EdU incorporation.The Ki67 þ and FOXA2 þ cell ratio on week 8 significantly decreased compared to that on week 2 post-transplantation (5.68 % ± 1.44 % and 0.60 % ± 0.09 % at weeks 2 and 8, respectively, n ¼ 3) (Supplementary Fig. S1AeC).Correspondingly, EdU þ cell numbers also reduced by week 8 compared to those at week 2 posttransplantation (4.56 % ± 0.77 % and 0.54 % ± 0.14 % at weeks 2 and 8, respectively, n ¼ 2e3) (Supplementary Fig. S1D and E).In addition, human nuclear protein (hNuc) þ and TH þ neurons were detected both 2 and 8 weeks after transplantation.The TH þ neuron ratio at week 8 increased compared to that at week 2 (2.60 % ± 0.48 % and 7.53 % ± 0.65 % at weeks 2 and 8, respectively, n ¼ 3e5) (Fig. 2FeH).These results indicated that grafted hiPSC-derived mDA progenitors exited the cell cycle and gave rise to TH þ mDA neurons in the X-SCID rat brain.

a-synuclein PFF incorporation into SNCA À/À hiPSC-derived mDA neurospheres
To investigate a-synuclein transmission into the hiPSC-derived mDA neurons, we first synthesized recombinant human a-synuclein PFFs under a cell-free system [24].After the 5-day incubation of recombinant human a-synuclein with rotation, we observed an increased fluorescence intensity of the amyloid-specific dye, ThT (Fig. 3A), and the CD spectra of a-synuclein changed from a single negative peak below 200 nm to a negative peak at approximately 220 nm (Fig. 3B), suggesting the random-coil-to-b-sheet-rich amyloid structure formation upon incubation.Furthermore, we confirmed thin and straight fibril formation by the AFM imaging of the a-synuclein solution (Fig. 3C).
Next, a-synuclein PFFs were exposed to SNCA À/À hiPSC-derived mDA neurospheres to verify intracellular a-synuclein PFF incorporation.SNCA À/À hiPSCs were generated using the CRISPR-Cas9 technology, never expressing endogenous a-synuclein protein after differentiation into mDA neurons [21].Moreover, we verified that the mDA neuronal differentiation efficiency was not affected by the SNCA null mutation.We generated mDA neurospheres from SNCA À/À hiPSCs as described previously [21] and exposed them to a-synuclein PFFs on Day 42 when MAP2 þ and TH þ neurons emerged (Fig. 4AeC).Fourteen days after the a-synuclein PFF exposure, we observed human a-synuclein in the SNCA À/À hiPSCderived TH þ neurons on Day 56 (Fig. 4D and E).Since SNCA À/À hiPSC-derived TH þ neurons lack the intrinsic a-synuclein protein expression, the immunoreactive a-synuclein signals should belong to the incorporated a-synuclein PFFs.Furthermore, our Z-stack imaging allowed us to detect human a-synuclein PFFs in TH þ cell bodies (Fig. 4F-F'") and their neurites (Fig. 4G-G'").

Inoculated a-synuclein propagation into grafted hiPSC-derived mDA neurons
To induce a-synuclein PFF propagation in the brain by resembling an established model, we inoculated a-synuclein PFFs in the striatum    of healthy mice [29].Sixteen weeks after the inoculation, human asynuclein PFFs were widely distributed in the mouse brain, e.g., in the prefrontal cortex (PFC), striatum/corpus callosum (CC), parietal cortex, and hippocampus, far from the injected site (Supplementary Fig. S2AeA'").In addition, we also detected human a-synucleinimmunoreactivity in the substantia nigra pars compacta (SNC) but not in that of noninoculated animals (Supplementary Fig. S2B and C).
In addition, we detected phosphorylated a-synuclein in the TH þ neurons in the SNC of the inoculated mice but not in that of noninoculated animals (Supplementary Fig. S2D, E, and E').
Finally, we investigated whether inoculated a-synuclein PFFs would be propagated into grafted hiPSC-derived mDA neurons.In this experiment, we first transplanted SNCA À/À hiPSC-derived mDA neuronal progenitors into the striatum of X-SCID rats.Four weeks after transplantation, a-synuclein PFFs were inoculated into the ipsilateral cerebral cortex, and the animals were subsequently sacrificed six weeks after inoculation (Fig. 5A).During our histological analysis, we observed hNCAM þ grafts in the striatum 10 weeks after transplantation (Fig. 5B).In addition, the inoculated human a-synuclein protein was spread from the injected site 6 weeks after inoculation (Fig. 5C and C 0 ).SNCA À/À hiPSC-derived mDA neurons also survived in the graft (Fig. 5D and D 0 ).We detected human a-synuclein-immunoreactivity in the grafted SNCA À/À hiPSC-derived TH þ neurons (Fig. 5EeE 00 ).Our Z-stack images revealed that a-synuclein PFFs were incorporated into the grafted SNCA À/À hiPSC-derived TH þ neurons (Fig. 5E'").These results indicated that inoculated human a-synuclein PFFs were transmitted and incorporated into grafted SNCA À/À hiPSC-derived mDA neurons.

Discussion
In this study, we managed to provide experimental evidence that grafted hiPSC-derived mDA neurons incorporated a-synuclein protein transmitted from the host brain.X-SCID rats were intracerebrally inoculated with human a-synuclein PFFs after the intrastriatal transplantation of the SNCA À/À hiPSC-derived cells that never expressed endogenous a-synuclein.We used an anti-human a-synuclein antibody that specifically recognizes human a-synuclein and does not bind with rodent a-synuclein [30].These experimental tools enabled us to successfully detect inoculated human a-synuclein PFF propagation into the grafted SNCA À/À hiPSC-derived DA neurons and exclude the detection of intrinsic asynuclein expression of human iPSC-derived neurons and the rodent brain.
Inclusion of a-synuclein, a main LB component is a neuropathological feature of PD [1].Familial PD is reportedly coupled with increased SNCA gene copy numbers and missense mutations involved in DA neuronal cell death in the SNC, which is another pathological feature of PD [31e35].Additionally, postmortem studies of patients with PD revealed that the aggregates of a-synuclein extended through the brain via interconnected neuronal pathways according to clinical symptom staging [2].Moreover, synucleinopathy could be modeled by recombinant a-synuclein PFF inoculation into the animal brain and exposing it to cultured neurons [29,36e38].In fact, we observed that intrastriatal inoculation of human a-synuclein PFFs led to their propagation through the brain and induced a-synuclein phosphorylation in healthy mouse SNC TH þ neurons.Postmortem studies of the patients with PD who received fetal mesencephalic transplantation revealed LB acquisition in longterm grafts, potentially contributing to worsened clinical outcomes [11e15].Therefore, several experimental approaches aimed at revealing the molecular mechanisms of synucleinopathy in the grafts, suggesting host-to-graft propagation [16e18].Without a doubt, this study demonstrated that inoculated human a-synuclein PFFs propagated in the brain and were incorporated into the grafted SNCA À/À hiPSC-derived mDA neurons.Since SNCA À/À hiPSCderived mDA neurons in the grafts never expressed intrinsic asynuclein, the human a-synuclein immunoreactivity in the grafted cells could only be derived from the host brain.
A former study described that exogenous a-synuclein PFF seeding recruits unfolded endogenous a-synuclein to initiate the pathological aggregate formation and induces a-synuclein phosphorylation in the cells [37].No such a-synuclein-related pathological changes occurred in the case of incorporation into SNCA À/À cells, which were not expressing endogenous a-synuclein unlike their wild-type counterparts [29,38].In coherence with previous reports, we did not detect any phosphorylated asynuclein in SNCA À/À hiPSC-derived mDA neurons either in vivo or in vitro (data not shown), although we observed inoculated asynuclein incorporation in the grafted SNCA À/À hiPSC-derived mDA neurons.To unravel the pathological changes of a-synuclein following the incorporation of the inoculated a-synuclein PFFs into the grafted hiPSC-derived mDA neurons, the grafts of SNCA þ/þ hiPSC-derived mDA neurons should be conducted, since pathological changes of endogenous a-synuclein are induced by exogenous a-synuclein PFFs after incorporation into the cells.Indeed, various studies revealed that a-synuclein pathology in the grafted cells appear after the incorporation of exogenous asynuclein [16e18].
Preclinical studies using positron emission tomography (PET) provided noninvasive imaging of dopamine reuptake in grafted hiPSC-derived DA neurons, dividing cell proliferation, and immune cell inflammation response to monitor the cellular aspects in longterm follow-up [5,8,39].Recently, several highly potent PET tracers were developed to monitor synucleinopathy [40e44].Since asynuclein propagation and aggregation in the grafts might diminish cell transplantation therapeutic efficacy, pathological feature monitoring in the grafts displaying synucleinopathy might provide beneficial follow-up to support long-term symptomatic relief from the grafted neurons for better cell transplantation therapeutic outcomes.Moreover, several a-synuclein oligomer-binding compounds have recently emerged as disease-modifying therapeutic candidates to reduce synucleinopathy [45e50], potentially applicable for improvement of a-synuclein propagation into the grafted hiPSC-derived mDA neurons to maintain the clinical outcomes as a disease-modified therapy in long-term follow-up.
In conclusion, we successfully proved that intracerebral inoculated a-synuclein PFFs are propagated and incorporated from the host into grafted SNCA À/À hiPSC-derived mDA neurons that lacked endogenous a-synuclein expression.Our results contribute to the common understanding of the underlying molecular mechanisms related to LB-like a-synuclein deposit formation in grafted mDA neurons and the symptomatic therapeutic strategy targeting asynuclein.

Fig. 3 .
Fig. 3. Human a-synuclein PFF characterization.(A) ThT fluorescence emission spectra excited at 440 nm for a-synuclein before (blue) and after (red) incubation at 37 C for five days.(B) FarUV CD spectra before (blue) and after (red) incubation at 37 C for five days.(C) AFM image of a-synuclein fibrils.Scale bar: 500 nm.

Table 1
List of antibodies.