Identifying Exosomes as a Messenger Unit During Heterochronic Parabiosis for Amelioration of Huntington’s Disease

Background:Huntington’s disease (HD) starts its pathology long before clinical manifestation, however, there is no therapy to cure it completely and only a few studies have been reported for delaying the progression of HD. We demonstrated the blood sharing effect by heterochronic parabiosis in HD and explored the underlying mechanism for transferring positive factors in the young blood serum.A shared blood circulation by heterochronic parabiosis has improved behavioral performance and HD pathology through mediation of mitochondria dysfunction and cell death. Furthermore, the messenger unit for the effective components in young blood is identied for the rst time to the best of our knowledge. Methods:R6/2 mice were surgically connected with young wild-type mice (n=13), old wild-type mice (n=8), or R6/2 mice (n=6) to examine the effect of heterochronic parabiosis.Parabionts composed of 5- to 6-week-old transgenic and wild-type mice were observed for 6 weeks in a single cage. The in vitro cellular model of HD cells were treated by the 200 μg/mlblood serum of the young or old mice, and by the exosomes isolated from thereof. Thein vitro cellular model of HD were developed by differentiating neural stem cells cultured from SVZ of the brain. young blood serum thein vitro cellular model HD improved mutant Huntingtin aggregation mitochondria biogenesis cell death (p53 Bax p<0.005, make R6/2 mice survive over 12 month in the case of control group, and over 17 month in the case of heterochronic parabiosis group, successively producing the HD R6/2 animal model for the heterochronic parabiosis for the rst time.By the heterochronic parabiosis of R6/2 mice, we foundthat young blood has positive factors in improving the HD pathology, and the identical results could be obtained by processingthe exosomes extracted from the young seruminto in vitro model.We discovered that the exosomes serve as a messenger unit for the positive factor of young serum during the heterochronic parabiosis.It could lead to a development of potential small molecule in exosome interventions, and a group of soluble factors in exosome targeting several pathways may help therapeutic benets for HD.

Several studies in other neurodegenerative diseases like AD have shown that exposure to a young blood circulation through heterochronic parabiosis, which is a surgical union of 2 organisms of different phenotype that leading to the formation of a vascular anastomosis and a shared circulatory system between two mice, [9] reverses cognitive de cits that is observed with normal aging. Although the idea of adopting heterochronic parabiosisto the HD seems pretty straightforward, however, it has not been reported so far inR6/2 mice. It is becausethe mouse model for HD, are highly vulnerable to stress and at 3 to 4 months of age, they develop trimming and cause death by muscle loss. [10,11] Also, a thorough understanding for the transportation mechanism of positive factors from the young blood to the old duringheterochronic parabiosis is necessary in order to adoptthe heterochronic parabiosis as the cure for HD.However, previous studies have only focused on which factors of 'young blood' have a positive effect on neurodegenerative disease. [9,[12][13][14]There wasonly vague understandings like the diffusion of blood that how the positive factors are transported, because the transportation process is nanoscale, which makes it di cult to identify the messenger unit.
We have focused on exosomes which are the smallest membranous vesicles (40-100nm) that has cargo ability for intercellular matter exchange. [15][16][17]Exosomes are generated via the inward budding of endosomes, to form multivesicular bodies (MVBs) that fuse with the membranes to release exosomes into the surrounding environment. [18,19]Exosomes, depending on their parental origin, contain a variety of proteins, lipids, non-coding RNAs, mRNA, and miRNA, collectively termed as "cargo" contains. Due to their cargo ability, exosomes represent a novel form of intracellular communication among cells without cell-to-cell direct contact. Exosomes are selectively taken up by the surrounding or distal cells and can reprogram the recipient cells due to their active cargo content. [20,21] By minimizing the damage of parabiosis surgery, we were able to make R6/2 mice survive over 12 month in the case of control group, and over 17 month in the case of heterochronic parabiosis group, successively producing the HD R6/2 animal model for the heterochronic parabiosis for the rst time.By the heterochronic parabiosis of R6/2 mice, we foundthat young blood has positive factors in improving the HD pathology, and the identical results could be obtained by processingthe exosomes extracted from the young seruminto in vitro model.We discovered that the exosomes serve as a messenger unit for the positive factor of young serum during the heterochronic parabiosis.It could lead to a development of potential small molecule in exosome interventions, and a group of soluble factors in exosome targeting several pathways may help therapeutic bene ts for HD.

Methods
Experimental model of HD All experimental animal procedures performed were approved by the Institutional Animal Care and Use Committee (IACUC, Approval number: 16-0043-C2A1) of Seoul National University Hospital, which was accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. Transgenic R6/2 (B6CBA-Tg(HDexon1)62Gpb/1J, 111 CAGs) and their WT littermates used in this study were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Mice used in this study were zQ175 KI heterozygous (CHDI-81003003) or WT littermates on a C57BL/6J background strain obtained from the CHDI colony at Jackson Laboratories (Bar Harbor, ME) or bred in-house at PsychoGenics, Inc. (Tarrytown, NY). ZQ175 mice, originating from the CAG 140 mice (from germline CAG expansion) were heterozygous and wild-type mice were generated by crossing heterozygous ZQ175 mice on a C57BL/6J background.The R6/2 transgenic mice model expresses exon 1 of a human mHtt and is the most widely used animal model for studying HD. These mice were obtained by crossing ovarian transplant hemizygote females with B6CBAF1/J males. R6/2 Miceand ZQ175 were bred at the Seoul National University Hospital under speci c pathogen-free conditions and mice homozygous offspring of heterozygous matings were identi ed by polymerase chain reaction (PCR) typing of tail-tip genomic DNA. The mice were house in groups with ad libitum access to food and water and a 12 hours light / 12 hours dark cycle. Mouse body weight was recorded weekly.

Procedures for parabiosis
Parabiosis were subjected to parabiotic surgery using methods adapted from JOVE, Bunster and Meyer.

BrdU administration and immunohistochemistry
To demonstrate a connected circulation between prabionts, BrdU was injected into one mice. The formation of shared blood circulation between the parabiontic animals was tested by injection of BrdU (150mg/kg, Sigma-Aldrich) to the intraperitoneal administration of one of the parabionts after 2weeks post-surgically and the pair was killed after 4 weeks. Mice were anesthetized and perfused through the heart with 10ml of cold saline and 4% paraformaldehyde in 0.1 M PBSat 12weeks of age. Brains were removed from the skull, cryoprotected in 30% sucrose at 4°C, and sectioned 20 μm. Free-oating sections were washed and followed by incubation in 1.5 M hydrogen chloride at 37°C for 30 min. After, the sections were washed in PBS with three times and blocked with normal goat serum, then stained with the BrdU antibody (1:300, Abcam, Cambridge, MA, USA). On the following day, the sections were washed in PBS with three times and incubated with Cy3 conjugated anti-rat IgG(1:100; Jackson immune Research Laboratories) for 2hours. BrdU (red) or DAPI (blue)-stained cells were identi ed using an inverted microscope (BX61, Olympus Corporation, Tokyo, Japan).

Weight measurement
Mice from the R6/2×TG2 KO line and from the zQ175×TG2 KO line were weighed every week for 6 weeks and for 16weeks, respectively.

Preparation of in vitrocellular model for HD
A colony was maintained by breeding ovarian transplant R6/2 females (B6CBATg(HDexon1)62Gpb/1J) obtained from the Jack-son Laboratories with B6CBAF1/J males, based on a C57BL/6 background. F1 offspring of the rst mating generated mice with either the wild-type or R6/2 genotype. Mice were housed under standard conditions (12 h light cycle from 08:00 h to 20:00 h) with adlibitum access to food and water. All animal experiments were studied with the approval of the Institutional Animal Care and Use Committee (IACUC, Approval number: 13-0058-C2A1) of Seoul National University Hospital. We developed an in vitro HD model by culturing neural stem cells from R6/2 mice. In brief, after sacri ced by CO 2 gas, brain tissues of 9-week-old mice were used for primary culture and neural stem cells were isolated by dissection and trypsin treatment. Cells were incubated in culture medium consisting of DMEM/F12 (Invitrogen, Carlsbad, CA, USA), 1% P/S (penicillin-streptomycin), 2% B27 Supplement (Gibco BRL, Carlsbad, CA, USA), 10 ng/mL EGF (Invitrogen, Carlsbad, CA, USA) and 10 ng/mL bFGF (Invitrogen, Carlsbad, CA, USA) at 37°C in a 95% O 2 , 5% CO 2 humidi ed atmosphere. neural stem cells were differentiated in the differentiation medium, which was composed of DMEM/F12, 1% PSA, 2% B27, and 5% FBS.

Isolation serum exosome and treatment of young and old serum-exo
To deplete exosomes from the mice serum, all centrifugation steps were performed at 4 °C. Exosomes were isolated by Exo-quick exosome precipitation solution (System Biosciences, Mountain View, CA), according to manufacturer's speci cations. Brie y, the serum 250μl was mixed thoroughly with 63μl of Exo-Quick exosome precipitation solution and centrifuged at 1500g for 30 min. The supernatant was then removedand centrifuged at 1500g for 5 min after adding buffer. The remaining exosome pellets were then suspended in 100μl PBS. Exosome concentration was measured using BCA protein assay kitfor treatment. HD cells were treated with 200 μg/ml of young or old serum-exo and young or old serum at 2 days of differentiation and incubated for 3 days. Control groups were treated with same volume of PBS.

Tissue preparation and Fluorescent Immunohistochemistry
For immunohistochemistry,mice were anesthetized and perfused through the heart with 10ml of cold saline and 4% paraformaldehyde in 0.1 M PBS at 12weeks of age. Brains were removed from the skull, cryoprotected in 30% sucrose at 4°C, and sectioned 20 μm. Free-oating sections were washed and blocked with normal goat serum, then stained with the Em48 antibody (1:300, Millipore, Billerica, MA, USA).On the following day, the sections were washed in PBS with three times and incubated with Cy3 conjugated anti-mouse IgG (1:100; Jackson immune Research Laboratories) for 2hours. EM48 (red) or DAPI (blue)-stained cells were identi ed using an upright microscope (Ni-E, Nikon Corporation, Tokyo, Japan). [23,24] DCX and BRDU Immunohistochemistry Free-oating sections were washed and followed by incubation in 1.5 M hydrogen chloride at 37°C for 30 min. After, the sections were washed in PBS with three times and blocked with normal goat serum, then stained with the DCX antibody (1:200; Santa Cruz, CA, USA) and BrdU antibody (1:300, Abcam, Cambridge, MA, USA). On the following day, the sections were washed in PBS with three times and incubated with FITC conjugated anti-rabbit IgG and Cy3 conjugated anti-rat IgG (1:100; Jackson immune Research Laboratories) for 2hours. DCX ( tc) and BrdU (red) or DAPI (blue)-stained cells were identi ed using an inverted microscope (BX61, Olympus Corporation, Tokyo, Japan).

Protein extraction and western blot analysis
Brains of R6/2 mice and ZQ175 were isolated, immediately frozen on liquid nitrogen, and stored at -80°C until protein extraction. Cultured HD cells were washed and harvested in PBS (phosphate buffered saline, WelGene, Daegu, Korea)using a cell scraper. Protein extracts were prepared using RIPA buffer (Radio immunoprecipitation assay buffer, Thermo-Scienti c, Waltham, MA, USA)containing freshly added protease inhibitor and phosphatase inhibitor (Roche, NJ, USA).The protein content was determined using a BCA (Bicinchoninic acid assay) protein assay kit (Pierce, Rockford, IL, USA). Forty micrograms of Cell survival assay Cell survival rate was measured by a colorimetric assay using the WST-1 (Roche, Mannheim, Germany) according to manufacturer's instruction. Brie y, cells were seeded in 96-well plates and incubated with young and old serum-exo 200 μg/ml for 72 hours. After 72 hours, WST-1 reagent was added to each well, and cells were incubated at 37 °C and 5% CO2 for 2 h. Absorbance was measured using a plate reader at 450 nm (reference 650 nm) and the result shown represent the averages of four independent experiments.

Flow cytometry
To analyze the apoptosis population of neuronal stem cells, ow cytometry using annexin V-FITC and propidium iodide (PI) staining was used. Neuronal stem cells were washed and harvested in PBS (phosphate buffered saline, WelGene, Daegu, Korea) using a cell scraper. Cells were counted and 1 × 10 6 cells were suspended in 1ml cold binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl 2 ). Cells were aliquoted into 1.5 ml tube at 1 × 10 5 cells per tube, and were incubated with 10 μl of annexin V-FITC at room temperature for 30min and 2 μg/ml of PI at room temperature for 10 min. After incubation, 400 μl of binding buffer was added and ow cytometric analysis was performed (FACS Calibur, BD Bioscience, CA, USA). FITC and PI uorescences were passed through 520 and 630 nm bandpass lters, respectively, and the data were analyzed using Flowing Software (www. owingsoftware.com).

Statistical analysis
All values indicated in the gures are presented as mean ± standard error. Results of western blot were analyzed using Student's t-test. A 2-tailed probability value below 0.05 was considered statistically signi cant. Data were analyzed by SPSS version 17.0 (SPSS Inc., USA).

Construction of Parabiosis Animal Model for HD and Modulation of HD Pathology
The R6/2 mice were surgically joined either with another R6/2 mice or with their corresponding wild-type littermate.In addition, WT-WT serve as controls to ensure that the surgical procedures did not cause ectopic mineralization (Fig. 1a).To successively perform the parabiosis of R6/2 mice, minimizing the damage of parabiosis and the stress were essential due to the high vulnerability of R6/2 mice. The binding site and method had a critical in uence on the survival of R6/2 parabionts, and we were able to obtain stable R6/2 parabionts for the rst time.Suturing the abdomen and the back of the mice was performed through clamping and tying their forelimbs with threads. More details about the methods of parabiosis are demonstrated in the Supplement(Additional le 1: Figure S1, Additional le 2: Figure S2, Additional le 3: Video 1, and Additional le 4: Video 2).The mice were observed periodically as described in materials and methods. No obvious signs of stress were noted during observations, after 6 weeks after surgery.
To demonstrate a connected circulation between parabionts, BrdU was injected into Het-WT. Brain from the injected mice and the attached mice in all groups of parabionts showed similar BrdU signals, indicating a joined circulatory system (Fig. 1b). The shared circulation was also demonstrated by examining the genomic DNA of blood cells in paired mice at four weeks after parabiotic surgery. By comparing the genotype of DNA from the Het-WT and the Iso-HD, the two bands that can be seen in Iso-HD werealso observed in Het-WT (Fig. 1c).
To evaluate the functional consequences of heterochronic parabiosis, we measured weight loss and lifespan. Phenotypes were examined from the parabionts paired for sixweeks. We also recorded survival,weight loss and lifespan of each group among the same composition of females and males.In contrast to theWT(Old)-HD, WT(Young)-HD showed increased survival (Fig. 1d). Heterochronic parabiosis with the young mice delayed the onset of mortality from 84 to 100 days, the average survival from 102 to 123 days, and the maximum survival from 118 to 145 days (P < 0.05). It also reduced tremor (data not shown), improved movement measured(Additional le 5 -7: Video3 -5), and improved clasping test(Additional le 8 -10: Video6 -8) at 9 and 12 weeks of age.Also, WT(Young)-HD showed delayed progression of weight loss at 12 weeks old (p<0.01) while HD-HD showed gradual weight loss from 10 to 12 weeks of age (Fig. 1e), whileR6/2 mice (with CAG repeats of between 154 and 200) die typically at around 3-4 months of age.The improvement on the behavior (Additional le 11 -13: Video9 -11) and the weight loss (Additional le 14: Figure S3)were also shown in ZQ175 mice.
The R6/2 mice showed mHtt aggregation in striatum and cortex during disease progression. To examine histological changes of the brain, mHtt aggregation were evaluated at 12 weeks of age. To evaluate effect on mHtt aggregation, brain was sectioned and sliced tissues were stained with an EM48 antibody which detects aggregation of mHtt, and Het-HD showed reduced mHtt aggregation in the striatum and cortex (Fig. 1f). Also, we extracted proteins from R6/2 mice brain and mHtt aggegation were measured by western blotanalysis (Fig. 1g),and WT(Young)-HD showed reduced mHtt aggregationin the brain.

Modulation of Pathological Phenotypes of HD by Parabiosis
Dysfunction of CREB-PGC-1a pathway has been regarded as the key molecule for HD progression. To examine effects of Heterochronic parabiosis on this pathway, 12 weeks old R6/2 mice were paired and western blot analysis was performed aftersix weeks from the surgery. Het-HD showed increased expression of p-CREB and PGC-1a (p<0.05 vs. R6/2 control) compared to Iso-HD (Fig. 2a). To investigate whether heterochronic parabiosisprotects against apoptosis, we examined the levels of apoptosis-related proteins by western blotting. p53, Bax, and cleaved caspase-3 levels were lower in the Het-HD than Iso-HD (Fig. 2b).
Also, IHC and western blot were performed to examine the improvement of cognitive function. It was found that DCX and BrdU, which are the representative signals for neurogenesis, were increased in Het-HD (Fig. 2c), and the same result was con rmed by western blot (Fig. 2d). It proves that young wild-type blood is also effective in improving cognition. Also, the same result was obtained for the modulation of pathological phenotypes of HD by parabiosis in the case of ZQ175 mice (Additional le 15 -16:

Amelioration of mHtt Aggregation by Exosome Treatment
To construct the in vitro cellular model for HD, neural stem cells were separated from the SVZ which was isolated from the R6/2 mice brain. Neural stem cells showed a spherical shape after the separation, and the expression of mHtt aggregation protein was not observed. The mHtt aggregation protein can be seen from 5 to 7 days after the differentiation of the cell, which is expressed as red in the cell nucleus (Additional le 17: FigureS6).To harvest the exosomes from the blood serum, thereare two representative methods for exosome isolation: serial ultracentrifugation and Exo-Quick reagent. Western bolt analyses were performed to determine the expression of exosome-speci c markerswhich are CD9, CD63 (tetraspanin proteins) and HSP70. The isolated products expressed all the markers, thus con rming the presence of exosomes (Additional le 18: FigureS7).
As shown earlier in Fig. 1d, paring the HD mice with theyoung wild-type increased survival, whereasparing with old wild-type did not. To investigate the underlying mechanismof age-dependent effect of the parabiosis with WT, the exosomal protein and RNA concentration in young blood and old blood were analyzed. Proteins and total RNA were extracted by protein extraction buffer and total RNA isolation kit, respectively, after isolating exosomes. Higher amount of exosomes, exosomal protein, and RNA levels were observed in young blood (Fig. 3a).
To investigate whether young serum-exosomes has a protective role in HD, we treated an in vitrocellular model for HD, which showed mHtt aggregations in nucleus after day 7 of induction, with young serumexosomes. Young serum-exosomes (200μg/mL) was applied to the cells for 3 days after inducing mHtt aggregation. At day 7, the control and young serum-exosomes groups were xed with 4% paraformaldehyde and stained with the Em48 antibody to detect mHtt aggregates, with DAPI as a counter stain (Fig. 3b). We counted DAPI(+) and Em48(+) cells in the HDand HD+Young-exo. The ratios of doublepositive cells to DAPI(+) cells were 20.8±2.3% for HD and 12.1±0.3% for HD+Young-exo. To con rm the reduction of mHtt aggregates, aggregates were also quanti ed by western blot (Fig. 3c). In HD+Youngexo, levels of mHtt aggregates in cells were signi cantly decreased.

Modulation of Molecular Pathology of HD by Exosome Treatment
To examine the effects of young serum-exosomeson the p-CREB-PGC1a pathway, cells were treated with control medium or young serum-exosomes for three days after days 2 of differentiation. Treatment with young serum-exosomes promoted expression of p-CREB and PGC1a (Fig. 4a). To examine theprotection against apoptosis, the levels of apoptosis-related proteins were evaluated by western blotting. p53, Bax, and cleaved caspase-3 levels were lower in the HD+Young-exo than in the HD (Fig. 4b). Also, to con rm the anti-apoptotic effect of young serum-exosomes, neural stem cellswere differentiated and treated by the young serum-exosomes for 3days, and ow cytometry analysis was performed using annexin-V and propidium iodide (Fig. 4c). Cell population wasanalyzed as viable/early-apoptotic/lateapoptotic/necrosis, and this calculation was conducted using the annexin-V and propidium iodide positive cell count. More necrotic population and less viable population were shown in HD,however, HD+Young-exo showed signi cant reduction of the apoptotic/necrotic cell population and an increase in the viable cell population.Taken together, young serum-exo treatment resulted in more cell survival and less cell death, accompanied by a reduction of mHtt aggregation protein, and apoptotic signaling. To examinethe cell survival effects, we investigated the WST-1. The result showed that treatment of young serum-exosomes signi cantly increases cell survival (Fig. 4d). Young serum-exosomes improves mitochondrial activation and cell survival while old serum-exosomes does not (Additional le 19: Figure  S8).

Discussion
In this study, we demonstrated thatheterochronic parabiosis of HD mice with the young wild-type modulates the body weight loss, mHtt aggregation, mitochondrial dysfunction, cell death, and cognitive impairment. [1,10] These are the representative pathologies of HD, thus extending the survival of HD mice. The positive effect ofheterochronic parabiosis originates from the shared blood circulation, [25,26] just like the other neurodegenerative diseases ameliorated by heterochronic parabiosis. [9,27,28]It is worth noting that the R6/2 mice die typically at around 3-4 months of ageaccompanied by trimming and muscle loss [10,11] and highly vulnerable to stress. Therefore minimizing the damage of parabiosis surgery were indispensableto make stable parabiotic pairs. We were able to obtain stable R6/2 parabionts for the rst timeby optimizing the binding site and method, which had a critical in uence on the survival of R6/2 parabionts.
As we have already demonstrated thatthe positive factorsexistin the young blood affecting the transgenic ones, [14]it should also exist in the messenger unit in the shared circulatory system. We identi ed that the exosomes are messenger units for transferring positive factors inside the blood by the in vitro cellular model for HD.Young blood serum as well as serum exosomesalso modulated mHtt aggregation, mitochondrial dysfunction, cell death, and cell viability.
Although there are several limitations that further study is required to identify the positive factor itselfthat are responsible for elucidating the mechanisms of exosome treatment. [16,29] However, we demonstrated that exosome showed more than equivalent effect compared to serum, which could lead to the development of the intravenous administration of serum exosome in humans, which is a low risk procedure already offered as a therapy with limited complications, [30,31] and a group of soluble factors in exosome targeting several pathways may help therapeutic bene ts.Therefore, itis feasible to test the e cacy of young serum exosome in patients with HD and possibly other forms of mitochondria dysfunction and neurodegeneration.

Conclusion
In summary, our results show that the overall pathology of HD is improved by the shared blood circulation through parabiosis, furthermore, we demonstrated that the exosomes are messenger units for transferring positive factors inside the blood by the in vitro cellular model for HD. Thus, the therapeutic potential of the exosomes from young serum has been con rmed through our study, and young serum-exo can be a valuable tool for treating HD. -Consent for publication Not applicable.
-Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information les.

-Competing interests
The authors declare that they have no competing interests. -Authors' contributions ML designed and conceptualized study, analyzed the data,and drafted the manuscript for intellectual content. WI and MK interpreted the data, and revised the manuscript for intellectual content. All authors read and approved the nal manuscript.
-Acknowledgements Not applicable.   Amelioration of mHtt aggregation by exosome treatment.a Comparison of the amount of exosomes, exosomal proteins, and RNA levels of blood serum from young and old wild-type. Young serum-exosome and Old serum-exosome indicates the exosomes derived from the blood serum from the young mice and old mice, respectively. b, cImmunohistochemistry and western blot of cells from demonstratemHtt aggregates in cells. CTL indicates the cells from the wild-type mice, and HD indicates the cells from the HD mice. HD+Young and HD+Young-exo indicates the HD cells treated by blood serum and exosomes derived by blood serum from the young wild-type mice, respectively. HD+Old and HD+Old-exo indicates Page 20/22 the HD cells treated by blood serum and exosomes derived by blood serum from the old wild-type mice, respectively.

Figure 4
Modulation of molecular pathology of HD by exosome treatment.a, b Improvement of mitochondrial dysfunction and modulation cell death, respectively. c Analysis for cell death.