Intrastriatal injection of α-synuclein can lead to widespread synucleinopathy independent of neuroanatomic connectivity
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Prionoid transmission of α-synuclein (αSyn) aggregates along neuroanatomically connected projections is posited to underlie disease progression in α-synucleinopathies. Here, we specifically wanted to study whether this prionoid progression occurs via direct inter-neuronal transfer and, if so, would intrastriatal injection of αSyn aggregates lead to nigral degeneration.
To test prionoid transmission of αSyn aggregates along the nigro-striatal pathway, we injected amyloidogenic αSyn aggregates into two different regions of the striatum of adult human wild type αSyn transgenic mice (Line M20) or non-transgenic (NTG) mice and aged for 4 months.
M20 mice injected in internal capsule (IC) or caudate putamen (CPu) regions of the striatum showed florid αSyn inclusion pathology distributed throughout the neuraxis, irrespective of anatomic connectivity. These αSyn inclusions were found in different cell types including neurons, astrocytes and even ependymal cells. On the other hand, intra-striatal injection of αSyn fibrils into NTG mice resulted in sparse αSyn pathology, mostly localized in the striatum and entorhinal cortex. Interestingly, NTG mice injected with preformed human αSyn fibrils showed no induction of αSyn inclusion pathology, suggesting the presence of a species barrier for αSyn fibrillar seeds. Modest levels of nigral dopaminergic (DA) neuronal loss was observed exclusively in substantia nigra (SN) of M20 cohorts injected in the IC, even in the absence of frank αSyn inclusions in DA neurons. None of the NTG mice or CPu-injected M20 mice showed DA neurodegeneration. Interestingly, the pattern and distribution of induced αSyn pathology corresponded with neuroinflammation especially in the SN of M20 cohorts. Hypermorphic reactive astrocytes laden with αSyn inclusions were abundantly present in the brains of M20 mice.
Overall, our findings show that the pattern and extent of dissemination of αSyn pathology does not necessarily follow expected neuroanatomic connectivity. Further, the presence of intra-astrocytic αSyn pathology implies that glial cells participate in αSyn transmission and possibly have a role in non-cell autonomous disease modification.
Keywordsα-synuclein Amyloid Prion Striatum Dopaminergic Neurodegeneration Astrocyte
glial fibrillary acidic protein
ionized calcium-binding adapter molecule 1
αSyn phosphorylated on Serine 129
Intracellular accumulation of misfolded α-synuclein (αSyn) is a neuropathological hallmark of α-synucleinopathies [1, 2, 3]. Inclusions comprised of amyloidogenic αSyn aggregates, phosphorylated at Serine129 [4, 5, 6] are often found in neuronal cell bodies termed Lewy bodies (LB) or in neuronal projections, called Lewy neurites [1, 2, 3]. Nevertheless, how αSyn pathology initiates and spreads in the nervous system is still an area of intense research and debate.
In recent years, prion-like conformational templating has emerged as a plausible major mechanism that may explain the stereotypic progression of pathogenic αSyn into different brain regions [7, 8, 9, 10]. Cell culture studies have shown that endogenous αSyn can be conformationally altered in the presence of exogenous pathologic αSyn to aggregate into inclusions [11, 12, 13, 14], akin to classical prions [15, 16]. In vivo evidence for this prion-like characteristic of αSyn pathogenesis emerged from studies involving direct intracerebral injections of preformed αSyn amyloid fibrils in αSyn transgenic mice [17, 18]. More recently, studies have shown that peripheral (intramuscular, intraperitoneal or intravenous) injections of αSyn amyloid fibrils into rodents can induce progressive formation of αSyn pathology in the CNS [19, 20, 21, 22]. However, the extent of induction and spread of seeded αSyn pathology in wild type nontransgenic (NTG) mice brains, on the other hand is more disparate; for example, while our group has observed that molecular templating and dissemination of pathogenic αSyn is an imprecise event in NTG mice , others have reported robust and widespread αSyn pathologies following exogenous αSyn challenge in NTG mice [14, 24, 25, 26]. Interestingly, injecting different molecular strains of αSyn can result in distinctive pathologies in rodents, possibly reflective of the spectrum of disease pathologies seen in different types of α-synucleinopathies [20, 27, 28]. Such dependence of seeded pathology on the presence of distinct conformer strains has been shown to exist in classical prion diseases . Overall, these studies have established that αSyn can spread in the CNS by prion-like templating mechanisms [7, 8, 9, 10].
Several recent publications have reported that following intracerebral injection of preformed αSyn fibrils, induction of intra-neuronal αSyn pathology leads to neurodegeneration, especially in the dopaminergic (DA) neurons of the nigrostriatal pathway [20, 24, 30]. This is reminiscent of selective neurodegeneration in α-synucleinopathies, such as Parkinson’s disease . Understanding how intercellular transmission of pathological αSyn along specific neuroanatomic projections leads to selective neurodegeneration in Parkinsonisms and related Lewy body dementias will allow us to design better therapeutics against these diseases. To test whether intrastriatal injection of pathological αSyn aggregates leads to selective nigral degeneration and further whether this is dependent on overexpression of human αSyn transgene, we administered preformed αSyn aggregates into the internal capsule (IC) or the caudate putamen (CPu) areas of either Line M20 (transgenic mice expressing human wild type (WT) αSyn ) or NTG mice. Our results indicate that while induction of αSyn pathology was sparse in NTG mice, M20 mice showed extensive Lewy-type inclusion pathology widely distributed throughout the brain, irrespective of anatomic connectivity. Surprisingly, we did not observe any αSyn inclusion pathology in DA neurons and only modest levels of DA neurodegeneration were noticed in M20 mice injected in the IC. We observed induction of gliosis that is associated with incipient αSyn pathology and interestingly, massive levels of pathological αSyn inclusions were detected within astrocytes in M20 mice. Overall, our results suggest that a combination of glial and neuronal inclusion pathology may result in distinctive αSyn strains that display widespread patterns of dissipation throughout the CNS without obvious direct neuroanatomic connectivity.
Expression and purification of recombinant αSyn protein
The bacterial expression plasmid pRK172 cDNA construct encoding full-length WT human or mouse αSyn was used to express these proteins in E. coli BL21 (DE3) and they were purified to homogeneity by size exclusion (Superdex 200 gel filtration) followed by ion exchange (Mono Q) chromatographies as previously described .
Fibril preparation of recombinant αSyn for mouse brain injection
Human αSyn recombinant protein or mouse αSyn recombinant protein was assembled into filaments by incubation at 37 °C at 5 mg/ml in sterile PBS (Invitrogen) with continuous shaking at 1050 rpm (Thermomixer R, Eppendorf, Westbury, NY). αSyn amyloid fibril assembly was monitored as previously described with K114 fluorometry . αSyn fibrils were diluted to a concentration of 2 mg/mL in sterile PBS and gently sonicated at room temperature in a water bath sonicator for 1 h. This resulted in fragmentation of αSyn amyloid aggregates into shorter fibrils [17, 22]. These fibrils were imaged using transmission electron microscopy (EM) (Additional file 1: Figure S1), and were validated for induction of intracellular amyloid inclusion formation in cell culture as previously described . For EM imaging, fibrils were adsorbed to 300-mesh carbon-coated copper grids, washed, stained with 1% uranyl acetate, and imaged at 100,000× magnification using a Hitachi H7600 transmission electron microscope (Hitachi).
Mouse husbandry and stereotactic injections
The following antibodies were used to detect pathological αSyn phosphorylated at Ser129: 81A (B. Giasson; 1:10,000 for immunohistochemistry and 1:3000 for immunofluorescence; ) and EP1536Y (1:1000; AbCam, Cambridge, MA). The latter antibody specifically reacts with pSer129-αSyn and does not cross-react with phosphorylated low-molecular-mass neurofilament subunit . Other antibodies used in this study are: Syn506, a conformation specific anti-αSyn antibody that preferentially detects αSyn in pathological inclusions (B. Giasson; 1:1000; ); anti-p62/sequestosome antibody (SQSTM1) (Proteintech, Chicago, IL); anti-glial fibrillary acidic protein (GFAP) (Dako, Carpentaria, CA); anti-Iba-1 antibody (1:1000; Wako, Richmond, VA) and anti-tyrosine hydroxylase (TH) antibody (Millipore, Billerica, MA).
Immunohistochemical and immunofluorescence analyses
Mouse brains and spinal cords were collected following intra-cardiac perfusion with PBS/heparin. Tissues were fixed with 70% ethanol/150 mM NaCl for 48 h followed by paraffin processing. Paraffin-embedded tissue sections were deparaffinized and hydrated through a series of graded ethanol solutions followed by washing with 0.1 M Tris, pH 7.6. The sections were blocked with 2% FBS in 0.1 M Tris, pH 7.6. Immunohistochemical detection was done using avidin-biotin complex (ABC) system (Vectastain ABC Elite Kit, Vector Laboratories, Burlingame, CA) and immunocomplexes were visualized with the chromogen 3,3′-diaminobenzidine (DAB). Sections were counterstained with hematoxylin. Slides were scanned using an Aperio ScanScope CS (Aperio Technologies Inc., Vista, CA) and images acquired using the ImageScopeTM software (Aperio Technologies Inc.). Quantification of immunostaining was done using the Pixel count Program (ImageScope, Aperio Technologies). For immunofluorescence detection, sections were incubated with secondary antibodies conjugated to Alexa fluor 594 or Alexa fluor 488 (Invitrogen, Eugene, OR) followed by Sudan Black treatment and staining with DAPI (Invitrogen, Eugene, OR). The sections were coverslipped with Fluoromount-G (Southern Biotech, Birmingham, AL) and visualized using an Olympus BX51 microscope mounted with a DP71 Olympus digital camera.
Stereological analysis of TH neurons in the substantia nigra
TH-positive neurons in the substantia nigra (SN) were counted by staining paraffin-embedded sections with an anti-TH polyclonal antibody (Millipore, Billerica, MA). Stereological counting of DA neurons was performed by counting and tallying TH-immunopositive neurons on every tenth section based on a previously published report .
Induction and spread of pathological αSyn in M20 mice does not strictly follow neuroanatomic projections
Since the IC provides connectivity between various brain areas supporting multiple, diverse, and dissociable functional regions, we next investigated whether injecting human αSyn fibrils directly into the CPu resulted in transmission of induced αSyn pathology into discrete areas linked by direct projections from this area (Cohort 3). The CPu area receives inputs from the cortex and sends projections directly into the globus pallidus, thalamus, and SN. Mice were examined for αSyn inclusion pathology following injection of human αSyn fibrils into the CPu. These M20 mice showed essentially a similar distribution of αSyn pathology as observed in Cohort 1 and Cohort 2 mice (Figs. 1 and 2c), indicating that templated induction of αSyn pathology follows similar patterns of distribution whether initiated in a white matter enriched area (IC) or a projection rich area (CPu). All M20 cohorts (Cohorts 1, 2 and 3) showed extensive perikaryal as well as axonal pathology; additionally, Cohort 3 mice showed pathology throughout the cortex especially in the visual and entorhinal cortex (Figs. 1 and 2c, Additional file 2: S2). On the whole, Cohort 3 of M20 mice showed relatively more robust and widespread distribution of αSyn inclusion pathology than mice injected with human αSyn fibrils or mouse αSyn fibrils in the IC (Cohorts 1, 2) (Fig. 2a-c). An important distinction between these cohorts is that the Cohort 3 mice seemed to develop relatively more robust αSyn pathology in and around the area of injection, i.e., striatum, than Cohort 1 and 2 mice, which had limited αSyn pathology proximal to the area of injection. We also observed robust induction of αSyn inclusion pathology in the ependymal cells abutting the lateral ventricles in the human αSyn fibril injected M20 mice in Cohorts 1 and 3 (Additional file 3: Figure S3).
Induction and spread of pathological αSyn in NTG mice is sparse and restricted
Intra-striatal administration of αSyn aggregates does not necessarily induce degeneration of dopaminergic neurons in the substantia nigra
Characterization of astrocytosis and microgliosis in αSyn seeded mice
Line M20 mice injected in IC (Cohorts 1 and 2) showed much more variability in astrogliosis across individual mice, in spite of consistently uniform distribution of αSyn pathology in these mice. In particular, Cohort 1 mice showed no induction of gliosis in and around the area of injection (i.e., striatum) (Fig. 7a), whereas there was increased astrogliosis and microgliosis in hippocampus and entorhinal cortex, albeit with more mouse-to-mouse variability; therefore, in a facile sense, induction of astrogliosis cannot be directly explained by the accretion of induced αSyn pathology (Additional file 5: Figure S5). Interestingly, there is also discordance between GFAP and Iba-1 immunostaining that is strikingly evident in the M20 mice injected in the IC (Cohorts 1 and 2) – for example, there was generally higher levels of astrogliosis in Cohort 2 compared to limited alterations in microgliosis (Fig. 7a and c; Additional file 5: Figure S5).
The extent of astrogliosis and microgliosis in NTG mice was variable throughout brain regions in the αSyn injected cohorts. Overall, in NTG mice injected in the IC (Cohorts 4 and 5) or CPu (Cohort 6), we observed sparse astrogliosis (Fig. 7b and d, Additional file 6: Figure S6). For example, Cohort 5 of NTG mice with no induction of αSyn inclusion pathology (i.e., injected in IC with human αSyn fibrils) expectedly does not show increased astrogliosis. Interestingly, with respect to mouse αSyn fibril injected cohorts, mice injected in the white matter rich IC area (Cohort 4) display higher (but variable) astrocytosis than in mice injected in the CPu (Cohort 6) (Fig. 7b, Additional file 6: Figure S6). Surprisingly, in spite of the presence of αSyn pathology, microgliosis was significantly downregulated in the striatum, hippocampus and entorhinal cortex of NTG mice injected with ms αSyn fibrils in the CPu (Cohort 6; Fig. 7d, Additional file 6: Figure S6).
Induction of robust αSyn pathology within nigral astrocytes but not TH neurons in M20 mice
In Cohort 3, astrocyte resident pSer129-αSyn pathology was also observed in several other brain areas that had induced αSyn pathology, such as ventral midbrain, hippocampus and cortex in these mice (Additional file 7: Figure S7b). We also observed a similar distribution of astrocytic pSer129-αSyn pathology in Cohort 1 of M20 mice injected with human αSyn fibrils, though individual mice in this cohort showed variable induction of astrogliosis (Additional file 7: Figure S7A). In the NTG cohorts displaying induced αSyn pathology (Cohorts 4 and 6), we did not observe any astrocytic αSyn pathology in the ventral midbrain, entorhinal cortex or hippocampus (Additional file 8: Figure S8A–B).
Templated conformational alterations in αSyn has been shown to induce LB-like pathology in mice, suggesting that this maybe a significant disease mechanism in human α-synucleinopathies [7, 8, 9, 10]. While some reports have indicated that induced αSyn pathology spreads along neuroanatomic connections in a prionoid fashion [18, 20, 24, 25, 26, 40], we and others have previously reported that propagation of seeded αSyn pathology in mice can follow atypical or non-prionoid characteristics [17, 21, 41, 42]. Here, using human WT αSyn transgenic mice (M20) and NTG mice, we show that 1) injection of αSyn aggregates into two different areas of striatum results in widespread αSyn inclusion pathology in M20 mice, irrespective of neuroanatomic connectivity; 2) templated αSyn pathology following intra-striatal injection is not transmitted to DA neurons and causes moderate to no degeneration in DA neurons; 3) induction of αSyn pathology can be an inefficient process and spares the SN-resident TH-immunopositive neurons in NTG mice; and 4) αSyn laden astrocytes were readily observed in brain regions of M20 mice with robust αSyn pathology, suggesting that non-neuronal cells can also contribute to the dissemination of αSyn pathology. Overall, our observations indicate that, in addition to inter-neuronal transfer of seeded αSyn pathology along anatomic connections, simple diffusion or glia-mediated regional transfer may also affect the extent and distribution of pathologies in mouse models of α-synucleinopathies. In our study, a significant proportion of αSyn pathological inclusions induced by the administration of exogenous αSyn fibrils were localized within astrocytes throughout the CNS, in stark comparison to other published studies that have shown minimal glial involvement [18, 26]. It is possible that this unique property of our αSyn preparations along with potential maturation of αSyn pathology within the astroglia may generate unique αSyn conformers with distinctive strain-like properties that are preferentially transmitted via glial cells relative to direct inter-neuronal transfer. Induction of α-synucleinopathy by such astrocyte-discriminative αSyn conformers could be used to mechanistically understand the disease etiology in α-synucleinopathies with massive glial involvement, such as in multiple system atrophy [43, 44]. Whether the αSyn fibrils used in our studies indeed possess a preference for enhanced glial transmission relevant to disease pathogenesis in distinct spectra of α-synucleinopathies and how this impacts the progression and outcome of different subtypes of α-synucleinopathies will be examined in future studies.
Critical parameters that govern dissemination of αSyn seeds: concept of endogenous barriers
In the prion field, permissive transmission depends on overcoming the species barrier, which is determined by a range of possible conformers of a particular prion, its sequence, as well as its interaction with cellular co-factors such as chaperones [45, 46, 47]. One such endogenous ‘barrier’ relevant to α-synucleinopathies may be the subtle differences in the sequence of mouse and human αSyn (7/140 amino acids difference)  or their differential interactions with cellular co-factors. Based on this premise, mouse and human αSyn may have differential abilities in recruiting endogenous αSyn into pathological aggregates . In support of this theory, we observed that in NTG mice, while mouse αSyn aggregates resulted in induction of αSyn pathology, human αSyn fibrils were ineffective. Therefore, it is likely that a ‘species barrier’ may determine the structural templating of αSyn and its dissemination in the brain. However, this notion is inconsistent with previous observations in NTG C57BL/6 J mice that intra-nigral injections of human or mouse αSyn aggregates result in identical patterns of pSer129-αSyn pathology . Additionally, we found that the templated induction of pathological αSyn by mouse αSyn fibrils is an inefficient process in NTG mice compared to M20 mice. Since exogenous mouse αSyn aggregates do not necessarily recruit endogenous αSyn readily into pathological aggregates, this data suggests that the induction of αSyn pathology is a non-stochastic process dependent on the overall threshold barrier of αSyn protein. Therefore, it is possible that endogenous barriers such as a thresholding effect may regulate the induction and prionoid dissemination of αSyn pathology.
The ‘species barrier’ concept that can help explain why human WT αSyn fibrils are inefficient in seeding NTG mice is supported by the fact that there are sequence differences between human and mouse αSyn, albeit only 7 amino acids . Interestingly, one of these alterations is an Ala in human αSyn compared to a Thr in mouse αSyn at residue 53, thus, mouse αSyn intrinsically has the Ala53Thr substitution that cause disease in human. In vitro mouse αSyn has a greater propensity to fibrilize into amyloid than human αSyn, akin to AlaThr53 human αSyn . Although both mouse and human αSyn fibrils can cross-seed monomers of each other to polymerize into fibrils, homotypic seeding is usually more efficient in vitro . Similarly, they both can initiate the aggregation of each other in vivo, but again homotypic seeding is more efficient . However, as shown in extensive studies comparing the influences of amino acid alterations in mouse and human αSyn on the seeded-induction of aggregation, differences observed by in vitro cross-seeding do not always directly correlate with in vivo seeding efficiency . The seeded induction of αSyn aggregation in vivo can be influenced by many differential factors unique to the αSyn seeds including cellular uptake, aggregate stability and cellular binding partners (eg. other proteins and lipids), in addition to subtle differences in the compatibility of unique conformers in initiating the recruitment and aggregation of intrinsic αSyn.
Mechanisms of de novo emergence of prions are still elusive. The aggregation of classical prions has been determined to be a non-stochastic process, whereby threshold of the prion protein determines the efficiency of prionoid transformation and propagation. Indeed, a criterion for prions is that these are formed more efficiently upon over-expression of the native protein. This was reported in the case of SUP35 and URE2 genes, where overproduction of each of these genes induce de novo formation of the respective prions [50, 51]. Indeed, in our present study as well as in previous studies, we have observed that templated induction of αSyn pathology is more efficient in mice overexpressing human αSyn than NTG mice [17, 23]. This suggests similarities between formation of classical prions and αSyn prionoid species. Overall, our study shows that the efficiency of induction of αSyn aggregation depends on the amount on available αSyn protein in the cell, suggesting that threshold of transgenic protein levels may be a determining factor for ensuring efficient prionoid induction of αSyn pathology.
In this present study and earlier studies , we have ascertained that M20 mice injected with αSyn aggregates show dramatic induction of astrogliosis and microgliosis. In sharp contrast to the observations by Rey et al. , we observed copious amounts of αSyn pathology co-localized with astrocytes, irrespective of the area of injection. This implies that, in addition to transneuronal transport, astrocytes and possibly other immune cells may also play key roles in the dissemination of pathological αSyn seeds. This has future therapeutic implications as glial cells can effectively act to reduce interneuronal transmission and more importantly clear extracellular αSyn seeds (reviewed in ). Indeed, it has been shown that both astrocytes and microglia can internalize and effectively degrade αSyn aggregates [53, 54, 55, 56]. Collectively, these data suggest that glial cells that normally do not express or express very low levels of αSyn are not prone to seeding in some settings and this may play an important role in creating an endogenous barrier to the prionoid propagation of αSyn.
Does trans-neuronal propagation of templated αSyn pathology cause DA neurodegeneration?
The etiology of DA neurodegeneration in the SN of patients with LB diseases remain unknown. One theory is that trans-neuronal transmission of seeded αSyn pathology along the neuroanatomically connected nigro-striatal pathway causes significant DA neuronal death [18, 20, 24]. However, in our study, we observed only a 20–25% DA neurodegeneration in M20 mice following injection into the IC with either mouse or human αSyn fibrils. No DA neurodegeneration was noted in NTG mice or in M20 mice injected into the CPu, an area that is directly linked to the SN. Additionally, we were intrigued by our observations that DA neurons were impervious to seeded αSyn pathology in both NTG and M20 mice. We reasoned that this observation can be attributed to multiple complex scenarios such as (1) an initial wave of pathological αSyn inclusion pathology lead to DA neurodegeneration followed by astrocytic engulfment of released materials or (2) astrocytes are able to engulf extracellular αSyn directly during the templated induction and dissemination process . In an effort to localize seeded αSyn pathology in the SN, we conducted immunofluorescent co-localization studies using antibodies against non-neuronal cells and found that a majority of these inclusions are present in astrocytes, but not in the nigral TH-immunopositive DA neurons. Since we did not observe any significant nigral DA neurodegeneration in some of these mice (Cohort 3 of M20 mice), our observations suggest that astrocytes likely engulf and scavenge pathological αSyn during the propagation process. Indeed, this is consistent with observations that even direct intra-nigral injection of αSyn aggregates does not necessarily cause nigral DA neurodegeneration [20, 25] but would be contrary to observations from other groups [18, 24]. Interestingly, whether glial cells, such as astrocytes, can have a disease modifying effect in α-synucleinopathies by acting as αSyn scavengers or as unwitting vehicles that aid in dissemination of αSyn, remains to be investigated.
Multiple independent reports, including this present study, show that αSyn aggregates display prionoid characteristics. Additionally, we show that contrary to expected routes of transmissibility exclusively following neuroanatomic connectivity, prionoid αSyn can be widely disseminated throughout the brain by various cellular networks - neurons as well as astrocytes. Therefore, our current findings, when aligned with the present literature, suggest that the mechanism influencing prionoid propagation of αSyn are complex and not always predictable by direct inter-neuronal connectivity. In fact, glial transmission of αSyn may also have a major impact on the pathological outcome in α-synucleinopathies. These findings are similar to disease pathogenesis by classical prion protein, where different strains of misfolded prions can have contextually-dependent transmission properties in the neuraxis [15, 16, 47]. Importantly, these properties open up potential therapeutic opportunities targeting such extracellular transfer of αSyn by utilizing endogenous immune and non-immune barriers.
This work was supported by grants from the National Parkinson Foundation (NPF-UN203) and NIH/NINDS (NS089622). Publication of this article was funded in part by the University of Florida Open Access Publishing Fund. The authors declare no competing financial interests.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its additional files.
PC and BG conceived and coordinated the study, analyzed the data and wrote the manuscript; PC performed mouse injections; ZS performed immunostaining, drafted images for publication and participated in data analysis; MMB and VH performed tissue harvesting and immunostaining; NJR performed electron microscopy; TEG participated in data interpretation. All authors read and approved the final manuscript.
None of the authors have any competing interests in the manuscript.
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All animal experiments were carried out in accordance with the University of Florida IACUC for the care and use of laboratory animals.
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