Localization of ¸-synuclein protein in the central nervous system of the short-lived fish, Nothobranchius furzeri Tyler A.Spaans & Tyrone Genade Department of Biology, Northwestern College, 101 7th St SW, Orange City, Iowa, 51041, email:
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Abstract Nothobranchius furzeri are short-lived fish from Zimbabwe and Mozambique (Africa) that inhabit temporary water bodies. Their captive lifespan is 12 to 40 weeks. Previous research has demonstrated motor deficits [1] that parallels MPTP and PARKIN-PINK1knock-out-mutant induced motor deficits in Medaka (Oryzias latipes, ricefish) [2]. Western blotting of brain extracts showed an accumulation of monomeric and oligomeric ¸-synculein (asyn) protein. Zebrafish do not express asyn protein in their brains [3] making them of dubious utility as a model organism of age-related ¸-synucleinopathy and Medaka do not develop ¸-synucleinopathy spontaneously making N. furzeri a potentially useful model in the research of ¸-synucleinopathy. Using a polyclonal antibody developed against the amino acids 90–104 of the Medaka asyn protein we developed a central nervous system asyn localization map for N. furzeri to serve as a guide for studying ¸-synucleinopathy in this species. ¸-synuclein protein is present in the retina, telencephalon, diencephalon, mesencephalon, cerebellum, medulla oblongata and spinal cord of N. furzeri. None of the asyn-containing cell bodies are pigmented. Immunoreactivity corresponds with asyn expression reported for the carp brain [4]. The immunoreactivity pattern also correlates with the known expression of tyrosine-hydroxylase in Medaka [2] and Zebrafish [5, 6]. Specifically, asyn protein was observed in neurons in the periventricular nucleus of the posterior tuberculum which is associated with MPTP induced motor deficits in Medaka [2]. This map of asyn localization will serve as a guide to assess the development of ¸-synucleinopathy in N. furzeri.
Result 1. Images of retina stained with anti-Medaka-asyn antibody/FastRed. (A) Image of retina with 10ˆ objective, (B–C) with 40ˆ objective. Immunoreactivity distributes in three layers: the ganglion cell layer (green arrow, B), the inner nuclear layer (magenta arrow, B) and the outer plexiform layer (cyan arrow, B) where immunoreactivity is weak. An example of a immunoreactive ganglion cell is visible in image B. Most immunoreactive structures do not co-localize with the methyl green nuclear stain and could represent synaptic processes and dendrites. An example of an enlarged immunoreactive inner nuclear layer cell is shown in image C (green arrow). This result is in accord with previously reported results where asyn gene expression was detected in the inner nuclear layer as well as along the border of the inner nuclear and plexiform layers [9]. Photoreceptors, bipolar cells, GABAergic and dopaminergic cells (amacrine and horizontal cells) and retinal ganglion cells were labeled in all vertebrate species studied in [9]. In [10] large asyn immunoreactive neurons were observed on the border of the inner plexiform and nuclear layers in the retina of Parkinson’s Disease victims, just as observed here.
Methods Eight week old fish were killed and fixed in Davidson’s Fixative at 4‹C overnight and then passaged through ethanol into xylene and then parrafin. The heads of the fish were serially sectioned at 5 —m. Every 5th section was dewaxed and rehydrated. Sections were not subjected to antigen retrieval. Sections were blocked in 0.1% sodium azide diluted in methanol for 30 minutes at room temperature followed by incubation in antibody blocking solution: 1% BSA blocking solution with 0.1% Tween and 0.03% sodium azide in PBS for 30 minutes. The anti-Medaka-asyn (aMasyn) was diluted 1:2000 in antibody blocking solution and incubated overnight at room temperature. Sections were washed of primary antibody and then incubated in donkey-anti-rabbit alkaline-phosphatase coupled secondary antibody (ab97061) at 1:3000. Immunoreactivity was visualized using FastRed chromogen (ab64254). Sections were incubated with FastRed for 3 minutes. Sections were counterstained with Methyl Green, dehydrated and mounted. Sections were examined using a Zeiss Primo Star microscope. Images were captured with a Axiocam 105 color camera and ZEN 2 software by Zeiss. Image panels were constructed in Adobe PhotoShop CS2. Images were resized as well as the color levels and contrast adjusted for publication. The naming of anatomical structures mainly follows the N. furzeri brain atlas [7] and secondarily the Medaka brain atlas [8].
Acknowledgments Prof Ryosuke Takahashi of the Department of Neurology, Kyoto University Graduate School of Medicine, Kyoto, Japan for the anti-¸-synuclein antibody. Mr Chad Miller for technical assistance. Northwestern College for funding and support.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
Cellerino et al. Biol. Rev., 2016. 91(2):511–533. Matsui et al. Exp Neurobiol, 2012. 21(3):94–100. Sun & Gitler. Developmental Dynamics, 2008. 237(9):2490–2495. Vaccaro et al. J. Comp. Neurol., 2015. 523(7):1095–1124. Semenova et al. Histochem. Cell Biol., 2014. 142(6):619–633. Ma. J. Comp. Neurol., 1994. 344(2):242–255. D’angelo. Anat Rec (Hoboken), 2013. 296(4):681–691. Ishikawa et al. The Fish Biology Journal Medaka, 1999. 10:1–26. Martínez-Navarrete et al. Mol. Vis., 2007. 13:949. Bodis-Wollner et al. Ann. Neurol., 2014. 75(6):964–966. Flinn et al. Journal of Neurochemistry, 2008. 106(5):1991–1997. Lee et al. The Cerebellum, 2015. 14(5):516–527. Javitch et al. PNAS, 1984. 81(14):4591–4595. Braak et al. Cell Tissue Res., 2004. 318(1):121–134. Braak et al. Neurobiology of Aging, 2003. 24(2):197–211. Matsui et al. Hum. Mol. Genet., 2013. 22(12):2423–2434.
Northwestern College, Iowa
Result 2. Images of olfactory bulb and telencephalon. All images taken by 10ˆ objective. Immunoreactive cell bodies are found within and around the periphery of the rostral OB (A) but only within the internal cellular layer of the caudal OB. In the telencephalon (A-I) cell bodies are visible in the Dlv and Dm (green arrows, B). The diffuse staining is weak at first (B) but grows more intense (C). Cell bodies are at first restricted to the Dlv (green arrow, D) and then spread into the Dld (E). A non-immunoreactive zone emerges between the Dld (pink asterisk) & Dlv (magenta asterisk) and the Dc (green asterisk) & Dld (cyan asterisk) which represents the lateral forebrain bundle. Dll separates out as a distinct region (magenta asterisk in F to green arrow in H) with a different staining pattern to the neighboring region, Dld (cyan asterisk, E&G). Immunoreactivity is visible in the ventro-lateral telencephalon which expands across to the ventro-ventral telencephalon (E–I). Immunoreactive cell bodies become visible in the PPa (green arrow, F) as well as along the ventricle bordering the PPa and then the PM (cyan arrow, G). Cell bodies are also visible in the SC (green asterisk, G). The ED is also immunoreactive with distinct cell bodies (green arrow, G). Staining within an area below the Lfb (magenta asterisk, I) grows more intense, with distinct cell bodies becoming visible. The cell bodies of Ev are non-immunoreactive (green arrow). These results agree well with Vaccaro et al. [4] except that they did not report immunoreactive cell bodies in the lateral regions of the dorsal telencephalon, nor the ED. Formic Acid treated sections of the Nothobranchius OB that were reacted with the SNL-4 antibody against asyn demonstrated asyn-accumulation in OB neurons. Variable asyn immunoreactivity in these neurons suggests the accumulation asyn in certain neurons. D’Angelo wasn’t able to discern any specific nuclei in the dorsal telencephalon [7] but by using the asyn immunoreactivity we can see different regions. The presence of asyn-immunoreactive neurons in the neurogenic niche lining the ventricles of the telencephalon is interesting.
Result 3. Images of the diencephalon & mesencephalon. (A) In the preoptic region: intense staining is visible in the TL (A&B, green arrow) and adjacent nuclei (PPd & PPv, blue arrow in A&B), the posterior commisure is faintly stained. Cell bodies are stained in the pituitary (cyan arrow, A&B). Strong immunoreactivity is seen in the hypothalamus (magenta arrow, A&B). These constitute several distinct regions of staining composed of both immunoreactive nuclei as well as fibers. Large immunoreactive cell bodies are visible along the ventricle walls (yellow arrow, B) in the region of the TPp and PVO. Immunoreactivity is distributed in four layers of the OT (images C&D): border of the periventricular gray zone and the stratum album centrale (magenta arrows); the strata album centrale and griseum centrale (yellow arrows); strata griseum centrale and fibrosum et griseum superficiale (green arrows); and in the stratum opticum (cyan arrows). The staining is punctate in the stratum griseum superficiale while in the stratum album centrale staining is both punctate as well as fibrous (with varicose fibers), suggesting synapses and axons. Within the periventricular gray zone there are also immunoreactive nuclei (blue arrows, D). Deeper into the diencephalon a distinct immunoreactive nucleus appears which could correspond with the CPN (magenta arrow, E). A fiber tract is stained in the hypothalamus (cyan arrow, E&F). These fibers appear to originate from nuclei in the TPp and PVO (green arrows, F). Immunoreactive cell bodies are also present in the lateral (small yellow arrow, E) and dorsal hypothalamus (blue arrows, E&F) as well as anterior tuberal nucleus (small green arrow, E). At the level of the NG (blue asterisks) strong immunoreactivity is visible in the ventral and diffuse inferior lobe of hypothalamus (green arrow, G&H). Weak and strongly immunoractive cell bodies are visible in the caudal hypothalamus (green arrow, I). Three other nuclei are immunoreactive: Nmlf (magenta arrow, H), NIII (yellow arrow, H) as well as immunoreactive cell bodies adjacent to the Nllf (cyan arrow, H). Our results are in accord with Vaccaro et al. [4] except that there are clearly immunoreactive cell bodies in the periventricular gray zone. We could also delineate asyn immunoreactivity to at least four layers. The extensive staining in the preoptic regions has been correlated with tyrosine-hydroxylase immunoreactivity [4, 5]. Immunoreactive cells in the TPp and PVO are expected. Research in Medaka and zebrafish have identified these as being essential to the motor systems [2, 11]. The loss of these neurons is associated with a Parkinsonism-like phenotype in Medaka fish. As for Vaccaro et al. we find asyn immunoreactive neurons in NIII, Nmlf, Nllf and the hypothalamus.
Result 4. Images of cerebellum and rostral medulla. Immunoreactivity is evident in the Va (green arrow, A&B) and cell bodies are seen along the ventricle walls (cyan arrow, A). Immunoreactive cell bodies are seen in the LV of the dorsal midbrain tegmentum (yellow arrow, B&D). Cell bodies and fibers are visible along the border of the DIL and ventral tegmentum (magenta arrows, B&C). There is diffuse immunoreactivity throughout the area comprising the reticular formation (blue asterisks, B, E, G&H). Immunoreactive cell bodies and fibers are visible in the EG (yellow arrow) and CC (magenta arrow, E, G&H). The CI is intensely immunoreactive and included both immunoreactive cell bodies and fibers (blue arrow, E&F). The immunoreactivity in the CI extends rostrally into the nucleus of raphe (not shown). Ventral to the CI are immunoreactive neurons in the ttbc. Immunoreactive cell bodies and fibers are visible in the gl of the cerebellum (green arrow, E, G–I). A band of immmunoreactive cells and fibers emerge between the cerebellum and EG (cyan arrow, G, H&I) and go on to form a border. Immunoreactive cell bodies are also present along the ventricle. Vaccaro et al. [4] report extensive immunoreactivity in the reticular formation, EG and CC and sparse cellular immunoreactivity in the cerebellum. The immunoreactive cell bodies visible in the gl follows the same pattern as reported for mice [12]: with increasing immunoreacitivty from rostral to caudal end. In mice it was the VGluT1 excitatory terminals and unipolar brush cells that were immunoreactive. The row of immunoreactive cell bodies cutting across the cerebellum is possibly the posterior lateral fissure in which case the structure between the cerebellum and GE is the vestibulocerebellum. The CI of fish is known as the interpeduncular nucleus of human anatomy. This nucleus is involved in the regulation of sleep and pain sensitivity. In the rat this brain region is richly supplied with receptors to MPTP [13] and displays Lewy Body pathology in Parkinson’s Disease [14]. The CI forms part of the raphe system which degenerates prior to the manifestation Parkinsonism symptoms [15].
Tyler A.Spaans & Tyrone Genade
Braak et al [14, 15] argue for a slow staging of Parkinson’s Disease with the vagal nuclei being the first CNS victims of the disease. There is extensive asyn immunoreactivity in the N. furzeri vagal nuclei, especially the large vagal (NXm) motor neurons. Vaccaro et al. [4] only showed immunoreactive cell bodies in the lateral and ventral horns of the spinal cord where we also see immunoreactive cell bodies in the Dorsal Horn.
What do the results mean? I
Abbreviations asyn, ¸-synuclein; aMasyn, anti-Medaka-¸-synuclein antibody; CC, cerebellar crest; CI, corpus interpeduncular; CPN, central pretectal nucleus; DAB, diaminobenzidine; ED, dorsal entopeduncular nucleus; EG, granular eminentiae; Ev, ventral entopeduncular nucleus; Dc, central zone of dorsal telencephalon; DIL, diffuse inferior lobe of hypothalamus; Dld, dorso-lateral zone of dorsal telencephalon; Dll, latero-lateral zone of dorsal telencephalon; Dlv, ventro-lateral zone of dorsal telencephalon; Dm, medio-dorsal zone of telencephalon; gl, glomerular layer; llf, lateral longitudinal fascicle; LV, lateral nucleus of valvula; mlf, medial longitudinal fascicle; NIII, nucleus of III nerve; NIXm, nucleus of nerve IX (motor branch); Nllf, nucleus of lateral longitudinal fascicle; mllf, nucleus of medial longitudinal fascicle; NG, glomerular nucleus; PM, magnocellular preoptic nucleus; PPa, anterior preoptic nucleus; PPd, dorsal periventricular pretectal nucleus; PPv, ventral periventricular pretectal nucleus; PVO, paraventricular organ; TL, longitudinal tori; Tpp, periventricular nucleus of posterior tuberculum; ttbc, cruciate tecto-bulbar tract; OB, olfactory bulb; OT, optic tectum; SC, superchiasmic nucleus; Va, valvula of cerebellum.
Result 5. Images of sections through the caudal medulla oblongata and spinal cord. A small cluster of immunoreactive cell bodies are present in the dorsal medulla between the central griseum and the CC (green arrow, A&C), which may represent the nucleus of solitary fascicle. There is cellular, diffuse and fibrous immunoreactivity in the ventral medulla, corresponding with the reticular formation (blue asterisks, A&C). Large immunoreactive cell bodies are visible in NIXm (blue arrow, A&B) as well as along the nerve issuing from NIXm (yellow arrow, A&B). The nuclei in the ventral medulla aggregate into the inferior olive (magenta arrow, C). Caudally, immunoreactivity becomes more intense in the lobus vagi (green asterisks, D&G) with a few dorsally located immunoreactive cell bodies (cyan arrow, G). Immunoreactive cell bodies and fibers are evident in the descending octavus nucleus and caudal nucleus of lateral line (magenta asterisk, D). Large immunoreactive neurons of the NXm are apparent (green arrow, D&E) with their large immunoreactive axons running radially out towards the ventral medulla’s reticular formation. As the medulla transitions to the spinal cord the well defined zones of immunoreactivity in the reticular formation merge with the descending octavus nucleus and caudal nucleus of lateral line and then medially and upwards to merge with lobus vagi (cyan arrows, H). Immunoreactive cell bodies are distributed throughout the area. In the spinal cord (I) there are a small number of immunoreactive cell bodies present in the Dorsal Horn (yellow arrow) and the Medial Lateral Motor Column (green arrow).
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Asyn expression in the N. furzeri brain is similar to that of other animals studied: ? Asyn is present in the same cell types of the N. furzeri retina as the human retina, especially, dopaminerigic cells; and shows similar Parkinsonism cellular pathology. ? Asyn is expressed in discrete brain regions and in cell types (e.g. TPp & NXm) which are implicated in ¸-synucleinopathy. ? Asyn is expressed in neurons associated with tyrosine-hydroxylase expression. ? Asyn expression is graded in the cerebellum as observed in the mouse. None of the cell bodies containing asyn are pigmented. Zebrafish do not express asyn, but use a modified ‚-synuclein instead [4] which doesn’t form synucleinopathies. Parkinsonism pathology has to be induced in Medaka by double knock-out of PINK and PARKIN [16]. N. furzeri expressed both asyn but seems to develop Parkinsonism pathology spontaneously in an age-related manner making them potentially useful models of ¸-synucleinopathy. We now have a localization map which we can use to study asyn pathology in the N. furzeri central nervous system.
August 21, 2017
