Distribution patterns of the zebrafish neuronal intermediate filaments inaa and inab

It has been reported that the neuronal intermediate filament (IF) α-internexin may plays a role in the formation of the neuronal cytoskeleton during mammalian development. From a phylogenetic viewpoint, zebrafish express inaa and inab as homologs of mammalian α-internexin. However, the distribution patterns of the inaa and inab proteins throughout zebrafish development have not been well-characterized. We generated antibodies specific for zebrafish inaa and inab and analyzed the distribution of these two proteins in developing zebrafish. Inaa was identified in the major subdivisions of embryonic and larval brains as early as 1 day postfertilization (dpf), including the telencephalon, optic tectum, and cerebellum, and inab was also detected in the same regions from 3 dpf to the adult stage. Moreover, we demonstrated for the first time that inaa was distinctively expressed in the photoreceptor-like cells of the pineal gland, where inab was sparsely detected. Besides, the expression of inaa in male adult fish was found to be stable under different photoperiod conditions. Thus, we suggest that inaa is one of useful markers for studies of zebrafish cone photoreceptors not only in the retina but also in the pineal gland. In conclusion, we report that the distribution patterns of inaa and inab are phylogenetically conserved in the telencephalon, optic tectum, and cerebellum. Moreover, inaa and inab had different expression patterns in the pineal gland and retina during zebrafish development. Both inaa and inab are neuronal IFs and their functional roles may be different in various aspects of zebrafish neuronal development.     

Molecular cloning and characterization of chicken neuronal intermediate filament protein α‐internexin

α‐Internexin is one of the neuronal intermediate filament (IF) proteins, which also include low‐, middle‐, and high‐molecular‐weight neurofilament (NF) triplet proteins, designated NFL, NFM, and NFH, respectively. The expression of α‐internexin occurs in most neurons as they begin differentiation and precedes the expression of the NF triplet proteins in mammals. However, little is known about the gene sequence and physiological function of α‐internexin in avians. In this study we describe the molecular cloning of the mRNA sequence encoding the chicken α‐internexin (chkINA) protein from embryonic brains. The gene structure and predicted amino acid sequence of chkINA exhibited high similarity to those of its zebrafish, mouse, rat, bovine, and human homologs. Data from transient‐transfection experiments show that the filamentous pattern of chkINA was found in transfected cells and colocalized with other endogenous IFs, as demonstrated via immunocytochemistry using a chicken‐specific antibody. The expression of chkINA was detected at the early stage of development and increased during the developmental process of the chicken. chkINA was expressed widely in chicken brains and colocalized with NF triplet proteins in neuronal processes, as assessed using immunohistochemistry. We also found that chkINA was expressed abundantly in the developing cerebellum and was the major IF protein in the parallel processes of granule neurons. Thus, we suggest that chkINA is a neuron‐specific IF protein that may be a useful marker for studies of chicken brain development. J. Comp. Neurol. 521:2147–2164, 2013. © 2012 Wiley Periodicals, Inc.     

Basophilic inclusions and neuronal intermediate filament inclusions in amyotrophic lateral sclerosis and frontotemporal lobar degeneration

Basophilic inclusions (BIs) and neuronal intermediate filament inclusions (NIFIs) are key structures of basophilic inclusion body disease and neuronal intermediate filament inclusion disease (NIFID), respectively. BIs are sharply‐defined, oval or crescent neuronal intracytoplasmic inclusions that appear pale blue‐gray in color with HE staining and purple in color with Nissl but are stained poorly with silver impregnation techniques. Immunohistochemically BIs are negative for tau, trans‐activation response DNA 43 (TDP‐43), α‐synuclein, neurofilament (NF) and α‐internexin, positive for p62, and variably ubiquitinated. Noticeably, BIs are consistently fused in sarcoma (FUS) positive. NIFIs are by definition immuno‐positive for class IV IFs including three NF triplet subunit proteins and α‐internexin but negative for tau, TDP‐43, and α‐synuclein. In NIFID cases several types of inclusions have been identified. Among them, hyaline conglomerate‐like inclusions are the only type that meets the above immunohistochemical features of NIFIs. This type of inclusion appears upon HE staining as multilobulated, faintly eosinophilic or pale amphophilic spherical masses with a glassy appearance. These hyaline conglomerates appear strongly argyrophilic, and robustly and consistently immuno‐positive for IFs. In contrast, this type of inclusion shows no or only occasional dot‐like FUS immunoreactivity. Therefore, BIs and NIFIs are distinct from each other in terms of morphological, tinctorial and immunohistochemical features. However, basophilic inclusion body disease (BIBD) and NIFID are difficult to differentiate clinically. Moreover, Pick body‐like inclusions, the predominant type of inclusions seen in NIFID, are considerably similar to the BIs of BIBD in that this type of inclusion is basophilic, poorly argyrophilic, negative for IFs and intensely immuno‐positive for FUS. As BIBD and NIFID share FUS accumulation as the most prominent molecular pathology, whether these two diseases are discrete entities or represent a pathological continuum remains a question to be answered.     

Restricted expression of the neuronal intermediate filament protein plasticin during zebrafish development

In the adult goldfish visual pathway, expression of the neuronal intermediate filament (nIF) protein plasticin is restricted to differentiating retinal ganglion cells (RGCs) at the margin of the retina. Following optic nerve injury, plasticin expression is elevated transiently in all RGCs coincident with the early stages of axon regeneration. These results suggest that plasticin may be expressed throughout the nervous system during the early stages of axonogenesis. To test this hypothesis, we analyzed plasticin expression during zebrafish (Danio rerio) neuronal development. By using immunocytochemistry and in situ hybridization, we found that plasticin is expressed in restricted subsets of early zebrafish neurons. Expression coincides with axon outgrowth in projection neurons that pioneer distinct axon tracts in the embryo. Plasticin is expressed first in trigeminal, Rohon-Beard, and posterior lateral line ganglia neurons, which are among the earliest neurons to initiate axonogenesis in zebrafish. Plasticin is expressed also in reticulospinal neurons and in caudal primary motoneurons. Together, these neurons establish the first behavioral responses in the embryo. Plasticin expression also coincides with initial RGC axonogenesis and progressively decreases after RGC axons reach the tectum. At later developmental stages, plasticin is expressed in a subset of the cranial nerves. The majority of plasticin-positive neurons are within or project axons to the peripheral nervous system. Our results suggest that plasticin subserves the changing requirements for plasticity and stability during axonal outgrowth in neurons that project long axons. J. Comp. Neurol. 399:561–572, 1998. © 1998 Wiley-Liss, Inc.     

Cytoskeletal changes during development and aging in the cortex of neurofilament light protein knockout mice

The neurofilament light (NFL) subunit is considered as an obligate subunit polymer for neuronal intermediate filaments comprising the neurofilament (NF) triplet proteins. We examined cytoskeletal protein levels in the cerebral cortex of NFL knockout (KO) mice at postnatal day 4 (P4), 5 months, and 12 months of age compared with age‐matched wild‐type (WT) mice of a similar genetic background (C57BL/6). The absence of NFL protein resulted in a significant reduction of phosphorylated and dephosphorylated NFs (NF‐P, NF‐DP), the medium NF subunit (NFM), and the intermediate filament α‐internexin (INT) at P4. At 5 months, NF‐DP, NFM, and INT remained significantly lower in knockouts. At 12 months, NF‐P was again significantly decreased, and INT significantly increased, in KOs compared with wild type. In addition, protein levels of class III neuron‐specific β‐tubulin and microtubule‐associated protein 2 were significantly increased in NFL KO mice at P4, 5 months, and 12 months, whereas β‐actin levels were significantly decreased at P4. Immunocytochemical studies demonstrated that NF‐DP accumulated abnormally in the perikarya of cortical neurons by 5 months of age in NFL KO mice. Neurons that lacked NF triplet proteins, such as calretinin‐immunolabeled nonpyramidal cells, showed no alterations in density or cytoarchitectural distribution in NFL KO mice at 5 months relative to WT mice, although calretinin protein levels were decreased significantly after 12 months in NFL KO mice. These findings suggest that a lack of NFL protein alters the expression of cytoskeletal proteins and disrupts other NF subunits, causing intracellular aggregation but not gross structural changes in cortical neurons or cytoarchitecture. The data also indicate that changes in expression of other cytoskeletal proteins may compensate for decreased NFs. J. Comp. Neurol. 521:1817–1827, 2013. © 2012 Wiley Periodicals, Inc.     

Heterogeneous inclusions in neurofilament inclusion disease

Neurofilament inclusion disease (NFID) is a rare disease, whose pathogenesis remains to be elucidated. Immunoreactivity of ubiquitin‐binding protein p62 has been reported in various neurodegenerative diseases, but it has not been studied in NFID. In this report we show p62 immunoreactivity in neuronal perikaryon of three cases of NFID. We found inclusions in NFID to be heterogenous based on immunoreactivity for α‐internexin, phosphorylated neurofilament‐H, p62 and ubiquitin. Moreover, we showed both p62‐ and α‐internexin‐immunoreactive inclusions within the perikarya of the same neuron. Electron microscopy findings support the notion that inclusions in NFID are heterogenous. The present study extends the list of proteins that have been identified as components of neuronal inclusions in NFID, and may help account for the pathogenesis of NFID.     

Genesis of rods in the zebrafish retina occurs in a microenvironment provided by polysialic acid‐expressing Müller glia

Polysialic acid (polySia) is a posttranslational modification of the neural cell adhesion molecule NCAM, which in the vertebrate brain is dynamically regulated during development and crucially involved in developmental and adult neurogenesis. In the fish retina, new neurons are persistently generated, but the possible contribution of polySia has not yet been addressed. Here we used immunohistochemistry with NCAM‐ and polySia‐specific antibodies to study spatiotemporal expression patterns of NCAM and polySia in the developing and mature zebrafish retina. As early as 2.3 days postfertilization (dpf), NCAM but not polySia was detected on cell somata and fibers of the developing retina. At 4.3 dpf polySia immunoreactivity first appeared in the ventral retina and was localized to the nascent outer nuclear layer (ONL). In mature zebrafish, polySia immunoreactivity in the ONL extended to the entire retina. Colocalization with rhodopsin‐EGFP in transgenic zebrafish or the Müller glia‐specific protein cellular retinaldehyde‐binding protein (CRALBP) revealed that polySia immunoreactivity was confined to the compartment of radial Müller glia processes crossing the ONL and to a small band of processes positioned proximal to the horizontal cell layer of the mature retina. As shown by 5‐bromo‐2‐deoxyuridine (BrdU) labeling, both newly generated rod precursors within the mature ONL and precursors of the marginal zone were polySia‐negative. Thus, polySia‐negative rod precursors of the mature zebrafish retina face a polySia‐NCAM‐positive microenvironment presented by radial Müller glia. In view of the prominent role of polySia in other neurogenic systems, this pattern indicates that polySia provides environmental cues that are relevant for the generation of new rods. J. Comp. Neurol. 518:636–646, 2010. © 2009 Wiley‐Liss, Inc.     

Increased expression of multiple neurofilament mRNAs during regeneration of vertebrate central nervous system axons

Characteristic changes in the expression of neuronal intermediate filaments (nIFs), an abundant cytoskeletal component of vertebrate axons, accompany successful axon regeneration. In mammalian regenerating PNS, expression of nIFs that are characteristic of mature neurons becomes suppressed throughout regeneration, whereas that of peripherin, which is abundant in developing axons, increases. Comparable changes are absent from mammalian injured CNS; but in goldfish and lamprey CNS, expression of several nIFs increases during axon regrowth. To obtain a broader view of the nIF response of successfully regenerating vertebrate CNS, in situ hybridization and video densitometry were used to track multiple nIF mRNAs during optic axon regeneration in Xenopus laevis. As in other successfully regenerating systems, peripherin expression increased rapidly after injury and expression of those nIFs characteristic of mature retinal ganglion cells decreased. Unlike the decrease in nIF mRNAs of regenerating PNS, that of Xenopus retinal ganglion cells was transient, with most nIF mRNAs increasing above normal during axon regrowth. At the peak of regeneration, increases in each nIF mRNA resulted in a doubling of the total amount of nIF mRNA, as well as a shift in the relative proportions contributed by each nIF. The relative proportions of peripherin and NF-M increased above normal, whereas proportions of xefiltin and NF-L decreased and that of XNIF remained the same. The increases in peripherin and NF-M mRNAs were accompanied by increases in protein. These results are consistent with the hypothesis that successful axon regeneration involves changes in nIF subunit composition conducive to growth and argue that a successful injury response differs between CNS and PNS.     

Associations between intermediate filament proteins expressed in cultured dorsal root ganglion neurons

The developmental profile of the neurofilament (NF) triplet proteins, α-internexin and peripherin in cultured dorsal root ganglion neurons from gestation day 15 rat embryos was determined by Western blot analysis. At the outset (day 0 in culture), the neurons contained mostly α-internexin. A significant increase in peripherin levels was seen at days 1–2, in the mid-sized (NFM) and low molecular weight (NFL) NF subunits at days 2–3, and in the high molecular weight (NFH) NF subunit at days 5–6. Immunofluorescence microscopy showed that the five intermediate filament proteins were co-localized in all neuronal cell bodies and neurites. Analysis of Triton X-100 extracts from okadaic acid-treated dorsal root ganglion cultures revealed that peripherin and α-internexin followed the same fragmentation pattern observed with NFs. Interactions between the various neuronal intermediate filament proteins in these extracts were assessed by immunoprecipitation under native conditions using antibodies specific for the individual proteins. Co-immunoprecipitation of NFH with NFL, NFM with NFL, NFM with α-internexin, and α-internexin with peripherin demonstrated that the intermediate filament cytoskeleton in cultured sensory neurons is a highly integrated structure. J. Neurosci. Res. 47:300–310, 1997. © 1997 Wiley-Liss, Inc.     

Pten associates with important gene regulatory network to fine-tune Müller glia-mediated zebrafish retina regeneration

Unlike mammals, zebrafish possess a remarkable ability to regenerate damaged retina after an acute injury. Retina regeneration in zebrafish involves the induction of Müller glia-derived progenitor cells (MGPCs) exhibiting stem cell-like characteristics, which are capable of restoring all retinal cell-types. The induction of MGPC through Müller glia-reprograming involves several cellular, genetic and biochemical events soon after a retinal injury. Despite the knowledge on the importance of Phosphatase and tensin homolog (Pten), which is a dual-specificity phosphatase and tumor suppressor in the maintaining of cellular homeostasis, its importance during retina regeneration remains unknown. Here, we explored the importance of Pten during zebrafish retina regeneration. The Pten gets downregulated upon retinal injury and is absent from the MGPCs, which is essential to trigger Akt-mediated cellular proliferation essential for retina regeneration. We found that the downregulation of Pten in the post-injury retina accelerates MGPCs formation, while its overexpression restricts the regenerative response. We observed that Pten regulates the proliferation of MGPCs not only through Akt pathway but also by Mmp9/Notch signaling. Mmp9-activity is essential to induce the proliferation of MGPCs in the absence of Pten. Lastly, we show that expression of Pten is fine-tuned through Mycb/histone deacetylase1 and Tgf-β signaling. The present study emphasizes on the stringent regulation of Pten and its crucial involvement during the zebrafish retina regeneration.     

alpha-Internexin immunoreactivity reflects variable neuronal vulnerability in Alzheimer's disease and supports the role of the beta-amyloid plaques in inducing neuronal injury

This study investigated the role of alpha-internexin in the neuronal alterations associated with beta-amyloid plaque formation in Alzheimer's disease (AD). Cortical neurons could be defined by their variable content of neurofilament (NF) triplet and alpha-internexin proteins, with a distinct population of supragranular pyramidal cells containing alpha-internexin alone. Both NF triplet and alpha-internexin were localized to reactive axonal structures in physically damaged neurons in experimental trauma models. Similarly, NF triplet and alpha-internexin immunoreactive neurites were localized to plaques densely packed with beta-amyloid fibrils in preclinical AD cases, indicating that certain plaques may cause structural injury or impediment of local axonal transport. However, alpha-internexin, and not NF triplet, ring-like reactive neurites were present in end-stage AD cases, indicating the relatively late involvement of neurons that selectively contain alpha-internexin. These results implicate the expression of specific intermediate filament proteins in a distinct hierarchy of differential neuronal vulnerability to AD.     

Internexin, MAP1B, and nestin in cortical dysplasia as markers of developmental maturity

Cortical dysplasias (CD) are characterized histologically by disorganized cortical lamination and abnormally shaped neurons. We hypothesized that neurons within CD have failed to differentiate fully and may express proteins such as cytoskeletal elements characteristic of immature cells. Disrupted expression of certain cytoskeletal proteins, which have been implicated in neuronal polarity, process outgrowth, and migration, could result in disorganized cortical lamination. Thus, we probed two CD subtypes, focal CD (FCD) and hemimegalencephaly (HME), with antibodies specific for cytoskeletal proteins that are developmentally regulated in neural progenitor cells and neurons to define more fully the developmental phenotype of neurons within CD. Microtubule-associated protein 1B (MAP1B) and the intermediate filament (IF) protein nestin are enriched in neural progenitors, whereas MAP2B, phosphorylated and non-phosphorylated forms of medium (NFM) and high (NFH) molecular weight neurofilament (NF) proteins, as well as the light NF subunit (NFL) and the IF protein α internexin are expressed in developing and mature neurons. Immunolabeling for internexin and MAP1B was more abundant in the most abnormally shaped neurons that populated dysplastic regions than in adjacent regions exhibiting milder cytoarchitectural abnormalities or control cortex. Nestin immunoreactivity was noted in large dysplastic and heterotopic neurons within the deeper cortical layers of CD specimens but not in normal cortex. In contrast, neurons in CD specimens also expressed cytoskeletal markers characteristic of differentiated neurons such as NF subunits and MAP2B. These findings suggest that the cytoarchitectural abnormalities in CD may reflect pathophysiological changes in the developing brain that disrupt expression of several key components of the neuronal cytoskeleton and may contribute to impaired migration of cortical neurons. Received: 19 August 1996 / Revised, accepted: 19 November 1996     

Investigation of barrier characteristics in the hyaloid-retinal vessel of zebrafish

The blood-retinal barrier (BRB) is essential for the physiological integrity of the retinal vessels. In particular, ocular pathologies of retinal neovascularization could be causally related to the BRB breakdown. Zebrafish have emerged as an advantageous model for studying vascular development and characteristics. Here we investigated for the first time the barrier characteristics of the hyaloid-retinal vessel using fli1-EGFP transgenic zebrafish. By 7 dpf, the hyaloid-retinal vessel was formed between lens and retina, where intercellular junctional complexes were already present between endothelial cells. Interestingly, NG-2 expression, but not GFAP, was colocalized with EGFP-positive cells of the hyaloid-retinal vessel. Among endothelial tight junction proteins, claudin-5 was expressed on EGFP-positive cells of the hyaloid-retinal vessel, whereas occludin and ZO-1 were not observed on the vessel. In addition, the hyaloid-retinal vessel was so leaky that a mixture of fluorescein tracers (2,000-kDa FITC-dextran, 10-kDa rhodamine-dextran, and 350-Da DAPI) diffusely infiltrated into all retinal layers. Our results suggest that, unlike retinal vessels of higher vertebrates, the hyaloid-retinal vessel of zebrafish shows insufficient characteristics to meet a functional endothelium-based CNS barrier. Therefore, it might be not suitable to use the hyaloid-retinal vessel of zebrafish for studying BRB biogenesis.     

Constant dark-rearing effects on visual adaptation of the zebrafish ERG.

Anatomical and electrophysiological studies have shown that zebrafish retinal neurons develop in a sequential manner. Several studies have examined the impact of restricted rearing environments on zebrafish visual development, but the results have been mixed. The purpose of this study was to examine the development of light adaptation properties of the zebrafish electroretinogram (ERG), and examine the effects of constant dark rearing on retinal development. Subjects were zebrafish, Danio rerio, reared under normal lighting conditions and zebrafish reared in constant dark from fertilization to 6 days postfertilization (dpf). Increment threshold functions were obtained from a- and b-wave ERG responses from normally reared subjects at different ages and from animals exposed to early constant dark rearing. Dark-reared subjects were tested immediately following constant dark exposure (6-9 dpf) and after exposure to normal cyclic lighting (11-13 dpf). Adult zebrafish were significantly more sensitive at lower background illuminations than were larvae zebrafish. Also, constant dark rearing had a differential effect on the a- and b-wave response measures. Constant dark rearing raised b-wave threshold uniformly across background illuminations, while only producing higher a-wave thresholds at low levels of illumination. These results are consistent with findings in studies on zebrafish retinal development, and may help explain some of the discrepancies across studies examining the effects of restricted rearing.     

R-cadherin expression in the developing and adult zebrafish visual system.

Cell adhesion molecules in the cadherin family have been implicated in histogenesis and maintenance of cellular structure and function in several organs. Zebrafish have emerged as an important new developmental model, but only three zebrafish cadherin molecules have been identified to date (N-cadherin, paraxial protocadherin, and VN-cadherin). We began a systematic study to identify other zebrafish cadherins by screening zebrafish cDNA libraries using an antibody raised to the cytoplasmic domain of mouse E-cadherin. Here, we report a partial cDNA with extensive sequence homology to R-cadherin. Spatial and temporal expression of this putative zebrafish R-cadherin was examined in embryos and adults by Northern analysis, RNase protection, and in situ hybridization. R-cadherin message increased during embryogenesis up to 80 hours postfertilization (hpf) and persisted in adults. In the embryonic brain, R-cadherin was first expressed in groups of cells in the diencephalon and pretectum. In adult zebrafish brain, R-cadherin continued to be expressed in several specific regions including primary visual targets. In the retina, R-cadherin was first detected at about 33 hours postfertilization in the retinal ganglion cell layer and the inner part of the inner nuclear layer. Expression levels were highest during periods of axon outgrowth and synaptogenesis. Retrograde labeling of the optic nerve with 1,1'-dioctadecyl-3,3,3',3', tetramethylindocarbocyanine perchlorate (DiI) followed by in situ hybridization confirmed that a subset of retinal ganglion cells in the embryo expressed R-cadherin message. In the adult, R-cadherin expression continued in a subpopulation of retinal ganglion cells. These results suggest that R-cadherin-mediated adhesion plays a role in development and maintenance of neuronal connections in zebrafish visual system.     

Zebrafish Cx35: cloning and characterization of a gap junction gene highly expressed in the retina

The vertebrate connexin gene family encodes protein subunits of gap junction channels, which provide a route for direct intercellular communication. Consequently, gap junctions play a vital role in many developmental and homeostatic processes. Aberrant functioning of gap junctions is implicated in many human diseases. Zebrafish are an ideal vertebrate model to study development of the visual system as they produce transparent embryos that develop rapidly, thereby facilitating morphological and behavioral testing. In this study, zebrafish connexin35 has been cloned from a P1 artificial chromosome (PAC) library. Sequence analysis shows a high degree of similarity to the Cx35/36 orthologous group, which are expressed primarily in nervous tissue, including the retina. The gene encodes a 304-amino acid protein with a predicted molecular weight of approximately 35 kDa. Injection of zebrafish Cx35 RNA into paired Xenopus oocytes elicited intercellular electrical coupling with weak voltage sensitivity. In development, Cx35 is first detectable by Northern analysis and RT-PCR, at 2 days post-fertilization (2 dpf), and in the adult it is expressed in the brain and retina. Immunohistochemical analysis revealed that the Cx35 protein is expressed in two sublaminae of the inner plexiform layer of the adult retina. A similar pattern was seen in the 4 and 5 dpf retina, but no labeling was detected in the retina of earlier embryos.     

Lysosomal trafficking defects link Parkinson's disease with Gaucher's disease

Lysosomal dysfunction has been implicated in multiple diseases, including lysosomal storage disorders such as Gaucher's disease, in which loss‐of‐function mutations in the GBA1 gene encoding the lysosomal hydrolase β‐glucocerebrosidase result in lipid substrate accumulation. In Parkinson's disease, α‐synuclein accumulates in Lewy bodies and neurites contributing to neuronal death. Previous clinical and genetic evidence has demonstrated an important link between Parkinson's and Gaucher's disease, as GBA1 mutations and variants increase the risk of Parkinson's and Parkinson's patients exhibit decreased β‐glucocerebrosidase activity. Using human midbrain neuron cultures, we have found that loss of β‐glucocerebrosidase activity promotes α‐synuclein accumulation and toxicity, whereas α‐synuclein accumulation further contributes to decreased lysosomal β‐glucocerebrosidase activity by disrupting β‐glucocerebrosidase trafficking to lysosomes. Moreover, α‐synuclein accumulation disrupts trafficking of additional lysosomal hydrolases, further contributing to lysosomal dysfunction and neuronal dyshomeostasis. Importantly, promoting β‐glucocerebrosidase activity reduces α‐synuclein accumulation and rescues lysosomal and neuronal dysfunction, suggesting that β‐glucocerebrosidase may be an important therapeutic target for advancing drug discovery in synucleinopathies including Parkinson's disease. © 2016 International Parkinson and Movement Disorder Society.     

Distribution of specific intermediate-filament proteins in the goldfish retina

The intermediate-filament proteins expressed in the goldfish retina were investigated by immunohistochemistry and by immunoblotting. Polyclonal antibodies that previously had been raised against the goldfish optic nerve neurofilament (ON1 and ON2) and glial filament (ON3 and ON4) proteins were used in this study. Anti-ON1/ON2 antiserum reacted on a retinal immunoblot with two proteins having molecular weights and isoelectric points corresponding to those of ON1 and ON2. Histologically, the most pronounced anti-ON1/ON2 reactivity was observed in the ganglion cell layer of the goldfish retina. The anti-ON3/ON4 antiserum reacted with a single protein on a retinal immunoblot. This protein had a molecular weight and isoelectric point which corresponded to the goldfish optic nerve glial filament proteins. This anti-serum labeled horizontal cells in retina sections. Three previously unidentified goldfish visual-pathway intermediate-filament proteins sharing a molecular weight of 60K were observed on two-dimensional gels of retinal cytoskeletal proteins and on retinal immunoblots which were probed with a monoclonal antibody which recognizes an epitope common to all intermediate filament proteins. The possible existence of homologs of mammalian GFAP and vimentin in the goldfish retina was also explored. Antibodies directed against mammalian GFAP and vimentin labeled the Müller fibers and the cone horizontal cells, respectively. However, immunoblot analysis and a comparison of the two-dimensional gel electrophoresis patterns of goldfish retinal and rat spinal cord cytoskeletal proteins demonstrated a lack of goldfish proteins identical to the mammalian intermediate-filament proteins.