Localization of phosphotyrosine adaptor protein ShcD/SHC4 in the adult rat central nervous system

Background Mammalian Shc (Src homology and collagen) proteins comprise a family of four phosphotyrosine adaptor molecules which exhibit varied spatiotemporal expression and signaling functions. ShcD is the most recently discovered homologue and it is highly expressed in the developing central nervous system (CNS) and adult brain. Presently however, its localization within specific cell types of mature neural structures has yet to be characterized. Results In the current study, we examine the expression profile of ShcD in the adult rat CNS using immunohistochemistry, and compare with those of the neuronally enriched ShcB and ShcC proteins. ShcD shows relatively widespread distribution in the adult brain and spinal cord, with prominent levels of staining throughout the olfactory bulb, as well as in sub-structures of the cerebellum and hippocampus, including the subgranular zone. Co-localization studies confirm the expression of ShcD in mature neurons and progenitor cells. ShcD immunoreactivity is primarily localized to axons and somata, consistent with the function of ShcD as a cytoplasmic adaptor. Regional differences in expression are observed among neural Shc proteins, with ShcC predominating in the hippocampus, cerebellum, and some fiber tracts. Interestingly, ShcD is uniquely expressed in the olfactory nerve layer and in glomeruli of the main olfactory bulb. Conclusions Together our findings suggest that ShcD may provide a distinct signaling contribution within the olfactory system, and that overlapping expression of ShcD with other Shc proteins may allow compensatory functions in the brain.


Background
The Shc (Src homology and collagen) family of adaptor molecules plays an important role in mediating signaling events that link activated cell surface proteins such as receptor tyrosine kinases (RTKs) to intracellular signaling pathways [1]. Shc proteins are characterized by the presence of an amino-terminal phosphotyrosine binding (PTB) domain, a central collagen homology 1 (CH1) region which contains a series of phosphorylatable tyrosine residues, and a carboxy-terminal Src homology 2 (SH2) domain. The interaction capabilities of these domains position Shc proteins within a variety of signaling complexes that regulate processes such as cellular differentiation, proliferation, and migration [1,2].
In addition to sequence and structure divergence, members of the Shc family differ in their spatiotemporal expression. During brain development, ShcA is found within dividing neural stem/progenitor cells (NPCs), though this expression declines over time such that at maturity, it is primarily expressed outside of the central nervous system (CNS) [6,10]. In contrast, ShcB and ShcC are largely restricted to the CNS, and expressed in the mature adult brain [4][5][6]11], with ShcC gradually replacing ShcA as NPCs progress towards a postmitotic phenotype [12]. While much less is known about ShcD expression, it has been detected in multiple sub-regions of the adult mouse brain [8], in skin and melanocytes [7], and in the neuromuscular junction where it signals with the MuSK RTK [8]. In the developing mouse embryo, ShcD is present throughout the CNS, as well as in skeletal and cardiac muscle, epithelia of several organs, and multiple neural crest-derived tissues [13]. Despite the prominent expression of ShcD in the CNS, its precise distribution and cellular localization therein has yet to be determined. In this report, we have used immunohistochemistry and double staining approaches to examine the pattern of ShcD expression in the adult rat brain and spinal cord, and compared this profile with those of the neuronally enriched ShcB and ShcC proteins.

Cellular distribution of ShcD in the adult rat brain
To examine the neural localization of Shc proteins, sections were prepared from adult rat brain and stained using ShcB, ShcC or ShcD-specific antibodies which we have previously validated for immunohistochemistry [13]. ShcD distribution in the mature brain appears relatively widespread, with somata and dendrites of most principal cells displaying ShcD immunoreactivity (Fig. 1). ShcD staining is most prominent within the olfactory nerve layer, where axons of the olfactory sensory neurons travel en route from the nasal mucosa to the olfactory bulb before synapsing in the glomeruli. Increased expression is also detected in specific subregions of the cerebellum and hippocampus, as well as in the subventricular zone (SVZ). By contrast, ShcD staining is rather diffuse in axons of many fiber tracts. The immunostaining patterns observed for ShcB and ShcC were highly similar to those reported previously on rat brain tissue [11,12] and are compared in detail with ShcD below.

Cellular distribution of ShcD compared with ShcB and ShcC in the olfactory bulb
We next profiled expression of the three neural Shc proteins in specific anterior to posterior structures. ShcD shows the highest immunoreactivity in axons and terminals of the glomerular and olfactory nerve layers ( Fig. 2A, B and E). It can also be seen within all other layers of the olfactory bulb, including in distinct cell bodies of the mitral cell layer and central granule cell layer, and in external tufted cells between the external plexiform layer and the glomerular layer ( Fig. 2A, B and E). Faint diffuse staining of ShcB (Fig. 2C, F) is present throughout the olfactory bulb, although no specific immunoreactivity is found in the glomeruli or olfactory nerve layer. ShcC can be observed in the somata and dendrites of interneurons Fig. 1 Immunolocalization of ShcD in the adult rat brain. ShcD is broadly distributed throughout the brain, with intense staining in the olfactory bulb (ob), as well as regions of the hippocampus (hip) and cerebellum (med). Parasagittal section of an 8-week old male Sprague-Dawley rat is shown. No staining was apparent when the primary antibody was omitted from the reaction. Superior and inferior cerebellar peduncle (scp, icp), corticospinal tract (cst), longitudinal fasciculus of the pons (lfp), lateral ventricle (denoted by *), subventricular zone (svz; arrow), cortex (ctx), intrabulbar (ica) and anterior limb (aca) of the anterior commissure (ac), olfactory nerve layer (onl), glomerular layer (glm). Scale bar = 2 mm located in the granule cell layer (Fig. 2D) and periglomerular region (Fig. 2G), as well as in mitral cells (Fig. 2D).

Cellular distribution of ShcD compared with ShcB and ShcC in the cerebral cortex, hippocampus and dentate gyrus
Within the neocortex and hippocampus, Shc proteins display different intensities of staining, with ShcC appearing most darkly stained in these regions, followed closely by ShcD, with only faint staining for ShcB. In the neocortex, ShcB, ShcC and ShcD immunoreactivity is present within somata and dendrites of the large pyramidal neurons in layer V (internal pyramidal layer or ganglion cell layer) ( Fig. 3A-C). Staining is prominent in the cytoplasm of these cells, consistent with the role of Shc proteins as intracellular adaptors. In the hippocampus proper, Shc staining can be detected in pyramidal cells of CA1 and CA3 areas as well as in fibers of the stratum radiatum ( Fig. 3D-F). In the dentate gyrus, both ShcC and ShcD (but not ShcB) are readily detected in the granule cell layer ( Fig. 3G-I); however, ShcD appears to be more intensely stained in the sparsely scattered neurons within the hilus, and even further enriched in distinct cells comprising the subgranular zone (SGZ) (Fig. 3G).

Cellular distribution of ShcD compared with ShcB and ShcC in the cerebellum
In the cerebellar cortex, all Shc proteins are present, though they exhibit distinct staining patterns across the three layers. ShcD immunoreactivity is weak but detectable within the inner granule cell layer, the central Purkinje cell layer and interneurons of the outer molecular layer ( Fig. 4A). ShcC immunoreactivity is similarly apparent throughout all three layers of the cerebellum, though the staining is much darker than that of ShcD, particularly in Purkinje cell somata, apical dendrites and associated spines (Fig. 4C). By contrast, only Purkinje cell somata and dendrites show ShcB staining (Fig. 4B).

Cellular distribution of ShcD compared with ShcB and ShcC in the brainstem
In the brainstem, differential staining of Shc proteins can again be observed, with intense ShcC reactivity, as well as robust levels of ShcD which are comparable to ShcB

Cellular distribution of ShcD compared with ShcB and ShcC in the spinal cord
Within the spinal cord, ShcD distribution appeared widespread (   neurons was diffuse compared to the intense ShcB and ShcC labeling of ventral motor neurons and surrounding neuropil (Fig. 6D-F). Similarly, both ShcB and ShcC but not ShcD could be observed in the ventral motor nerves (Fig. 6G-I). High ShcD immunoreactivity is present within the axon tracts within the white matter (Fig. 6A).

Cellular distribution of ShcD compared with ShcB and ShcC in the trigeminal ganglion
In the trigeminal ganglion, prominent staining for all Shc proteins can be observed within the cell bodies of sensory neuron clusters ( Fig. 7A-C) as well as in axons ( Fig. 7D-F), although the labeling patterns are quite variable. Notably, subpopulations of cells within the ganglion are immunonegative for ShcB and ShcC, and a distinctive ring devoid of immunoreactivity was observed surrounding ShcB and C positive cells, but not around ShcD positive cells (Fig. 7B, C). Furthermore, ShcC appears to localize within discrete puncta which are particularly evident along the axon (Fig. 7I), while ShcB and ShcD remain diffuse (Fig. 7G, H).

Localization of ShcD within neurons and neural progenitor cells
Finally, to precisely determine the identity of cells in the adult nervous system which express ShcD, we performed double staining of ShcD with cell type-specific markers. Dual staining of ShcD with neuronal marker NeuN in the cortex revealed that ShcD is present in mature neurons (Fig. 8A). Additionally, expression of ShcD in neural progenitors was assessed using NPC marker Nestin. Near adjacent coronal sections of the SVZ stained for ShcD or Nestin demonstrate overlapping fluorescent localization patterns (Fig. 8B), and prominent ShcD expression in cells lining the lateral ventricle. Co-staining of ShcD with Nestin in the SGZ of the dentate gyrus supports more directly the possibility of ShcD expression in NPCs (Fig. 8C).

Discussion
The present immunohistochemical study confirms the widespread presence of ShcB and ShcC in adult neurons, and outlines anatomic and cell type-specific ShcD distribution within the adult CNS for the first time. These expression profiles are summarized in Table 1. In some neuronal populations, all three Shc proteins coexist, while others show restricted expression of only one or two Shc proteins. Cytoplasmic localization was most commonly observed for all Shc proteins. Intriguingly, we uncovered distinct differences in the regional distribution of ShcB, ShcC and ShcD, discussed below, which highlights the complexity of these important neural adaptor proteins.

ShcB, ShcC and ShcD show overlapping expression in motor and sensory neurons
Most of the examined regions showed overlapping expression of all three Shc proteins. Along with ShcD, ShcB and ShcC were present in the adult brainstem and spinal cord, consistent with previous reports [11], and intense staining for all proteins was particularly evident in motor neurons. The nerves in the brainstem show minimal staining for all three neural Shc proteins, while the motor nerves of the spinal cord showed little to no ShcD staining and much less intense ShcB staining. Despite their prominent expression, there have been no reported functions for Shc proteins in motor neurons, though expression of ShcC has been shown to decline upon nerve injury [14]. Similarly, all Shc proteins were found within cell bodies and axons of subsets of sensory neurons in the trigeminal ganglion. ShcB and ShcC are reportedly excluded from supporting glial cells in sensory ganglia [11,12], and although we were unable to specifically discern glial cells in this study, ShcD transcripts are readily detected in a subtype of multipotent glial cells known as oligodendrocyte precursor cells [15]. Interestingly, ShcC staining in sensory neurons was enriched in puncta around the nucleus and along axons, as reported previously in other neuron subtypes [12]. This punctate pattern might reflect the presence of signaling endosomes that perpetuate Trk signaling from the axon to the cell body through retrograde transport [16]. Indeed, ShcC can associate with Trk RTKs [17,18], and other Shc molecules have been associated with regulation of RTK endocytosis [9,19]. Functionally, ShcB and ShcC have been implicated in survival of subpopulations of post-mitotic neurons in other ganglia of the mammalian nervous system, though no overt phenotypes were reported [20]. Notably, ShcD is highly expressed during embryogenesis in both dorsal root and trigeminal ganglia [13], thus it is possible that ShcD could compensate for loss of ShcB and/or ShcC in these and other structures.

ShcC and ShcD predominate in large projection neurons and interneurons
Neural Shc protein expression was particularly evident in large projection neurons in the cerebellum and hippocampus, although the staining patterns differed for the three proteins. In the cerebellum, all Shc proteins were present in Purkinje cells, ranging in intensity from ShcC (highest), to ShcB, then ShcD (lowest). ShcB staining was restricted to this cell layer, while ShcC and ShcD were also found in the granule cell and molecular layers. The functional role for Shc proteins in this area of the nervous system remains to be determined. Similarly, in the hippocampus, all Shc proteins were present in pyramidal neurons, as well as in smaller-sized neurons, although ShcB was overall barely detectable compared to ShcC and ShcD. ShcC was highly enriched in granule cells of the dentate gyrus, consistent with its involvement in hippocampal long-term potentiation [21]. By contrast, ShcD staining was markedly intense in cells dispersed throughout the SGZ and hilus of the dentate gyrus, including Nestin-positive NPCs. The dentate gyrus is one of three neurogenic niches in the adult brain, and the SGZ represents the source of granule cell precursors [22]. NPCs differentiate in the SGZ, then migrate and integrate into the granule cell layer [23,24]. Although the physiological function of ShcD is presently unknown, ShcD is upregulated in the early phase of neural differentiation of embryonic stem cells in vitro [25], and it can also promote migration of melanoma cells [7]. It will thus be of future interest to determine whether ShcD influences cellular processes linked to neurogenesis.

ShcD is uniquely expressed throughout the olfactory bulb
The most overt differences in Shc protein expression occur in the olfactory bulb. Here, intense ShcD staining was observed in the glomerular and olfactory nerve layers, as well as in projection neurons (mitral cells and tufted cells) and interneurons (granule cells). We were unable to confirm ShcD expression in periglomerular interneurons due to the intense overall ShcD staining in glomeruli. Consistent with previous reports [11], ShcB was virtually undetectable across the olfactory bulb, while ShcC was found in mitral cells, granule cells, and periglomerular cells. The olfactory bulb is responsible for processing olfactory inputs, and it also represents a region of new neuron incorporation as the terminal site for NPCs born in the SVZ and olfactory epithelium (OE) neurogenic niches [26][27][28][29]. Consistent with their expression patterns, Shc proteins have been previously implicated in aspects of NPC signaling. ShcC is broadly detected in interneurons, where it promotes survival and maturation of neural progenitors [12], while ShcA selectively controls NPC proliferation in the adult SVZ [30]. These opposing roles for ShcA and ShcC in neurogenesis may reflect differences in their signaling properties, including the reduced ability of ShcC to activate Erk compared to ShcA [31]. Intriguingly, ShcD is expressed similarly to ShcA in the SVZ, though ShcD demonstrates additional localization to cells lining the lateral ventricle. In the olfactory system, ShcD is found in an overlapping pattern with ShcC in the main olfactory bulb, and uniquely in the OE and glomeruli, and it has a distinct function in suppressing Erk activation downstream of TrkB [32]. Given the established role of TrkB in migration [33] and differentiation [34] of NPCs, along with its high expression in OE-derived NPCs [35], it is tempting to speculate that ShcD could play a novel role in growth factor-mediated signaling in NPCs.

Conclusions
In closing, the Shc family has evolved from a single locus in less complex organisms to four distinct loci (ShcA-D) in mammals [1,36]. While these related adaptors can exhibit compensatory actions, our findings extend the notion that differences in expression patterns and signaling properties likely promote non-redundant functions of Shc proteins within the adult mammalian CNS.

Animals
Adult male Sprague-Dawley rats (200-300 g; Charles River, QC) were housed under standard conditions with free access to food and water. A total of ten rats were used in this study; 5 rats were used for IHC, three rats were used for IF, and two rats were used in antibody control experiments. 8 weeks old rats were used in all figures, except for Fig. 8 where 12-14 weeks old rats were used. Animals were euthanized by intraperitoneal sodium pentobarbital injection (120 mg/kg) or CO 2 inhalation (plexiglass chamber with a flow rate of 6.0 L/min (30% air volume) for at least 5 min) followed by decapitation. All procedures were carried out in accordance with the guidelines established by the Canadian Council on Animal Care, and approved by the University of Guelph Animal Care Committee (Guelph, ON) under animal utilization protocol #06006R.

Table 1 Summary of Shc expression patterns in the rat central nervous system
Expression levels were attributed as follows: -no expression; + low expression; ++ moderate expression; +++ high expression a As per Ponti et al. [11] ShcB ShcC ShcD  1000) was generated against the unique CH2 region. Its specificity has been confirmed by preadsorption with the original antigen prior to immunohistochemistry [8,13], by immunoblot against lysates prepared from cells expressing other Shc proteins [8], and by immunoblot on brain lysates prepared from ShcD knockout mice [32].

Immunoperoxidase histochemistry
Animals were deeply anaesthetized with sodium pentobarbital (120 mg/kg) and slowly perfused through the ascending aorta with 60 mL of ice cold, artificial cerebrospinal fluid (85 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate monobasic, 25 mM NaHCO 3 , 25 mM glucose, 75 mM sucrose, 0.5 mM CaCl 2 , 4 mM MgCl 2 , 100 µM kynurenic acid), saturated with 95% O 2 /5% CO 2 . This solution has been shown previously to aid in preserving the integrity of cell membranes in the CNS (adapted from [37]). Following washout of the blood, animals were slowly perfused with 60 mL of an ice cold solution containing 2% paraformaldehyde (PFA) in 100 mM phosphate buffer (pH 6.5), followed by 120 mL of 2% PFA in 100 mM phosphate buffer (pH 8.5) (adapted from the method described by [38]). The brains and spinal cords were then removed from the skull and post-fixed overnight in 2% PFA in 100 mM phosphate buffer (pH 8.5) at 4 °C, sectioned in the coronal or parasagittal plane at 50 μm on a vibrating microtome (Vibratome 3000 Plus) and stored in Millonig's buffer (pH 7.4) containing 0.04% sodium azide. Sections were processed according to the method described by [38]. Briefly, free-floating sections were placed into 6-well culture plates (