Regulator of G protein signaling 14 (RGS14) is expressed pre- and postsynaptically in neurons of hippocampus, basal ganglia, and amygdala of monkey and human brain

Abstract

Regulator of G protein signaling 14 (RGS14) is a multifunctional signaling protein primarily expressed in mouse pyramidal neurons of hippocampal area CA2 where it regulates synaptic plasticity important for learning and memory. However, very little is known about RGS14 protein expression in the primate brain. Here, we validate the specificity of a new polyclonal RGS14 antibody that recognizes not only full-length RGS14 protein in primate, but also lower molecular weight forms of RGS14 protein matching previously predicted human splice variants. These putative RGS14 variants along with full-length RGS14 are expressed in the primate striatum. By contrast, only full-length RGS14 is expressed in hippocampus, and shorter variants are completely absent in rodent brain. We report that RGS14 protein immunoreactivity is found both pre- and postsynaptically in multiple neuron populations throughout hippocampal area CA1 and CA2, caudate nucleus, putamen, globus pallidus, substantia nigra, and amygdala in adult rhesus monkeys. A similar cellular expression pattern of RGS14 in the monkey striatum and hippocampus was further confirmed in humans. Our electron microscopy data show for the first time that RGS14 immunostaining localizes within nuclei of striatal neurons in monkeys. Taken together, these findings suggest new pre- and postsynaptic regulatory functions of RGS14 and RGS14 variants, specific to the primate brain, and provide evidence for unconventional roles of RGS14 in the nuclei of striatal neurons potentially important for human neurophysiology and disease.

Keywords: Amygdala; Basal ganglia; Hippocampus; Human; Nucleus; RGS14.

Figure 1. Polyclonal RGS14 antibody recognizes epitopes within the carboxy-terminal half of the protein

(A) Full-length and truncated forms of FLAG-RGS14 are listed, and their corresponding protein sequences are depicted by cartoon. The human immunogen used to generate this polyclonal antibody is outlined by the dashed box. (B) Constructs in A were cloned into pcDNA3.1, expressed in HEK 293 cells and immunoblotted for RGS14 (left) or FLAG (right). Full-length RGS14 is recognized by the antibody (lane 1). While the front half of the protein (lanes 2–3) is not recognized by the antibody, the back half of RGS14 (lanes 4–6) is, indicating that the epitope(s) are located within the C-terminal end of the protein (from R1 domain to the GPR motif). Expression of each construct is verified by immunoblotting for the FLAG tag, which is universal to all the constructs (right). The epitopes recognized by the polyclonal antibody match the immunogen depicted by the dashed box in (A).

Figure 2. RGS14 antibody specifically recognizes RGS14, but not other RGS proteins

Lysates from hippocampus or HEK 293 cells transfected with RGS2, 4, 10, 12, 14, or 16 were immunoblotted for recognition by the RGS14 antibody or their respective tag. Lane 1: The RGS14 antibody recognizes a band corresponding to full-length RGS14 in the mouse hippocampus. Lanes 2–7: FLAG-RGS14, FLAG-RGS10, GFP-RGS12 trans-spliced (TS), RGS2-HA, RGS4-HA, and RGS16-HA were immunoblotted for RGS14 (top), or their respective tags (bottom). The RGS14 antibody recognized full-length, recombinant FLAG-RGS14 (lane 2), but not the other RGS (lanes 3–7), including RGS10 and RGS12, which are in the same R12 family as RGS14 and thus have a closely related sequence. Immunoblotting for each respective tag confirmed expression of each construct.

Figure 3. RGS14 antibody recognizes endogenous RGS14 in the rodent and primate brain

Hippocampal (Hp) and Striatal (Str) punches taken from an adult wild type mouse and a rhesus monkey were lysed and processed through the Bradford assay to quantify protein concentration. The striatal punches in the monkey were taken from the caudate nucleus (CD). Fifty micrograms of protein was loaded into each lane and separated on an 11% acrylamide gel. (A) RGS14 was detected by immunoblotting with 1:1000 diluted RGS14 antibody. Mouse hippocampal and striatal lysates contain full-length RGS14. Monkey hippocampal lysate contains full-length RGS14, and caudate lysate contains full-length RGS14 and three putative splice variants. The putative splice variants match predicted molecular weights: isoform 1 (canonical/full-length; UniProt O43566-7; GenBank EAW85012.1); isoform 2 (UniProt O43566-4; GenBank AAM12650.1); isoform 3 (UniProt O43566-5; GenBank: AAY26402.1); isoform 4 (UniProt O43566-6; GenBank: BAC85600.1). Their predicted protein sequences (Zhao et al. 2013) are represented by cartoons. (B) RGS14 antibody was pre-adsorbed with purified rat RGS14 and purified human RGS14 proteins (10 μg protein: 1 μg antibody). Membranes were subjected to the same antibody dilution (1:1000) and the same immunoblot protocol. Stained RGS14 bands are absent following pre-adsorption, indicating that the antibody is specific and that all detected bands are RGS14.

Figure 4. RGS14 immunoreactivity is expressed in discrete regions throughout the monkey brain

(A–F) Coronal sections through the rostrocaudal extent of the monkey brain show strong RGS14 immunostaining in the caudate nucleus (CD), putamen (PUT), nucleus accumbens (Acc), internal and external globus pallidus (GPi, GPe), substantia nigra (SN), and hippocampus (Hp), while moderate staining was found in the amygdala (Am). Apart from caudal hippocampal labeling, brainstem sections posterior to that displayed in panel F were devoid of RGS14 immunostaining.

Figure 5. RGS14 labeling in monkey brain is specific

(A–D) RGS14 immunoreactivity was ablated in all regions of the monkey brain after incubation with RGS14 antibody pre-adsorbed with the synthetic immunogenic peptide against which it was made (A versus B; C versus D). Note that brain sections depicted in A and C are also used in Figure 4.

Figure 6. RGS14 is expressed in CA1 and CA2 pyramidal cell bodies and neuropil in the monkey hippocampus

(A) Light microscopy reveals dense RGS14 labeling of pyramidal cell bodies in CA2. By contrast, there is virtually no labeling in CA3. CA1 displays a lighter RGS14 labeling than CA2, which is preferentially localized in the neuropil. (B and C) Electron micrographs of RGS14-positive terminals (Ter) forming asymmetric axo-dendritic (B) or axo-spinous (C) synapses in CA1. In each micrograph, RGS14-immunoreactive axon terminals (Ter), spines (Sp) and dendrites (Den) are indicated, while u.Ter, u.Sp, and u.Den mark unlabeled corresponding elements. The red arrowheads point at asymmetric synapses that involve RGS14-containing terminals, which likely originate from CA2-CA1 axonal projections. (D) Post-synaptic RGS14 labeling in CA1 dendrites and spines.

Figure 7. RGS14 is localized in dendrites and spines of GABAergic projection neurons in the monkey striatum

A) A light micrograph shows RGS14-immunoreactive cell bodies alongside a dense neuropil labeling in the monkey striatum. The inset shows a high power view of RGS-positive striatal cell bodies that morphologically resemble those of striatal projection neurons. (B–C) Electron micrographs of RGS14-labeled dendrites (Den) and spines (Sp) in the monkey putamen. Note in the lower part of B, the spines coming off a dendritic process, further confirming the RGS14 expression in spiny projection neurons. (D) Electron micrograph of an RGS14-positive axon terminal that forms a symmetric axo-dendritic synapse (red arrow) with an unlabeled dendrite (u.Den) in the putamen. Although the source of these terminals was not characterized, their ultrastructural features suggest that they may originate from recurrent collaterals of GABAergic striatofugal axons (Wilson and Groves 1980).

Figure 8. RGS14 localizes in the cytosol and the nucleus of striatal projection neurons

Electron micrographs of RGS14-immunoreactive dendrites (Den) and spines (Sp) (A–B), and neuronal cell bodies (C–D) in the monkey striatum as revealed with the pre-embedding immunogold method. Note that most gold particles are largely located within the cytosol of immunoreactive structures, with rare instances of plasma membrane labeling. In (A), the red arrowheads indicate examples of RGS14 labeling in spines. In (B), the red arrowhead indicates an example of RGS14 labeling in dendrites. In (C) and (D), the staining is associated with the endoplasmic reticulum (red arrowhead) and vesicular organelles in the cytoplasm of immunoreactive cell bodies, while some gold particles are also found in the nucleus of these labeled neurons (yellow arrowheads).

Figure 9. RGS14 is localized presynaptically in striatopallidal GABAergic terminals

(A) Light micrograph of RGS14-immunoreactive neuropil in both the internal and external globus pallidus (GPi, GPe) displays the “woolly fibers” pattern of labeling previously used to describe the massive striatal GABAergic innervation wrapped around pallidal dendrites (Haber and Nauta 1983). (B–D) Electron micrographs of RGS14 labeling in GPe and GPi confirm that most of the immunoreactivity is found in axon terminals (Ter) that display the ultrastructural features of striatal GABAergic terminals and form symmetric synapses (blue arrowheads) with unlabeled dendrites (u.Den) of pallidal neurons. Note that red arrowheads depict asymmetric synapses formed by unlabeled terminals (u.Ter). Labeled unmyelinated axons (Ax) are also found throughout the neuropil in both pallidal segments (C).

Figure 10. RGS14 is localized pre-synaptically in the monkey substantia nigra pars reticulata

(A) Light micrograph showing dense RGS14 neuropil labeling in the pars reticulata (SNr) of the monkey substantia nigra, while the pars compacta (SNc) is completely devoid of immunoreactivity. As in GPe and GPi, the SNr labeling is made up of rich plexuses of varicosities that display a woolly fibers-like pattern of innervation of dendritic processes. (B–C) At the electron microscope level, the immunostaining is mainly confined to pre-synaptic terminals (Ter) that display the ultrastructural features of striatonigral GABAergic boutons and form symmetric axo-dendritic synapses (blue arrowhead in C). (D) illustrates a subset of RGS14-immunoreactive terminals that forms asymmetric axo-dendritic synapses (red arrowheads). The amygdala is a potential source of these putative excitatory terminals.

Figure 11. RGS14 is expressed throughout the monkey amygdala

(A) Low power view of the distribution of RGS14 immunoreactivity in the monkey amygdala. (B–C) Light microscope images show discrete RGS14 staining of cell bodies and neuropil in the basomedial (BM) and basolateral (BL) nuclei, but not the lateral (LA) nuclei. (D–E) RGS14 labeling in the central lateral (CeL) and amygdalostriatal region (AStr). The dense band of labeling adjacent to the CeL corresponds to a sub-region of the AStr (arrows in E and F) that contains a larger neuronal density than neighboring AStr regions as revealed by NeuN immunostaining (F).

Figure 12. RGS14 expression in human hippocampus is comparable to that of monkey and mouse

Human hippocampi were double stained with Nissl reagent and RGS14 immunolabeling. (A) Low power view of RGS14 labeling in CA2 and CA1 subfields, but not in CA3, of human hippocampus. (B, D) Higher magnification of RGS14-immunoreactive pyramidal cell bodies in the CA2 (B) and CA1 (D) regions, as shown in the monkey and mouse hippocampus. The insets in the upper right corner of each panel show examples of cell body labeling. There is minimal neuropil labeling in both regions. (C) Micrograph showing thionin-stained CA3 neurons devoid of RGS14 immunoreactivity.

Figure 13. RGS14 expression in the human basal ganglia is consistent with labeling in the monkey basal ganglia

(A–B) Light micrographs of RGS14-positive neuronal cell bodies, double stained with Nissl reagent, within a lightly labeled neuropil in the human caudate nucleus (A) and putamen (B). The insets in the upper right corner of each panel show examples of cell body labeling. (C–D) Dense RGS14-immunoreactive woolly fibers-like neuropil in the human GPe and GPi. The pattern of RGS14 labeling in both the striatum and the globus pallidus in humans is consistent with that described in monkeys.

Figure 14. Circuitry diagram of RGS14 protein expression in the primate brain

Circuit diagram of RGS14-positive neurons and their connections within the basal ganglia and hippocampus networks. Red arrowheads indicate pre-synaptic RGS14 labeling in terminals. Red dots depict post-synaptic RGS14 labeling in cell bodies, dendrites, and spines. Axonal projections are designated by black lines.