Expression of RGS4 in principal dendrites. ( A , B ) The apical and one basal pyramidal dendrite (ap-den, bas-den) are shown emerging from the soma; the plasma membrane has been outlined. Note an increase in RGS4 expression in the tapering ap-den . ( C ) Particles label the cytoplasm of the apical dendrite and may appear opposite symmetric synapses of varicosities en passant (arrowheads in inset); arrows point to a microtubule running longitudinally inside the stem of the axon. ( D ) The size of particle clusters (arrows) is indicative of RGS4 levels and is markedly larger in ap-den than in small dendrites and axons in the adjoining neuropil. ax, axon; den, dendrite. Scale bars: ( A , B ) 1 l m; ( C , D ) 500 nm; ( C , inset) 200 nm.

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Mapping the Regulator of G Protein Signaling 4 (RGS4): Presynaptic and Postsynaptic Substrates for Neuroregulation in Prefrontal Cortex

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Feb 2009

Regulator of G protein signaling 4 (RGS4) regulates intracellular signaling via G proteins and is markedly reduced in the prefrontal cortex (PFC) of patients with schizophrenia. Characterizing the expression of RGS4 within individual neuronal compartments is thus key to understanding its actions on individual G protein-coupled receptors (GPCRs). He...

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... in B ), peaked in the stem, and remained high along its proximal path. Label filled the entire cytoplasm (Fig. 3 C , D ). Although we did find examples of RGS4 localization directly across from symmetric synapses of varicosities en passant (e.g., Fig. 3 C , inset), these may have been a reflection of the overall high expression levels of the protein in proximal dendrites rather than a specific association with synaptic membranes. Tagged RGS4 under the electron microscope could be traced for several micrometers in the apical dendrite and occasionally into second-order branches, though immunoreactivity dimin- ished with distance from the soma (Fig. 4 A , graph). In section planes capturing apical dendrites along a considerable length, immunoreactivity ended abruptly such that virtually no particles could be found distal to that point. The point where RGS4 could no longer be detected was measured at 7--18 l m from the soma. However, analysis was hindered by the fact that immunoreactive dendrites were often truncated as they left the section plane, suggesting that labeling in certain apical shafts may have extended farther (compare to light microscopic findings in Fig. 1 B ). In apical dendrites where labeling ended before splitting into second-order branches, particle clusters were nonetheless present in the branching point (Fig. 4 B ). The occurrence of RGS4 in bifurcating dendrites may be of particular significance for signal initiation/transduction (see Discussion), especially as numerous axons populate the branch points (Fig. 4 B ). Distal dendrites ( < 0.6 l m in diameter; minor axis) were proportionately the most prominent RGS4 immunoreactive structures in the neuropil (Table 1; Supplementary Fig. 3 A -- G ). They typically contained a few small particle clusters in the cytoplasm, apart from those sections corresponding to synaptic profiles. Indeed, RGS4 in distal dendrites was directly associated with synapses (Fig. 5 A -- E ), which was further confirmed in longitudinally sectioned stems to exclude bias (Supplementary Fig. 3 A ). Synapse-associated particles were separated by 15--35 nm from the postsynaptic membrane, and rarely were embedded in the postsynaptic density (PSD) per se. Note that the label is distributed across from both central and peripheral portions of the synaptic disk, and involves asymmetric and to a lesser extent symmetric axodendritic synapses, exemplified in Figure 5 A ,C, respectively. Seventy-six percent of RGS4- labeled axodendritic synapses were type I, asymmetric junctions (see Table 2). However, RGS4 expression in type II, symmetric synapses could be underestimated as we excluded synapses captured at oblique planes, and those that could not be unequivocally categorized in a single section. In favorable section planes, immunoreactive spines arose from labeled, presumably pyramidal dendrites (Supplementary Fig. 3 C , D ). RGS4 in postsynaptic spines (Table 2) exhibited 2 distinct distribution patterns that were uniquely correlated with inputs from glutamatergic-like (Glut-like) versus non- glutamatergic--like (non-Glut--like) axons (see RGS4 in Axons for definition). Please note that this analysis is not to imply that a given category of spines would receive exclusively symmetric or asymmetric synapses. Rather, it focuses on RGS4 expression on individual axospinous synapses, and attempts to correlate the expression with the axon ultrastructure and synaptology. In this context, it is imperative also to note that axon varicosities other than glutamatergic may in fact establish asymmetric synapses in PFC (e.g., Aoki et al. 1998), whereas certain symmetric synapses may utilize glutamate as a cotrans- mitter (Boulland et al. 2008). Therefore, data should be interpreted with the clear understanding that a portion of axons featuring a Glut-like ultrastructure could potentially utilize another neurotransmitter/neuromodulator, whereas axons with non-Glut features might also engage glutamatergic signaling mechanisms. At asymmetric synapses from Glut-like axons, the particles typically labeled extrasynaptic spine membranes (Fig. 6 A ), and rarely appeared subjacent to the PSD. However, RGS4 was found at perisynaptic membranes flanking the asymmetric synapse (see below; see also Supplementary Fig. 3 B ). In those spines receiving symmetric synapses from non-Glut--like axons, RGS4 was found subjacent to the PSD, as in postsynaptic dendrites (Fig. 6 B ). It should be marked that perisynaptic expression of RGS4 was uniquely associated with spines receiving asymmetric synapses, and not found in spines receiving symmetric synapses or in dendrites receiving either type of synapses (compare the axospinous and axodendritic asymmetric synapses in Fig. 6 C ). Based on their ultrastructure and synaptology (see Peters et al. 1991 and specific criteria below), axon varicosities in the PFC neuropil were categorized as Glut-like and non-Glut--like (compare ax1 to ax2 in Fig. 7 C and in Supplementary Fig. 3 C , but see our statement in previous section). Prevalence of RGS4 expression (Table 1) as well as its subcellular distribution clearly differed in Glut-like and non-Glut--like axons. Non-Glut--like axons featured loose clusters of pleomorphic vesicles, including 80--120 nm clear and dense-cored vesicles. In general, they established symmetric synapses with a 10- to 15-nm-wide cleft and often narrow and multiple active zones. Such axons contained numerous immunoparticles that typically labeled the axoplasm in association with electron-opaque vesicular structures, possibly of the endosomal line (Fig. 7 A , B ). RGS4 particles were also found at extrasynaptic places along the axolemma but the bulk of immunoreactivity clearly appeared intracellularly. Yet in single sections, most RGS4 non-Glut--like varicosities were asynaptic (see Supplementary Fig. 3 C ). Serial sectioning revealed spines as their prime postsynaptic target (Fig. 7 A , B ), which is noteworthy given that dendritic stems would receive the majority of symmetric synapses (presumed c -aminobutyric acidergic [GABAergic]) in the neocortex (Colonnier 1981). One axodendritic synapse of a labeled non-Glut--like axon is shown in Figure 7 C as a part of a synaptic triad involving an immunonegative Glut-like axon. Glut-like axons featured 30-nm round vesicles docked onto the presynaptic grid and established asymmetric synapses with wide, 20-nm clefts and often a central perforation. They contained many fewer RGS4 particles when compared with the non-Glut-- like axons. Immunoparticles, however, were associated with the axolemma at perisynaptic locations and extrasynaptically (Fig. 7 D , E ); the axoplasm was infrequently labeled. Dendritic spines were prime postsynaptic targets for the RGS4-positive Glut-like axons, with shafts receiving fewer synapses. Axon intervaricose segments exhibited a sporadic RGS4 immunoreactivity. Thus, it is not clear whether RGS4 in the nonmyelinated axonal profiles (Fig. 7 F ) may in part represent a functional regulatory component (e.g., for preterminal GPCRs; see Aoki et al. 1998; Muly et al. 2003) or merely indicates protein captured en passant , as could likely be the case in the myelinated profile shown in Figure 7 G . Immunoparticles aligned with plasma membranes even when synapses were not present, as if the apposed profile directed the accumulation of RGS4. When we screened such appositions in serial sections, synapses were not found nor did RGS4- particles line up with membranes other than those apposing an axon (Fig. 8 A , B ), suggesting that RGS4 may assemble on the plane of the plasmalemma to potentially regulate focal stimulation of extrasynaptic receptors. In addition, RGS4 was detected in somata and major processes of astrocytes (see also Fig. 1 B ) but not found in slender astrocytic processes in the neuropil, including those that typically enwrap synapses. Hence, it is unlikely that RGS4 could function as regulator of a GPCR in these processes, such as the glial a 2A adrenoceptor ( a 2A -AR; Aoki et al. 1998; Wang et al. 2007). RGS4 on astrocytic membranes—apparently nonsynaptic—was found across from axons as well as postsynaptic profiles (Fig. 8 C ). In summary, we have shown that RGS4 exhibited very distinct distribution patterns on the basis of distance from the soma (i.e., proximal vs. distal dendrites), ultrastructure and synaptology (e.g., Glut-like vs. non-Glut--like axons), and presence of specialized plasmalemma (e.g., synaptic and SSC-lined membranes). The data are schematically summarized in Figure 9. As we discuss in the following sections, such patterns may subserve and likely reflect specificity of neuroregulation, for implicit in the proposed actions of RGS4 is its localization at or near plasma membranes where G proteins and GPCRs assemble. In somata, RGS4 was enriched near SSCs, the smooth reticulum that holds IP R-gated Ca 2 + stores (Delmas and Brown 2002; Berridge 2005). So far, SSC-lined membranes have been identified as DA D 5 receptor (D 5 R)--specific microdomains, linking nonsynaptic receptors to internal Ca 2 + stores in PFC pyramids (Paspalas and Goldman-Rakic 2004a). D 5 Rs couple to Gs to elevate cAMP but have also been implicated in triggering Ca 2 + signaling cascades via the Gq--phosphoinositide signal transduction system (Pacheco and Jope 1997; Jin et al. 2001). 2 + In fact, D 1 /D 5 Rs stimulate Ca release from IP 3 -sensitive stores in perikarya of dissociated cortical neurons (Lezcano and Bergson 2002). Thus, a selective placement of RGS4 in microdomains would potentially allow for regulation of signal transduction triggered by nonsynaptic D 5 Rs, namely phospholipase C activation, IP 3 formation, and IP 3 R-mediated Ca 2 + release from the SSC. Beyond the soma, RGS4 expression in the pyramidal apical dendrite was markedly higher than in any other cellular compartment, including basal dendrites. Intense labeling of the apical dendrite cannot represent newly ...

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... to RGS4 was mainly detected in pyramidal neurons in layers II/III ( Fig. 1 A , B ) and V. The nucleus was intensely labeled (see controls in Supplementary Fig. 1) and contrasted with the weakly reactive cytoplasm, especially when visualized with immunogold (e.g., Fig. 1 D ; compare with the peroxidase reaction filling the entire soma in Fig. 1 B ). Patches of immunoreactivity on perisomatic membranes (Fig. 1 D , E ) likely correspond to labeling of the subsurface cisterns (SSC) identified with electron microscopy (see RGS4 in the Soma and Principal Dendrites ). Labeled pyramidal neurons featured a prominent apical dendrite and short basal dendrites, though in most cells, basal branches were not reactive (Fig. 1 B ; see also Fig. 3 A , B ). RGS4 immunoreactivity of the apical dendrite decreased abruptly after 10--40 l m or about 1--3 soma lengths in the majority of neurons, and hence, we did not visualize the elaborate meshwork of distal apical dendrites invading layer I. The neuropil contained the highly reactive first-order dendrites and high-order branches as well as distinct punctate labeling throughout, including in layer I (Fig. 1 C ). However, the end- tufts of apical pyramidal dendrites below the pia were not labeled. Immunoreactive nonpyramidal neurons were clearly identified in layer I (Fig. 1 C ) as well as in layer VI (Fig. 1 F ). Their dendritic ramifications were not labeled or were only visualized within a short distance. A faint reaction was observed in somata of astrocytes in layer I (Fig. 1 B ). High RGS4 immunoreactivity marked the nucleoplasm; the nuclear envelope was labeled with particle clusters subjacent to nucleopores (Fig. 2 A ; Supplementary Fig. 2 A , B ). In compar- ison, the perikaryon displayed moderate immunoreactivity on smooth endomembranes and vesicles bordering Golgi complexes (data not shown). Rough reticular cisterns were often labeled, but overall RGS4 did not show enrichment in the rough endoplasmic reticulum (Fig. 2 A ). In general, the somatic plasma membrane was not labeled nor were axosomatic synapses ‘‘targeted’’ in any way by RGS4 particles. Rather, immunoparticles appeared subjacent to SSC- lined membranes and ‘‘particularly’’ next to the vacuolar rim of a cistern (Fig. 2 B ). It is important to note that SSCs are specialized smooth reticulum endomembranes that hold inositol trisphosphate receptor (IP 3 R)--gated Ca 2 + stores, and may function as junctional and signaling microdomains (see Delmas and Brown 2002; Paspalas and Goldman-Rakic 2004a; and Discussion below). RGS4 levels increased in the base of the tapering apical dendrite (Fig. 3 A ; compare to the weak expression in basal dendrite in B ), peaked in the stem, and remained high along its proximal path. Label filled the entire cytoplasm (Fig. 3 C , D ). Although we did find examples of RGS4 localization directly across from symmetric synapses of varicosities en passant (e.g., Fig. 3 C , inset), these may have been a reflection of the overall high expression levels of the protein in proximal dendrites rather than a specific association with synaptic membranes. Tagged RGS4 under the electron microscope could be traced for several micrometers in the apical dendrite and occasionally into second-order branches, though immunoreactivity dimin- ished with distance from the soma (Fig. 4 A , graph). In section planes capturing apical dendrites along a considerable length, immunoreactivity ended abruptly such that virtually no particles could be found distal to that point. The point where RGS4 could no longer be detected was measured at 7--18 l m from the soma. However, analysis was hindered by the fact that immunoreactive dendrites were often truncated as they left the section plane, suggesting that labeling in certain apical shafts may have extended farther (compare to light microscopic findings in Fig. 1 B ). In apical dendrites where labeling ended before splitting into second-order branches, particle clusters were nonetheless present in the branching point (Fig. 4 B ). The occurrence of RGS4 in bifurcating dendrites may be of particular significance for signal initiation/transduction (see Discussion), especially as numerous axons populate the branch points (Fig. 4 B ). Distal dendrites ( < 0.6 l m in diameter; minor axis) were proportionately the most prominent RGS4 immunoreactive structures in the neuropil (Table 1; Supplementary Fig. 3 A -- G ). They typically contained a few small particle clusters in the cytoplasm, apart from those sections corresponding to synaptic profiles. Indeed, RGS4 in distal dendrites was directly associated with synapses (Fig. 5 A -- E ), which was further confirmed in longitudinally sectioned stems to exclude bias (Supplementary Fig. 3 A ). Synapse-associated particles were separated by 15--35 nm from the postsynaptic membrane, and rarely were embedded in the postsynaptic density (PSD) per se. Note that the label is distributed across from both central and peripheral portions of the synaptic disk, and involves asymmetric and to a lesser extent symmetric axodendritic synapses, exemplified in Figure 5 A ,C, respectively. Seventy-six percent of RGS4- labeled axodendritic synapses were type I, asymmetric junctions (see Table 2). However, RGS4 expression in type II, symmetric synapses could be underestimated as we excluded synapses captured at oblique planes, and those that could not be unequivocally categorized in a single section. In favorable section planes, immunoreactive spines arose from labeled, presumably pyramidal dendrites (Supplementary Fig. 3 C , D ). RGS4 in postsynaptic spines (Table 2) exhibited 2 distinct distribution patterns that were uniquely correlated with inputs from glutamatergic-like (Glut-like) versus non- glutamatergic--like (non-Glut--like) axons (see RGS4 in Axons for definition). Please note that this analysis is not to imply that a given category of spines would receive exclusively symmetric or asymmetric synapses. Rather, it focuses on RGS4 expression on individual axospinous synapses, and attempts to correlate the expression with the axon ultrastructure and synaptology. In this context, it is imperative also to note that axon varicosities other than glutamatergic may in fact establish asymmetric synapses in PFC (e.g., Aoki et al. 1998), whereas certain symmetric synapses may utilize glutamate as a cotrans- mitter (Boulland et al. 2008). Therefore, data should be interpreted with the clear understanding that a portion of axons featuring a Glut-like ultrastructure could potentially utilize another neurotransmitter/neuromodulator, whereas axons with non-Glut features might also engage glutamatergic signaling mechanisms. At asymmetric synapses from Glut-like axons, the particles typically labeled extrasynaptic spine membranes (Fig. 6 A ), and rarely appeared subjacent to the PSD. However, RGS4 was found at perisynaptic membranes flanking the asymmetric synapse (see below; see also Supplementary Fig. 3 B ). In those spines receiving symmetric synapses from non-Glut--like axons, RGS4 was found subjacent to the PSD, as in postsynaptic dendrites (Fig. 6 B ). It should be marked that perisynaptic expression of RGS4 was uniquely associated with spines receiving asymmetric synapses, and not found in spines receiving symmetric synapses or in dendrites receiving either type of synapses (compare the axospinous and axodendritic asymmetric synapses in Fig. 6 C ). Based on their ultrastructure and synaptology (see Peters et al. 1991 and specific criteria below), axon varicosities in the PFC neuropil were categorized as Glut-like and ...

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... (Fig. 2 A ). In general, the somatic plasma membrane was not labeled nor were axosomatic synapses ‘‘targeted’’ in any way by RGS4 particles. Rather, immunoparticles appeared subjacent to SSC- lined membranes and ‘‘particularly’’ next to the vacuolar rim of a cistern (Fig. 2 B ). It is important to note that SSCs are specialized smooth reticulum endomembranes that hold inositol trisphosphate receptor (IP 3 R)--gated Ca 2 + stores, and may function as junctional and signaling microdomains (see Delmas and Brown 2002; Paspalas and Goldman-Rakic 2004a; and Discussion below). RGS4 levels increased in the base of the tapering apical dendrite (Fig. 3 A ; compare to the weak expression in basal dendrite in B ), peaked in the stem, and remained high along its proximal path. Label filled the entire cytoplasm (Fig. 3 C , D ). Although we did find examples of RGS4 localization directly across from symmetric synapses of varicosities en passant (e.g., Fig. 3 C , inset), these may have been a reflection of the overall high expression levels of the protein in proximal dendrites rather than a specific association with synaptic membranes. Tagged RGS4 under the electron microscope could be traced for several micrometers in the apical dendrite and occasionally into second-order branches, though immunoreactivity dimin- ished with distance from the soma (Fig. 4 A , graph). In section planes capturing apical dendrites along a considerable length, immunoreactivity ended abruptly such that virtually no particles could be found distal to that point. The point where RGS4 could no longer be detected was measured at 7--18 l m from the soma. However, analysis was hindered by the fact that immunoreactive dendrites were often truncated as they left the section plane, suggesting that labeling in certain apical shafts may have extended farther (compare to light microscopic findings in Fig. 1 B ). In apical dendrites where labeling ended before splitting into second-order branches, particle clusters were nonetheless present in the branching point (Fig. 4 B ). The occurrence of RGS4 in bifurcating dendrites may be of particular significance for signal initiation/transduction (see Discussion), especially as numerous axons populate the branch points (Fig. 4 B ). Distal dendrites ( < 0.6 l m in diameter; minor axis) were proportionately the most prominent RGS4 immunoreactive structures in the neuropil (Table 1; Supplementary Fig. 3 A -- G ). They typically contained a few small particle clusters in the cytoplasm, apart from those sections corresponding to synaptic profiles. Indeed, RGS4 in distal dendrites was directly associated with synapses (Fig. 5 A -- E ), which was further confirmed in longitudinally sectioned stems to exclude bias (Supplementary Fig. 3 A ). Synapse-associated particles were separated by 15--35 nm from the postsynaptic membrane, and rarely were embedded in the postsynaptic density (PSD) per se. Note that the label is distributed across from both central and peripheral portions of the synaptic disk, and involves asymmetric and to a lesser extent symmetric axodendritic synapses, exemplified in Figure 5 A ,C, respectively. Seventy-six percent of RGS4- labeled axodendritic synapses were type I, asymmetric junctions (see Table 2). However, RGS4 expression in type II, symmetric synapses could be underestimated as we excluded synapses captured at oblique planes, and those that could not be unequivocally categorized in a single section. In favorable section planes, immunoreactive spines arose from labeled, presumably pyramidal dendrites (Supplementary Fig. 3 C , D ). RGS4 in postsynaptic spines (Table 2) exhibited 2 distinct distribution patterns that were uniquely correlated with inputs from glutamatergic-like (Glut-like) versus non- glutamatergic--like (non-Glut--like) axons (see RGS4 in Axons for definition). Please note that this analysis is not to imply that a given category of spines would receive exclusively symmetric or asymmetric synapses. Rather, it focuses on RGS4 expression on individual axospinous synapses, and attempts to correlate the expression with the axon ultrastructure and synaptology. In this context, it is imperative also to note that axon varicosities other than glutamatergic may in fact establish asymmetric synapses in PFC (e.g., Aoki et al. 1998), whereas certain symmetric synapses may utilize glutamate as a cotrans- mitter (Boulland et al. 2008). Therefore, data should be interpreted with the clear understanding that a portion of axons featuring a Glut-like ultrastructure could potentially utilize another neurotransmitter/neuromodulator, whereas axons with non-Glut features might also engage glutamatergic signaling mechanisms. At asymmetric synapses from Glut-like axons, the particles typically labeled extrasynaptic spine membranes (Fig. 6 A ), and rarely appeared subjacent to the PSD. However, RGS4 was found at perisynaptic membranes flanking the asymmetric synapse (see below; see also Supplementary Fig. 3 B ). In those spines receiving symmetric synapses from non-Glut--like axons, RGS4 was found subjacent to the PSD, as in postsynaptic dendrites (Fig. 6 B ). It should be marked that perisynaptic expression of RGS4 was uniquely associated with spines receiving asymmetric synapses, and not found in spines receiving symmetric synapses or in dendrites receiving either type of synapses (compare the axospinous and axodendritic asymmetric synapses in Fig. 6 C ). Based on their ultrastructure and synaptology (see Peters et al. 1991 and specific criteria below), axon varicosities in the PFC neuropil were categorized as Glut-like and non-Glut--like (compare ax1 to ax2 in Fig. 7 C and in Supplementary Fig. 3 C , but see our statement in previous section). Prevalence of RGS4 expression (Table 1) as well as its subcellular distribution clearly differed in Glut-like and non-Glut--like axons. Non-Glut--like axons featured loose clusters of pleomorphic vesicles, including 80--120 nm clear and dense-cored vesicles. In general, they established symmetric synapses with a 10- to 15-nm-wide cleft and often narrow and multiple active zones. Such axons contained numerous immunoparticles that typically labeled the axoplasm in association with electron-opaque vesicular structures, possibly of the endosomal line (Fig. 7 A , B ). RGS4 particles were also found at extrasynaptic places along the axolemma but the bulk of immunoreactivity clearly appeared intracellularly. Yet in single sections, most RGS4 non-Glut--like varicosities were asynaptic (see Supplementary Fig. 3 C ). Serial sectioning revealed spines as their prime postsynaptic target (Fig. 7 A , B ), which is noteworthy given that dendritic stems would receive the majority of symmetric synapses (presumed c -aminobutyric acidergic [GABAergic]) in the neocortex (Colonnier 1981). One axodendritic synapse of a labeled non-Glut--like axon is shown in Figure 7 C as a part of a synaptic triad involving an immunonegative Glut-like axon. Glut-like axons featured 30-nm round vesicles docked onto the presynaptic grid and established asymmetric synapses with wide, 20-nm clefts and often a central perforation. They contained many fewer RGS4 particles when compared with the non-Glut-- like axons. Immunoparticles, however, were associated with the axolemma at perisynaptic locations and extrasynaptically (Fig. 7 D , E ); the axoplasm was infrequently labeled. Dendritic spines were prime postsynaptic targets for the RGS4-positive Glut-like axons, with shafts receiving fewer synapses. Axon intervaricose segments exhibited a sporadic RGS4 immunoreactivity. Thus, it is not clear whether RGS4 in the nonmyelinated axonal profiles (Fig. 7 F ) may in part represent a functional regulatory component (e.g., for preterminal GPCRs; see Aoki et al. 1998; Muly et al. 2003) or merely indicates protein captured en passant , as could likely be the case in the myelinated profile shown in Figure 7 G . Immunoparticles aligned with plasma membranes even when synapses were not present, as if the apposed profile directed the accumulation of RGS4. When we screened such appositions in serial sections, synapses were not found nor did RGS4- particles line up with membranes other than those apposing an axon (Fig. 8 A , B ), suggesting that RGS4 may assemble on the plane of the plasmalemma to potentially regulate focal stimulation of extrasynaptic receptors. In addition, RGS4 was detected in somata and major processes of astrocytes (see also Fig. 1 B ) but not found in slender astrocytic processes in the neuropil, including those that typically enwrap synapses. Hence, it is unlikely that RGS4 could function as regulator of a GPCR in these processes, such as the glial a 2A adrenoceptor ( a 2A -AR; Aoki et al. 1998; Wang et al. 2007). RGS4 on astrocytic membranes—apparently nonsynaptic—was found across from axons as well as postsynaptic profiles (Fig. 8 C ). In summary, we have shown that RGS4 exhibited very distinct distribution patterns on the basis of distance from the soma (i.e., proximal vs. distal dendrites), ultrastructure and synaptology (e.g., Glut-like vs. non-Glut--like axons), and presence of specialized plasmalemma (e.g., synaptic and SSC-lined membranes). The data are schematically summarized in Figure 9. As we discuss in the following sections, such patterns may subserve and likely reflect specificity of neuroregulation, for implicit in the proposed actions of RGS4 is its localization at or near plasma membranes where G proteins and GPCRs assemble. In somata, RGS4 was enriched near SSCs, the smooth reticulum that holds IP R-gated Ca 2 + stores (Delmas and Brown 2002; Berridge 2005). So far, SSC-lined membranes have been identified as DA D 5 receptor (D 5 R)--specific microdomains, linking nonsynaptic receptors to internal Ca 2 + stores in PFC pyramids (Paspalas and Goldman-Rakic 2004a). D 5 Rs couple to Gs to elevate cAMP but have also been implicated in triggering Ca 2 + signaling cascades via ...

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... RGS4 could no longer be detected was measured at 7--18 l m from the soma. However, analysis was hindered by the fact that immunoreactive dendrites were often truncated as they left the section plane, suggesting that labeling in certain apical shafts may have extended farther (compare to light microscopic findings in Fig. 1 B ). In apical dendrites where labeling ended before splitting into second-order branches, particle clusters were nonetheless present in the branching point (Fig. 4 B ). The occurrence of RGS4 in bifurcating dendrites may be of particular significance for signal initiation/transduction (see Discussion), especially as numerous axons populate the branch points (Fig. 4 B ). Distal dendrites ( < 0.6 l m in diameter; minor axis) were proportionately the most prominent RGS4 immunoreactive structures in the neuropil (Table 1; Supplementary Fig. 3 A -- G ). They typically contained a few small particle clusters in the cytoplasm, apart from those sections corresponding to synaptic profiles. Indeed, RGS4 in distal dendrites was directly associated with synapses (Fig. 5 A -- E ), which was further confirmed in longitudinally sectioned stems to exclude bias (Supplementary Fig. 3 A ). Synapse-associated particles were separated by 15--35 nm from the postsynaptic membrane, and rarely were embedded in the postsynaptic density (PSD) per se. Note that the label is distributed across from both central and peripheral portions of the synaptic disk, and involves asymmetric and to a lesser extent symmetric axodendritic synapses, exemplified in Figure 5 A ,C, respectively. Seventy-six percent of RGS4- labeled axodendritic synapses were type I, asymmetric junctions (see Table 2). However, RGS4 expression in type II, symmetric synapses could be underestimated as we excluded synapses captured at oblique planes, and those that could not be unequivocally categorized in a single section. In favorable section planes, immunoreactive spines arose from labeled, presumably pyramidal dendrites (Supplementary Fig. 3 C , D ). RGS4 in postsynaptic spines (Table 2) exhibited 2 distinct distribution patterns that were uniquely correlated with inputs from glutamatergic-like (Glut-like) versus non- glutamatergic--like (non-Glut--like) axons (see RGS4 in Axons for definition). Please note that this analysis is not to imply that a given category of spines would receive exclusively symmetric or asymmetric synapses. Rather, it focuses on RGS4 expression on individual axospinous synapses, and attempts to correlate the expression with the axon ultrastructure and synaptology. In this context, it is imperative also to note that axon varicosities other than glutamatergic may in fact establish asymmetric synapses in PFC (e.g., Aoki et al. 1998), whereas certain symmetric synapses may utilize glutamate as a cotrans- mitter (Boulland et al. 2008). Therefore, data should be interpreted with the clear understanding that a portion of axons featuring a Glut-like ultrastructure could potentially utilize another neurotransmitter/neuromodulator, whereas axons with non-Glut features might also engage glutamatergic signaling mechanisms. At asymmetric synapses from Glut-like axons, the particles typically labeled extrasynaptic spine membranes (Fig. 6 A ), and rarely appeared subjacent to the PSD. However, RGS4 was found at perisynaptic membranes flanking the asymmetric synapse (see below; see also Supplementary Fig. 3 B ). In those spines receiving symmetric synapses from non-Glut--like axons, RGS4 was found subjacent to the PSD, as in postsynaptic dendrites (Fig. 6 B ). It should be marked that perisynaptic expression of RGS4 was uniquely associated with spines receiving asymmetric synapses, and not found in spines receiving symmetric synapses or in dendrites receiving either type of synapses (compare the axospinous and axodendritic asymmetric synapses in Fig. 6 C ). Based on their ultrastructure and synaptology (see Peters et al. 1991 and specific criteria below), axon varicosities in the PFC neuropil were categorized as Glut-like and non-Glut--like (compare ax1 to ax2 in Fig. 7 C and in Supplementary Fig. 3 C , but see our statement in previous section). Prevalence of RGS4 expression (Table 1) as well as its subcellular distribution clearly differed in Glut-like and non-Glut--like axons. Non-Glut--like axons featured loose clusters of pleomorphic vesicles, including 80--120 nm clear and dense-cored vesicles. In general, they established symmetric synapses with a 10- to 15-nm-wide cleft and often narrow and multiple active zones. Such axons contained numerous immunoparticles that typically labeled the axoplasm in association with electron-opaque vesicular structures, possibly of the endosomal line (Fig. 7 A , B ). RGS4 particles were also found at extrasynaptic places along the axolemma but the bulk of immunoreactivity clearly appeared intracellularly. Yet in single sections, most RGS4 non-Glut--like varicosities were asynaptic (see Supplementary Fig. 3 C ). Serial sectioning revealed spines as their prime postsynaptic target (Fig. 7 A , B ), which is noteworthy given that dendritic stems would receive the majority of symmetric synapses (presumed c -aminobutyric acidergic [GABAergic]) in the neocortex (Colonnier 1981). One axodendritic synapse of a labeled non-Glut--like axon is shown in Figure 7 C as a part of a synaptic triad involving an immunonegative Glut-like axon. Glut-like axons featured 30-nm round vesicles docked onto the presynaptic grid and established asymmetric synapses with wide, 20-nm clefts and often a central perforation. They contained many fewer RGS4 particles when compared with the non-Glut-- like axons. Immunoparticles, however, were associated with the axolemma at perisynaptic locations and extrasynaptically (Fig. 7 D , E ); the axoplasm was infrequently labeled. Dendritic spines were prime postsynaptic targets for the RGS4-positive Glut-like axons, with shafts receiving fewer synapses. Axon intervaricose segments exhibited a sporadic RGS4 immunoreactivity. Thus, it is not clear whether RGS4 in the nonmyelinated axonal profiles (Fig. 7 F ) may in part represent a functional regulatory component (e.g., for preterminal GPCRs; see Aoki et al. 1998; Muly et al. 2003) or merely indicates protein captured en passant , as could likely be the case in the myelinated profile shown in Figure 7 G . Immunoparticles aligned with plasma membranes even when synapses were not present, as if the apposed profile directed the accumulation of RGS4. When we screened such appositions in serial sections, synapses were not found nor did RGS4- particles line up with membranes other than those apposing an axon (Fig. 8 A , B ), suggesting that RGS4 may assemble on the plane of the plasmalemma to potentially regulate focal stimulation of extrasynaptic receptors. In addition, RGS4 was detected in somata and major processes of astrocytes (see also Fig. 1 B ) but not found in slender astrocytic processes in the neuropil, including those that typically enwrap synapses. Hence, it is unlikely that RGS4 could function as regulator of a GPCR in these processes, such as the glial a 2A adrenoceptor ( a 2A -AR; Aoki et al. 1998; Wang et al. 2007). RGS4 on astrocytic membranes—apparently nonsynaptic—was found across from axons as well as postsynaptic profiles (Fig. 8 C ). In summary, we have shown that RGS4 exhibited very distinct distribution patterns on the basis of distance from the soma (i.e., proximal vs. distal dendrites), ultrastructure and synaptology (e.g., Glut-like vs. non-Glut--like axons), and presence of specialized plasmalemma (e.g., synaptic and SSC-lined membranes). The data are schematically summarized in Figure 9. As we discuss in the following sections, such patterns may subserve and likely reflect specificity of neuroregulation, for implicit in the proposed actions of RGS4 is its localization at or near plasma membranes where G proteins and GPCRs assemble. In somata, RGS4 was enriched near SSCs, the smooth reticulum that holds IP R-gated Ca 2 + stores (Delmas and Brown 2002; Berridge 2005). So far, SSC-lined membranes have been identified as DA D 5 receptor (D 5 R)--specific microdomains, linking nonsynaptic receptors to internal Ca 2 + stores in PFC pyramids (Paspalas and Goldman-Rakic 2004a). D 5 Rs couple to Gs to elevate cAMP but have also been implicated in triggering Ca 2 + signaling cascades via the Gq--phosphoinositide signal transduction system (Pacheco and Jope 1997; Jin et al. 2001). 2 + In fact, D 1 /D 5 Rs stimulate Ca release from IP 3 -sensitive stores in perikarya of dissociated cortical neurons (Lezcano and Bergson 2002). Thus, a selective placement of RGS4 in microdomains would potentially allow for regulation of signal transduction triggered by nonsynaptic D 5 Rs, namely phospholipase C activation, IP 3 formation, and IP 3 R-mediated Ca 2 + release from the SSC. Beyond the soma, RGS4 expression in the pyramidal apical dendrite was markedly higher than in any other cellular compartment, including basal dendrites. Intense labeling of the apical dendrite cannot represent newly synthesized protein en passant , for RGS4 levels rose to a peak proximally, and declined sharply as distance from the soma increased. Thus, it likely represents another substrate for neuroregulation. Interestingly, the apical dendritic field proximal to the pyramidal soma expresses the highest levels of serotonin 2A receptor (5HT 2A R) in PFC, supporting the notion that it is ‘‘the ‘hot’ spot for 5HT 2A R-mediated physiological actions relevant to normal and ‘psychotic’ functional states of the cerebral cortex’’ (Jakab and Goldman-Rakic 1998; reviewed in Aghajanian and Marek 2000). The 5HT 2A Rs couple to Gq to mobilize the phosphoinositide system, and several lines of evidence now implicate altered 5HT 2A R signaling in mental illness, including attention deficit hyperactivity disorder, affective and anxiety disorders, and schizophrenia and ...

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... neurons featured a prominent apical dendrite and short basal dendrites, though in most cells, basal branches were not reactive (Fig. 1 B ; see also Fig. 3 A , B ). RGS4 immunoreactivity of the apical dendrite decreased abruptly after 10--40 l m or about 1--3 soma lengths in the majority of neurons, and hence, we did not visualize the elaborate meshwork of distal apical dendrites invading layer I. The neuropil contained the highly reactive first-order dendrites and high-order branches as well as distinct punctate labeling throughout, including in layer I (Fig. 1 C ). However, the end- tufts of apical pyramidal dendrites below the pia were not labeled. Immunoreactive nonpyramidal neurons were clearly identified in layer I (Fig. 1 C ) as well as in layer VI (Fig. 1 F ). Their dendritic ramifications were not labeled or were only visualized within a short distance. A faint reaction was observed in somata of astrocytes in layer I (Fig. 1 B ). High RGS4 immunoreactivity marked the nucleoplasm; the nuclear envelope was labeled with particle clusters subjacent to nucleopores (Fig. 2 A ; Supplementary Fig. 2 A , B ). In compar- ison, the perikaryon displayed moderate immunoreactivity on smooth endomembranes and vesicles bordering Golgi complexes (data not shown). Rough reticular cisterns were often labeled, but overall RGS4 did not show enrichment in the rough endoplasmic reticulum (Fig. 2 A ). In general, the somatic plasma membrane was not labeled nor were axosomatic synapses ‘‘targeted’’ in any way by RGS4 particles. Rather, immunoparticles appeared subjacent to SSC- lined membranes and ‘‘particularly’’ next to the vacuolar rim of a cistern (Fig. 2 B ). It is important to note that SSCs are specialized smooth reticulum endomembranes that hold inositol trisphosphate receptor (IP 3 R)--gated Ca 2 + stores, and may function as junctional and signaling microdomains (see Delmas and Brown 2002; Paspalas and Goldman-Rakic 2004a; and Discussion below). RGS4 levels increased in the base of the tapering apical dendrite (Fig. 3 A ; compare to the weak expression in basal dendrite in B ), peaked in the stem, and remained high along its proximal path. Label filled the entire cytoplasm (Fig. 3 C , D ). Although we did find examples of RGS4 localization directly across from symmetric synapses of varicosities en passant (e.g., Fig. 3 C , inset), these may have been a reflection of the overall high expression levels of the protein in proximal dendrites rather than a specific association with synaptic membranes. Tagged RGS4 under the electron microscope could be traced for several micrometers in the apical dendrite and occasionally into second-order branches, though immunoreactivity dimin- ished with distance from the soma (Fig. 4 A , graph). In section planes capturing apical dendrites along a considerable length, immunoreactivity ended abruptly such that virtually no particles could be found distal to that point. The point where RGS4 could no longer be detected was measured at 7--18 l m from the soma. However, analysis was hindered by the fact that immunoreactive dendrites were often truncated as they left the section plane, suggesting that labeling in certain apical shafts may have extended farther (compare to light microscopic findings in Fig. 1 B ). In apical dendrites where labeling ended before splitting into second-order branches, particle clusters were nonetheless present in the branching point (Fig. 4 B ). The occurrence of RGS4 in bifurcating dendrites may be of particular significance for signal initiation/transduction (see Discussion), especially as numerous axons populate the branch points (Fig. 4 B ). Distal dendrites ( < 0.6 l m in diameter; minor axis) were proportionately the most prominent RGS4 immunoreactive structures in the neuropil (Table 1; Supplementary Fig. 3 A -- G ). They typically contained a few small particle clusters in the cytoplasm, apart from those sections corresponding to synaptic profiles. Indeed, RGS4 in distal dendrites was directly associated with synapses (Fig. 5 A -- E ), which was further confirmed in longitudinally sectioned stems to exclude bias (Supplementary Fig. 3 A ). Synapse-associated particles were separated by 15--35 nm from the postsynaptic membrane, and rarely were embedded in the postsynaptic density (PSD) per se. Note that the label is distributed across from both central and peripheral portions of the synaptic disk, and involves asymmetric and to a lesser extent symmetric axodendritic synapses, exemplified in Figure 5 A ,C, respectively. Seventy-six percent of RGS4- labeled axodendritic synapses were type I, asymmetric junctions (see Table 2). However, RGS4 expression in type II, symmetric synapses could be underestimated as we excluded synapses captured at oblique planes, and those that could not be unequivocally categorized in a single section. In favorable section planes, immunoreactive spines arose from labeled, presumably pyramidal dendrites (Supplementary Fig. 3 C , D ). RGS4 in postsynaptic spines (Table 2) exhibited 2 distinct distribution patterns that were uniquely correlated with inputs from glutamatergic-like (Glut-like) versus non- glutamatergic--like (non-Glut--like) axons (see RGS4 in Axons for definition). Please note that this analysis is not to imply that a given category of spines would receive exclusively symmetric or asymmetric synapses. Rather, it focuses on RGS4 expression on individual axospinous synapses, and attempts to correlate the expression with the axon ultrastructure and synaptology. In this context, it is imperative also to note that axon varicosities other than glutamatergic may in fact establish asymmetric synapses in PFC (e.g., Aoki et al. 1998), whereas certain symmetric synapses may utilize glutamate as a cotrans- mitter (Boulland et al. 2008). Therefore, data should be interpreted with the clear understanding that a portion of axons featuring a Glut-like ultrastructure could potentially utilize another neurotransmitter/neuromodulator, whereas axons with non-Glut features might also engage glutamatergic signaling mechanisms. At asymmetric synapses from Glut-like axons, the particles typically labeled extrasynaptic spine membranes (Fig. 6 A ), and rarely appeared subjacent to the PSD. However, RGS4 was found at perisynaptic membranes flanking the asymmetric synapse (see below; see also Supplementary Fig. 3 B ). In those spines receiving symmetric synapses from non-Glut--like axons, RGS4 was found subjacent to the PSD, as in postsynaptic dendrites (Fig. 6 B ). It should be marked that perisynaptic expression of RGS4 was uniquely associated with spines receiving asymmetric synapses, and not found in spines receiving symmetric synapses or in dendrites receiving either type of synapses (compare the axospinous and axodendritic asymmetric synapses in Fig. 6 C ). Based on their ultrastructure and synaptology (see Peters et al. 1991 and specific criteria below), axon varicosities in the PFC neuropil were categorized as Glut-like and non-Glut--like (compare ax1 to ax2 in Fig. 7 C and in Supplementary Fig. 3 C , but see our statement in previous section). Prevalence of RGS4 expression (Table 1) as well as its subcellular distribution clearly differed in Glut-like and non-Glut--like axons. Non-Glut--like axons featured loose clusters of pleomorphic vesicles, including 80--120 nm clear and dense-cored vesicles. In general, they established symmetric synapses with a 10- to 15-nm-wide cleft and often narrow and multiple active zones. Such axons contained numerous immunoparticles that typically labeled the axoplasm in association with electron-opaque vesicular structures, possibly of the endosomal line (Fig. 7 A , B ). RGS4 particles were also found at extrasynaptic places along the axolemma but the bulk of immunoreactivity clearly appeared intracellularly. Yet in single sections, most RGS4 non-Glut--like varicosities were asynaptic (see Supplementary Fig. 3 C ). Serial sectioning revealed spines as their prime postsynaptic target (Fig. 7 A , B ), which is noteworthy given that dendritic stems would receive the majority of symmetric synapses (presumed c -aminobutyric acidergic [GABAergic]) in the neocortex (Colonnier 1981). One axodendritic synapse of a labeled non-Glut--like axon is shown in Figure 7 C as a part of a synaptic triad involving an immunonegative Glut-like axon. Glut-like axons featured 30-nm round vesicles docked onto the presynaptic grid and established asymmetric synapses with wide, 20-nm clefts and often a central perforation. They contained many fewer RGS4 particles when compared with the non-Glut-- like axons. Immunoparticles, however, were associated with the axolemma at perisynaptic locations and extrasynaptically (Fig. 7 D , E ); the axoplasm was infrequently labeled. Dendritic spines were prime postsynaptic targets for the RGS4-positive Glut-like axons, with shafts receiving fewer synapses. Axon intervaricose segments exhibited a sporadic RGS4 immunoreactivity. Thus, it is not clear whether RGS4 in the nonmyelinated axonal profiles (Fig. 7 F ) may in part represent a functional regulatory component (e.g., for preterminal GPCRs; see Aoki et al. 1998; Muly et al. 2003) or merely indicates protein captured en passant , as could likely be the case in the myelinated profile shown in Figure 7 G . Immunoparticles aligned with plasma membranes even when synapses were not present, as if the apposed profile directed the accumulation of RGS4. When we screened such appositions in serial sections, synapses were not found nor did RGS4- particles line up with membranes other than those apposing an axon (Fig. 8 A , B ), suggesting that RGS4 may assemble on the plane of the plasmalemma to potentially regulate focal stimulation of extrasynaptic receptors. In addition, RGS4 was detected in somata and major processes of astrocytes (see also Fig. 1 B ) but not found in slender astrocytic processes in the neuropil, including those ...

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RGS4 Actions in Mouse Prefrontal Cortex Modulate Behavioral and Transcriptomic Responses to Chronic Stress and Ketamine

Article

Feb 2024
MOL PHARMACOL

The signal transduction protein, regulator of G protein signaling 4 (RGS4), plays a prominent role in physiologic and pharmacological responses by controlling multiple intracellular pathways. Our earlier work identified the dynamic but distinct roles of RGS4 in the efficacy of monoamine-targeting versus fast-acting antidepressants. Using a modified chronic variable stress (CVS) paradigm in mice, we demonstrate that stress-induced behavioral abnormalities are associated with the downregulation of RGS4 in the medial prefrontal cortex (mPFC). Knockout of RGS4 (RGS4KO) increases susceptibility to CVS, as mutant mice develop behavioral abnormalities as early as 2 weeks after CVS resting-state functional magnetic resonance imaging I (rs-fMRI) experiments indicate that stress susceptibility in RGS4KO mice is associated with changes in connectivity between the mediodorsal thalamus (MD-THL) and the mPFC. Notably, RGS4KO also paradoxically enhances the antidepressant efficacy of ketamine in the CVS paradigm. RNA-sequencing analysis of naive and CVS samples obtained from mPFC reveals that RGS4KO triggers unique gene expression signatures and affects several intracellular pathways associated with human major depressive disorder. Our analysis suggests that ketamine treatment in the RGS4KO group triggers changes in pathways implicated in synaptic activity and responses to stress, including pathways associated with axonal guidance and myelination. Overall, we show that reducing RGS4 activity triggers unique gene expression adaptations that contribute to chronic stress disorders and that RGS4 is a negative modulator of ketamine actions. SIGNIFICANCE STATEMENT: Chronic stress promotes robust maladaptation in the brain, but the exact intracellular pathways contributing to stress vulnerability and mood disorders have not been thoroughly investigated. In this study, the authors used murine models of chronic stress and multiple methodologies to demonstrate the critical role of the signal transduction modulator regulator of G protein signaling 4 in the medial prefrontal cortex in vulnerability to chronic stress and the efficacy of the fast-acting antidepressant ketamine.

Oligonol ameliorates liver function and brain function in the 5 × FAD mouse model: transcriptional and cellular analysis

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Oct 2023

Alzheimer's disease (AD) is a common neurodegenerative disease worldwide and is accompanied by memory deficits, personality changes, anxiety, depression, and social difficulties. For treatment of AD, many researchers have attempted to find medicinal resources with high effectiveness and without side effects. Oligonol is a low molecular weight polypeptide derived from lychee fruit extract. We investigated the effects of oligonol in 5 × FAD transgenic AD mice, which developed severe amyloid pathology, through behavioral tests (Barnes maze, marble burying, and nestle shredding) and molecular experiments. Oligonol treatment attenuated blood glucose levels and increased the antioxidant response in the livers of 5 × FAD mice. Moreover, the behavioral score data showed improvements in anxiety, depressive behavior, and cognitive impairment following a 2-month course of orally administered oligonol. Oligonol treatment not only altered the circulating levels of cytokines and adipokines in 5 × FAD mice, but also significantly enhanced the mRNA and protein levels of antioxidant enzymes and synaptic plasticity in the brain cortex and hippocampus. Therefore, we highlight the therapeutic potential of oligonol to attenuate neuropsychiatric problems and improve memory deficits in the early stage of AD.

Immune synaptopathies: how maternal immune activation impacts synaptic function during development

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May 2023
EMBO J

In the last two decades, the term synaptopathy has been largely used to underline the concept that impairments of synaptic structure and function are the major determinant of brain disorders, including neurodevelopmental disorders. This notion emerged from the progress made in understanding the genetic architecture of neurodevelopmental disorders, which highlighted the convergence of genetic risk factors onto molecular pathways specifically localized at the synapse. However, the multifactorial origin of these disorders also indicated the key contribution of environmental factors. It is well recognized that inflammation is a risk factor for neurodevelopmental disorders, and several immune molecules critically contribute to synaptic dysfunction. In the present review, we highlight this concept, which we define by the term "immune-synaptopathy," and we discuss recent evidence suggesting a bi-directional link between the genetic architecture of individuals and maternal activation of the immune system in modulating brain developmental trajectories in health and disease.

Bisphenol-A impairs synaptic formation and function by RGS4-mediated negative regulation of BDNF/NTRK2 signaling in the cerebral cortex

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Jul 2022
DIS MODEL MECH

Bisphenol-A (BPA) is a representative endocrine disruptor, which widely used in a variety of products including plastics, medical equipment and receipts. Hence, most people are exposed to BPA through skin, inhalation and digestive system in everyday life. Furthermore, BPA crosses the blood-brain barrier and is linked to multiple neurological dysfunctions found in neurodegenerative and neuropsychological disorder. However, the mechanisms underlying BPA-associated neurological dysfunctions remain poorly understood. Here, we report that BPA exposure alters synapse morphology and function in the cerebral cortex. Cortical pyramidal neurons treated with BPA showed reduced size and number of dendrites and spines. The density of excitatory synapses was also decreased by BPA treatment. More importantly, we found that BPA disrupted normal synaptic transmission and cognitive behavior. RGS4 and its downstream BDNF/NTRK2 pathway appeared to mediate the BPA effect on synaptic and neurological dysfunction. Our findings shed lights on molecular mechanistic insights into anatomical and physiological neurotoxic consequences related to a serious endocrine modifier.

A new aspect on the correlation of ten SNPs in MIR and their target genes in dopaminergic pathways in schizophrenia

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Mar 2022

Background Schizophrenia (SCZ) is a severe mental disorder in which people interpret reality abnormally. Different studies indicated a complex polygenic control over SCZ. In the present study, we investigated the potential correlation between ten SNPs among MicroRNA (MIR) and their target genes; rs369770942, rs143525573, rs200982455, rs530404895, rs753764536, rs374732351, rs4680, rs165599, rs340597269, and rs10759, and schizophrenia in the Iranian population. Results The results revealed that the T allele in rs200982455 increased the risk factor by 3.19 times. We obtained a significant association between rs165599 and schizophrenia in codominant, dominant, and overdominant inheritance models ( P = 0.016, P = 0.01, P = 0.004, respectively). Moreover, the risk of schizophrenia increased in the presence of the G allele in rs165599 up to 2.12, 2.35, and 2.28 times, respectively. The A allele in rs10759 increased the risk factor up to 1.05 times. Conclusion Our finding showed that some of the studied SNPs within the genes and MIRs involved in the dopaminergic pathway may consider as a biomarker in the diagnostic patterns in Schizophrenia.

Prenatal interleukin 6 elevation increases glutamatergic synapse density and disrupts hippocampal connectivity in offspring

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Nov 2021
IMMUNITY

Early prenatal inflammatory conditions are thought to be a risk factor for different neurodevelopmental disorders. Maternal interleukin-6 (IL-6) elevation during pregnancy causes abnormal behavior in offspring, but whether these defects result from altered synaptic developmental trajectories remains unclear. Here we showed that transient IL-6 elevation via injection into pregnant mice or developing embryos enhanced glutamatergic synapses and led to overall brain hyperconnectivity in offspring into adulthood. IL-6 activated synaptogenesis gene programs in glutamatergic neurons and required the transcription factor STAT3 and expression of the RGS4 gene. The STAT3-RGS4 pathway was also activated in neonatal brains during poly(I:C)-induced maternal immune activation, which mimics viral infection during pregnancy. These findings indicate that IL-6 elevation at early developmental stages is sufficient to exert a long-lasting effect on glutamatergic synaptogenesis and brain connectivity, providing a mechanistic framework for the association between prenatal inflammatory events and brain neurodevelopmental disorders.

The Antipsychotic Drug Clozapine Suppresses the RGS4 Polyubiquitylation and Proteasomal Degradation Mediated by the Arg/N-Degron Pathway

Article

Apr 2021
NEUROTHERAPEUTICS

Although diverse antipsychotic drugs have been developed for the treatment of schizophrenia, most of their mechanisms of action remain elusive. Regulator of G-protein signaling 4 (RGS4) has been reported to be linked, both genetically and functionally, with schizophrenia and is a physiological substrate of the arginylation branch of the N-degron pathway (Arg/N-degron pathway). Here, we show that the atypical antipsychotic drug clozapine significantly inhibits proteasomal degradation of RGS4 proteins without affecting their transcriptional expression. In addition, the levels of Arg- and Phe-GFP (artificial substrates of the Arg/N-degron pathway) were significantly elevated by clozapine treatment. In silico computational model suggested that clozapine may interact with active sites of N-recognin E3 ubiquitin ligases. Accordingly, treatment with clozapine resulted in reduced polyubiquitylation of RGS4 and Arg-GFP in the test tube and in cultured cells. Clozapine attenuated the activation of downstream effectors of G protein–coupled receptor signaling, such as MEK1 and ERK1, in HEK293 and SH-SY5Y cells. Furthermore, intraperitoneal injection of clozapine into rats significantly stabilized the endogenous RGS4 protein in the prefrontal cortex. Overall, these results reveal an additional therapeutic mechanism of action of clozapine: this drug posttranslationally inhibits the degradation of Arg/N-degron substrates, including RGS4. These findings imply that modulation of protein post-translational modifications, in particular the Arg/N-degron pathway, may be a novel molecular therapeutic strategy against schizophrenia.

The genie in the bottle-magnified calcium signaling in dorsolateral prefrontal cortex

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Dec 2020
MOL PSYCHIATR

Neurons in the association cortices are particularly vulnerable in cognitive disorders such as schizophrenia and Alzheimer’s disease, while those in primary visual cortex remain relatively resilient. This review proposes that the special molecular mechanisms needed for higher cognitive operations confer vulnerability to dysfunction, atrophy, and neurodegeneration when regulation is lost due to genetic and/or environmental insults. Accumulating data suggest that higher cortical circuits rely on magnified levels of calcium (from NMDAR, calcium channels, and/or internal release from the smooth endoplasmic reticulum) near the postsynaptic density to promote the persistent firing needed to maintain, manipulate, and store information without “bottom-up” sensory stimulation. For example, dendritic spines in the primate dorsolateral prefrontal cortex (dlPFC) express the molecular machinery for feedforward, cAMP–PKA–calcium signaling. PKA can drive internal calcium release and promote calcium flow through NMDAR and calcium channels, while in turn, calcium activates adenylyl cyclases to produce more cAMP–PKA signaling. Excessive levels of cAMP–calcium signaling can have a number of detrimental effects: for example, opening nearby K ⁺ channels to weaken synaptic efficacy and reduce neuronal firing, and over a longer timeframe, driving calcium overload of mitochondria to induce inflammation and dendritic atrophy. Thus, calcium–cAMP signaling must be tightly regulated, e.g., by agents that catabolize cAMP or inhibit its production (PDE4, mGluR3), and by proteins that bind calcium in the cytosol (calbindin). Many genetic or inflammatory insults early in life weaken the regulation of calcium–cAMP signaling and are associated with increased risk of schizophrenia (e.g., GRM3 ). Age-related loss of regulatory proteins which result in elevated calcium–cAMP signaling over a long lifespan can additionally drive tau phosphorylation, amyloid pathology, and neurodegeneration, especially when protective calcium binding proteins are lost from the cytosol. Thus, the “genie” we need for our remarkable cognitive abilities may make us vulnerable to cognitive disorders when we lose essential regulation.

In-depth transcriptomic analyses of LncRNA and mRNA expression in the hippocampus of APP/PS1 mice by Danggui-Shaoyao-San

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Nov 2020

Alzheimer's disease (AD) is an age-related neurodegenerative disease with a high incidence worldwide, and with no medications currently able to prevent the progression of AD. Danggui-Shaoyao-San (DSS) is widely used in traditional Chinese medicine (TCM) and has been proven to be effective for memory and cognitive dysfunction, yet its precise mechanism remains to be delineated. The present study was designed to investigate the genome-wide expression profile of long non-coding RNAs (LncRNAs) and messenger RNAs (mRNAs) in the hippocampus of APP/PS1 mice after DSS treatment by RNA sequencing. A total of 285 differentially expressed LncRNAs and 137 differentially expressed mRNAs were identified (fold-change ≥2.0 and P < 0.05). Partial differentially expressed LncRNAs and mRNAs were selected to verify the RNA sequencing results by quantitative polymerase chain reaction (qPCR). A co-expression network was established to analyze co-expressed LncRNAs and genes. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to evaluate the biological functions related to the differentially co-expressed LncRNAs, and the results showed that the co-expressed LncRNAs were mainly involved in AD development from distinct origins, such as APP processing, neuron migration, and synaptic transmission. Our research describes the lncRNA and mRNA expression profiles and functional networks involved in the therapeutic effect of DSS in APP/PS1 mice model. The results suggest that the therapeutic effect of DSS on AD involves the expression of LncRNAs. Our findings provide a new perspective for research on the treatment of complex diseases using traditional Chinese medicine prescriptions.

The chromatin basis of neurodevelopmental disorders: Rethinking dysfunction along the molecular and temporal axes

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Jan 2018
PROG NEURO-PSYCHOPH

The complexity of the human brain emerges from a long and finely tuned developmental process orchestrated by the crosstalk between genome and environment. Vis à vis other species, the human brain displays unique functional and morphological features that result from this extensive developmental process that is, unsurprisingly, highly vulnerable to both genetically and environmentally induced alterations. One of the most striking outcomes of the recent surge of sequencing-based studies on neurodevelopmental disorders (NDDs) is the emergence of chromatin regulation as one of the two domains most affected by causative mutations or Copy Number Variations besides synaptic function, whose involvement had been largely predicted for obvious reasons. These observations place chromatin dysfunction at the top of the molecular pathways hierarchy that ushers in a sizeable proportion of NDDs and that manifest themselves through synaptic dysfunction and recurrent systemic clinical manifestation. Here we undertake a conceptual investigation of chromatin dysfunction in NDDs with the aim of systematizing the available evidence in a new framework: first, we tease out the developmental vulnerabilities in human corticogenesis as a structuring entry point into the causation of NDDs; second, we provide a much needed clarification of the multiple meanings and explanatory frameworks revolving around "epigenetics", highlighting those that are most relevant for the analysis of these disorders; finally we go in-depth into paradigmatic examples of NDD-causing chromatin dysregulation, with a special focus on human experimental models and datasets.

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