Identification of hippocampal area CA2 in hamster and vole brain.

Prairie voles (Microtus ochrogaster) and Syrian, or golden, hamsters (Mesocricetus auratus) are closely related to mice (Mus musculus) and are commonly used in studies of social behavior including social interaction, social memory, and aggression. Hippocampal area CA2 is known to play a key role in these behaviors in mice and responds to social stimuli in rats, but CA2 has yet to be characterized in hamsters or voles, which are also used in studies of social behaviors. Here, we used immunofluorescence to determine whether CA2 could be molecularly identified in tissue from voles and hamsters. We found that  staining for many CA2 markers was similar in these three species, with labeling seen in neurons at the distal end of the mossy fibers . In contrast, although perineuronal nets (PNNs) surround CA2 cells in mice, PNN staining differed across species. In voles, both CA2 and CA3 were labeled, whereas in hamsters, labeling was seen primarily in CA3. These results demonstrate that CA2 can be molecularly distinguished from neighboring CA1 and CA3 areas in voles and hamsters with several antibodies commonly used in mice. However, PNN staining is not useful for identifying CA2 in voles or hamsters, suggestive of differing roles for either PNNs or for the hippocampal subregions in social behavior. These findings reveal commonalities across species in the molecular profile of CA2 and should facilitate future studies of CA2 in these species.

Identification of hippocampal area CA2 in hamster and vole brain.

Prairie voles (Microtus ochrogaster) and Syrian, or golden, hamsters (Mesocricetus auratus) are closely related to mice (Mus musculus) and rats (Rattus norvegicus, for example) and are commonly used in studies of social behavior including social interaction, social memory, and aggression. The CA2 region of the hippocampus is known to play a key role in social memory and aggression in mice and responds to social stimuli in rats, likely owing to its high expression of oxytocin and vasopressin 1b receptors. However, CA2 has yet to be identified and characterized in hamsters or voles. In this study, we sought to determine whether CA2 could be identified molecularly in vole and hamster. To do this, we used immunofluorescence with primary antibodies raised against known molecular markers of CA2 in mice and rats to stain hippocampal sections from voles and hamsters in parallel with those from mice. Here, we report that, like in mouse and rat, staining for many CA2 proteins in vole and hamster hippocampus reveals a population of neurons that express regulator of G protein signaling 14 (RGS14), Purkinje cell protein 4 (PCP4) and striatal-enriched protein tyrosine phosphatase (STEP), which together delineate the borders with CA3 and CA1. These cells were located at the distal end of the mossy fiber projections, marked by the presence of Zinc Transporter 3 (ZnT-3) and calbindin in all three species. In addition to staining the mossy fibers, calbindin also labeled a layer of CA1 pyramidal cells in mouse and hamster but not in vole. However, Wolframin ER transmembrane glycoprotein (WFS1) immunofluorescence, which marks all CA1 neurons, was present in all three species and abutted the distal end of CA2, marked by RGS14 immunofluorescence. Staining for two stress hormone receptors-the glucocorticoid (GR) and mineralocorticoid (MR) receptors-was also similar in all three species, with GR staining found primarily in CA1 and MR staining enriched in CA2. Interestingly, although perineuronal nets (PNNs) are known to surround CA2 cells in mouse and rat, we found that staining for PNNs differed across species in that both CA2 and CA3 showed staining in voles and primarily CA3 in hamsters with only some neurons in proximal CA2 showing staining. These results demonstrate that, like in mouse, CA2 in voles and hamsters can be molecularly distinguished from neighboring CA1 and CA3 areas, but PNN staining is less useful for identifying CA2 in the latter two species. These findings reveal commonalities across species in molecular profile of CA2, which will facilitate future studies of CA2 in these species. Yet to be determined is how differences in PNNs might relate to differences in social behavior across species.

BAG3 regulates the specificity of the recognition of specific MAPT species by NBR1 and SQSTM1.

Macroautophagy/autophagy receptors are essential for the recognition and clearance of specific cargos by selective autophagy, which is essential for maintaining MAPT proteostasis. Previous studies have implicated different autophagy receptors in directing distinct species of MAPT to autophagy, but the underlying mechanisms have not been fully investigated. Here we examine how the autophagy receptors NBR1 and SQSTM1 differentially associate with specific forms of MAPT. In primary neurons depletion of NBR1, unlike depletion of SQSTM1, significantly increased phosphorylated MAPT levels. The specificity of the interactions was confirmed using in vitro binding assays with purified proteins. We provide direct evidence that the co-chaperone BAG3 promotes the preferential association of NBR1 with monomeric MAPT and SQSTM1 with oligomeric MAPT. Using an in vitro affinity-isolation assay, we show that SQSTM1 only binds to monomeric MAPT when BAG3 is absent and fails to bind when BAG3 is present. The opposite is true of NBR1; its association with monomeric MAPT was dependent on the presence of BAG3. Interestingly, in Alzheimer disease brain the association of NBR1 with BAG3 was significantly decreased. In a mouse model, ablation of BAG3 in neural cells disrupted the association of NBR1 with phosphorylated MAPT and led to increased levels of phosphorylated and oligomeric MAPT. Overall, our results uncover a novel role for BAG3 in regulating the specificity of selective autophagy receptors in targeting different species of MAPT and provide compelling evidence that BAG3 plays a key role in maintaining MAPT proteostasis.Abbreviations: AD: Alzheimer disease; BAG3: BCL2-associated athanogene 3; BSA: bovine serum albumin; CERAD: Consortium to Establish a Registry for Alzheimer's Disease; ESCRT: endosomal sorting complexes required for transport; GST: glutathione S-transferases; MAPT: microtubule-associated protein tau; NBR1: NBR1, autophagy cargo receptor; NFT: neurofibrillary tangles; PMI: postmortem interval; SQSTM1: sequestosome 1.

BAG3 regulates the specificity of the recognition of specific MAPT species by NBR1 and SQSTM1.

Autophagy receptors are essential for the recognition and clearance of specific cargos by selective autophagy, which is essential for maintaining MAPT proteostasis. Previous studies have implicated different autophagy receptors in directing distinct species of MAPT to autophagy, but the underlying mechanisms have not been fully investigated. Here we examine how the autophagy receptors NBR1 and SQSTM1 differentially engage specific forms of MAPT and facilitate their clearance. In primary neurons depletion of NBR1, unlike depletion of SQSTM1, significantly increased phosphorylated MAPT levels. The specificity of the interactions were confirmed using in vitro binding assays with purified proteins. We provide direct evidence that NBR1 preferentially binds to monomeric MAPT, while SQSTM1 interacts predominantly with oligomeric MAPT, and that the co-chaperone BAG3 regulates the specificity of these interactions. Using an in vitro pulldown assay, we show that SQSTM1 only binds to monomeric MAPT when BAG3 is absent and fails to bind when BAG3 is present. The opposite is true of NBR1; its binding to monomeric MAPT was dependent on the presence of BAG3. Interestingly, in Alzheimer's disease brain the association of NBR1 with BAG3 was significantly decreased. In a mouse model, ablation of BAG3 in neural cells disrupted the association of NBR1 with phosphorylated MAPT and lead to increased levels of phosphorylated and oligomeric MAPT. Overall, our results uncover a novel role for BAG3 in regulating the specificity of selective autophagy receptors in targeting different species of MAPT and provide compelling evidence that BAG3 plays a key role in maintaining MAPT proteostasis.