Human Somatostatin SST4 Receptor Transgenic Mice: Construction and Brain Expression Pattern Characterization

Abstract

Somatostatin receptor subtype 4 (SST4) has been shown to mediate analgesic, antidepressant and anti-inflammatory functions without endocrine actions; therefore, it is proposed to be a novel target for drug development. To overcome the species differences of SST4 receptor expression and function between humans and mice, we generated an SST4 humanized mouse line to serve as a translational animal model for preclinical research. A transposon vector containing the hSSTR4 and reporter gene construct driven by the hSSTR4 regulatory elements were created. The vector was randomly inserted in Sstr4-deficient mice. hSSTR4 expression was detected by bioluminescent in vivo imaging of the luciferase reporter predominantly in the brain. RT-qPCR confirmed the expression of the human gene in the brain and various peripheral tissues consistent with the in vivo imaging. RNAscope in situ hybridization revealed the presence of hSSTR4 transcripts in glutamatergic excitatory neurons in the CA1 and CA2 regions of the hippocampus; in the GABAergic interneurons in the granular layer of the olfactory bulb and in both types of neurons in the primary somatosensory cortex, piriform cortex, prelimbic cortex and amygdala. This novel SST4 humanized mouse line might enable us to investigate the differences of human and mouse SST4 receptor expression and function and assess the effects of SST4 receptor agonist drug candidates.

Reassessment of SST4 Somatostatin Receptor Expression Using SST4-eGFP Knockin Mice and the Novel Rabbit Monoclonal Anti-Human SST4 Antibody 7H49L61

“…In the present investigation, a distinct immunostaining for SST4 was found in the cell bodies and the axons of cortical pyramidal cells, consistent with published data obtained using polyclonal antibodies in humans [ 31 ] and in rats [ 15 , 27 , 28 ] as well as in SST4 humanized mice [ 34 ]. SST4 has also been identified in neurons and satellite cells of trigeminal and dorsal root ganglia of mice and rats, respectively, by mRNA analysis or with polyclonal antibodies, and it was proposed that the analgesic effects of SST4 agonists are partially mediated by these receptors [ 22 , 23 , 29 ].…”

“…Second, with the novel monoclonal SST4 antibody 7H49L61 virtually the same staining pattern was observed in humans, although there may be some species differences, especially with respect to the pituitary and the pancreas, which need to be further evaluated. Third, both staining patterns correspond well to the published mRNA and protein expression data for mice, rats, and humans [ 15 , 26 , 27 , 28 , 29 , 31 , 34 ]. Given that monoclonal anti-mouse SST4 antibodies are currently not available, the SST4-eGFP knockin mouse line can serve as a useful tool for the detection of SST4 in mice at the cellular and subcellular level by direct fluorescence microscopy or immunohistochemical techniques using anti-GFP antibodies.…”

“…First, in immunoblots, 7H49L61 specifically detected its cognate receptor in crude extracts from SST4-transfected HEK-293 cells but not in extracts from mock-transfected cells. Second, 7H49L61 yielded highly efficient staining of distinct cell populations known to express SST4 [ 31 , 32 , 34 ] in formalin-fixed paraffin-embedded human tissue samples. Third, the SST4-immunosignals were completely abolished after pre-adsorption of the antibody with its immunizing peptide.…”

“…Using polyclonal antibodies, SST4 receptors have been discovered in the human cortex, hippocampus, nucleus ruber, globus pallidus, cerebellum, and medulla [ 31 ], as well as in the exocrine pancreas [ 32 , 33 ], the parathyroid glands, the bronchial glands, the fundic glands of the stomach, the Brunner’s glands in the duodenum, and the distal tubules of the kidney [ 32 ]. Additionally, very recently, in a SST4 humanized mouse line, SST4 expression has been disclosed in the cerebral cortex, the olfactory bulb, the hippocampus, the amygdala, and in the trigeminal ganglia [ 34 ].…”

Neurodegeneration in the centrally-projecting Edinger–Westphal nucleus contributes to the non-motor symptoms of Parkinson’s disease in the rat

“…In the rotenone model, four EWcp sections per animal were pretreated for RNAscope according to our recently developed protocol [ 37 ] optimized for 30 µm-thick PFA fixed sections. Subsequent steps of RNAscope protocol was performed according to the supplier’s suggestions.…”