Quantitative Immunohistochemistry to Measure Regional Expression of Nurr1 in the Brain and the Effect of the Nurr1 Heterozygous Genotype.

Evangel Kummari,Shirley X Guo-Ross,Jennifer Makenzie Nutter,Heath S Partington,Jeffrey B Eells

doi:10.3389/fnana.2021.563854

Evangel Kummari, Shirley X Guo-Ross + Show 3 more

Open Access

https://doi.org/10.3389/fnana.2021.563854

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Abstract

The transcription factor Nurr1 is a member of the steroid hormone nuclear receptor superfamily. Ablation of Nurr1 expression arrests mesencephalic dopamine neuron differentiation while attenuation of Nurr1 in the subiculum and hippocampus impairs learning and memory. Additionally, reduced Nurr1 expression has been reported in patients with Parkinson’s disease and Alzheimer’s disease. In order to better understand the overall function of Nurr1 in the brain, quantitative immunohistochemistry was used to measure cellular Nurr1 protein expression, across Nurr1 immunoreactive neuronal populations. Additionally, neuronal Nurr1 expression levels were compared between different brain regions in wild-type mice (+/+) and Nurr1 heterozygous mice (+/−). Regional Nurr1 protein was also investigated at various time points after a seizure induced by pentylenetetrazol (PTZ). Nurr1 protein is expressed in various regions throughout the brain, however, a wide range of Nurr1 expression levels were observed among various neuronal populations. Neurons in the parietal and temporal cortex (secondary somatosensory, insular, auditory, and temporal association cortex) had the highest relative Nurr1 expression (100%) followed closely by the claustrum/dorsal endopiriform cortex (85%) and then subiculum (76%). Lower Nurr1 protein levels were found in neurons in the substantia nigra pars compacta and ventral tegmental area (39%) followed by CA1 (25%) and CA3 (19%) of the hippocampus. Additionally, in the parietal and temporal cortex, two distinct populations of high and medium Nurr1 expressing neurons were observed. Comparisons between +/− and +/+ mice revealed Nurr1 protein was reduced in +/− mice by 27% in the parietal/temporal cortex, 49% in the claustrum/dorsal endopiriform cortex, 25% in the subiculum, 33% in substantia nigra pars compacta, 22% in ventral tegmental area, and 21% in CA1 region of the hippocampus. Based on these data, regional mechanisms appear to exist which can compensate for a loss of a Nurr1 allele. Following a single PTZ-induced seizure, Nurr1 protein in the dentate gyrus peaked around 2 h and returned to baseline by 8 h. Since altered Nurr1 expression has been implicated in neurologic disorders and Nurr1 agonists have showed protective effects, understanding regional protein expression of Nurr1, therefore, is necessary to understand how changes in Nurr1 expression can alter brain function.

Highlights

The nuclear receptor Nurr1 (NR4A2) is structurally similar to the steroid-thyroid hormone nuclear receptors
Nurr1 can function as monomer or form dimers or a heterodimer with other nuclear receptors, such as Nur77, NOR-1, retinoid X receptor (RXR), or retinoic acid receptor (RAR), to bind to DNA response elements, such as the Nur binding response element (NBRE) and Nur response elements (NurRE), to regulate target gene expression (Wallen-Mackenzie et al, 2003)
Nurr1 expression was observed in the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA), as has been previously reported

Summary

Introduction

The nuclear receptor Nurr (NR4A2) is structurally similar to the steroid-thyroid hormone nuclear receptors. The distribution of Nurr mRNA expression in the brain has been previously reported (Reviewed in Eells et al, 2000). Based mostly on in situ hybridization and some immunohistochemistry studies, the distribution of Nurr in the brain includes dopamine neurons in the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) (Backman et al, 1999), glutamatergic neurons in the claustrum (Watakabe et al, 2014), dorsal endopiriform cortex, subiculum (Moon et al, 2015, 2019), and in the deep layer (VIb) of frontal cortex (Arimatsu et al, 2003). Within the frontal cortex Nurr is a marker for cortical sub-plate neurons in layer VIb and co-localizes with Ca2+/calmodulin kinase IV expression (Arimatsu et al, 2003; Navarro et al, 2014). Expression was located in scattered neurons in the insular and secondary somatosensory cortex (Arimatsu et al, 2003) and hippocampus (Saucedo-Cardenas and Conneely, 1996)

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